Hot Topics in Cell Biology by Melinda Taylor

Hot Topics in Cell Biology

Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

Chartridge Books Oxford Hexagon House, Avenue 4, Station Lane Witney, Oxford OX28 4BN, UK Tel: +44 (0) 1865 598888 Chartridge Books Oxford is an imprint of Biohealthcare Publishing (Oxford) Limited.

First published in 2012 Copyright ﾂｩ2012: The editors and contributors, unless otherwise stated. ISBN paperback: 978-1-909287-00-6 ISBN digital (pdf): 978-1-909287-01-3 ISBN digital ebook (epub): 978-1-909287-02-0 ISBN digital ebook (mobi): 978-1-909287-03-7 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. All rights reserved. No part of this publication may be reproduced, stored in or introduced into a retrieval system, or transmitted, in any form, or by any means, (electronic, mechanical, photocopying, recording or otherwise) without the prior written permission of the Publishers. This publication may not be lent, resold, hired out or otherwise disposed of by way of trade in any form of binding or cover other than that in which it is published without the prior consent of the Publishers. Any person who does any unauthorised act in relation to this publication may be liable to criminal prosecution and civil claims for damages. The Publishers make no representation, express or implied, with regard to the accuracy of the information contained in this publication and cannot accept any legal responsibility or liability for any errors or omissions. The material contained in this publication constitutes general guidelines only and does not represent to be advice on any particular matter. No reader or purchaser should act on the basis of material contained in this publication without first taking professional advice appropriate to their particular circumstances. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book.

Produced from electronic copy supplied by the editors. Printed in the UK and USA. Cover image: Neurons differentiated from Parkinson's disease patient-specific induced pluripotent stem cells, before showing any obvious morphological phenotype, stained for TUJ1 (green) and tyrosine hydroxylase (red), and counterstained with DAPI (blue). Courtesy of ﾃ］gel Raya, author of the article in this book: Sﾃ｡nchez-Danﾃｩs et al.

List of contents FOREWORD

CONTRIBUTORS

INTRODUCTION An overview of cell biology in the 21st century. Rick Visser and José Becerra

1.1. Dynamic of cell compartments Dynamics of cell compartments and pathological implications. Inés M. Antón and

Jaime Renau-Piqueras

Non-Muscle Myosin II Integrates Cell Adhesion and Migration by Controlling the Compartmentalization of Adhesive Signaling. Alba Juanes-García and Miguel

Vicente-Manzanares

A 3D-functional-screening identifies new components of the machinery for epithelial lumen morphogenesis. Fernando Martín-Belmonte, Manuel Gálvez-

Santisteban, Alejo E. Rodriguez-Fraticelli, Silvia Vergarajáuregui, Inmaculada Bañón, and Ilenia Bernascone

1.2. The intracellular trafficking Some basic ideas about the intracellular membrane trafficking. Gustavo Egea and

María M. Malagón

Introducing the temporal dimension to the ultrastructural analysis of membrane budding in S. cerevisiae. Fatima-Zahra Idrissi, Isabel María Fernández-Golbano

and María Isabel Geli

1.3. Cell signalling Cell signalling and communication. Isabel Fabregat

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

MYADM, a member of the MAL protein family, regulates Rac1 targeting to membrane rafts and cell migration. Juan F. Aranda, Natalia Reglero-Real, Beatriz

Marcos-Ramiro, Ana Ruiz-Sáenz, Miguel Bernabé-Rubio, Isabel Correas, Jaime Millán, Miguel A. Alonso

1.4. Autophagy, apoptosis and cell homeostasis An overview of cell homeostasis. Isabel Varela-Nieto and Mª Angela Burrell

Differential regulation of autophagy, proliferation and cell survival during otic neurogenesis. María R. Aburto, Marta Magariños and Isabel Varela-Nieto

1.5. Cell biology of aging Overview of cell senescence and longevity. Guillermo López-Lluch

Mitochondrial homeostasis and healthy aging. Guillermo López-Lluch

1.6. Plant cell biology Cell biology of plant development and adaptation. Pilar S. Testillano and Dolores

Rodríguez

Root Behaviour: From Sensory Perceptions to Motoric Actions. František Baluška

Plant steroid hormones control cell cycle and differentiation in the Arabidopsis root.

Mary-Paz González-García, Josep Vilarrasa-Blasi and Ana I. Caño-Delgado

Nuclear reprogramming of plant differentiating cells: remodelling of nuclear domains and epigenetic regulation. Pilar S. Testillano and María-Carmen Risueño

Judit Berman, Changfu Zhu and Paul Christou

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

1.7. Methods in Cell biology The last advances in TEM for the in situ molecular recognition.

Carmen López-Iglesias, Lidia Delgado, Gema Martínez, Yolanda Muela, Nieves Hernández

Image analysis as a service for cell-based assays and tissue cultures.

Rafael Rodríguez

In Vivo non-Invasive Bioluminescence Imaging: a Short Course. Nuria Rubio,

Olaia Fernández-Vila, María Alieva, Julio Rodríguez-Bagó and Jerónimo Blanco

APPLIED CELL BIOLOGY Cell biology and beyond: Applications of cell biology. Leonor Santos-Ruiz

106

2.1. Cell biology of cancer Cancer cell biology: key points in future treatments of cancer.

Guillermo López-Lluch

112

Ribosome biogenesis, cell cycle progression and tumorigenesis: Key points in ribosomopathies and future treatment of cancer. Teng Teng and George Thomas

118

Epithelial plasticity and metastasis: New regulatory mechanisms. Amparo Cano

and Gema Moreno-Bueno

130

2.2. Cell Therapy and Tissue Engineering Regenerative Medicine in the Context of Cell Biology: Technical and Practical Approaches. José A. Andrades and María J. Gómez-Lechón

138

The Stem Cell And Its Neighborhood: The Niche Of Stem Cells In Animal And Plant Organisms. José Becerra

146

Efficiency and Biosafety in Cell Therapy. Agustin G. Zapata

151

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

New strategies for cardiac regeneration. Felipe Prósper

158

Stem cells in Skin Tisular Engineering. Sara Guerrero-Aspizua, Marta Carretero

and Marcela del Río

166

Modeling Parkinson’s disease using induced pluripotent stem cells. Adriana

Sánchez-Danés, Antonella Consiglio and Ángel Raya

181

2.3. Neurodegeneration and Cell Biology Neurodegeneration: A challenge for cell biology in the 21st century. Antonia

Gutierrez and Joan X. Comella

189

Neuron Biology In Health And Disease. José Carlos Dávila

195

Brain derived neurotrophic factor: Neuronal dysfunction versus cell death in Huntington’s Disease. Jordi Alberch, Josep M. Canals, Silvia Ginés and Esther

Pérez-Navarro

202

Oligomeric Amyloid-Beta And Neuronal Dysfunction In Alzheimer’s Disease.

Antonia Gutiérrez and Javier Vitorica

207

Stem Cells And Adult Neurogenesis In The Human Brain.

Mª Salomé Sirerol-Piquer and José Manuel García-Verdugo

214

2.4. Nanotechnology and Cell Biology: challenges and opportunities Functional Magnetic Nanoparticles For Life Sciences. Clara Marquina, Alejandro

Perez-Luque, Gerardo F. Goya, Rodrigo Fernandez-Pacheco, Laura Asin, Jesús M. De La Fuente, Maria-Carmen Risueño, Pilar S. Testillano and Manuel R. Ibarra

221

Tumor targeting of drug-loaded magnetic nanoparticles by an external magnetic field for in vivo cytokine delivery in cancer immunotherapy. Raquel Mejías and

Domingo F. Barber

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

227

Rational design and development of nanotechnology-based vasoactive intestinal peptide (VIP) applications in health research. Rebecca Klippstein, Soledad López-

Enríquez and David Pozo

242

COROLLARY Towards a Cell Evo-Devo. Manuel Marí-Beffa

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

252

Foreword Cell biology has advanced rapidly in the last decades, being

possibly

one

of the

biological

disciplines that has progressed most since the last half of the twentieth century. During this period, it has gained answers to important questions in Biology. Epigenetics, autophagy, induced pluripotent stem cells (iPS), sequencing technology, RNA interference, etc., are some of the today’s hot fields, experiencing a dayby-day progress. These and other major issues of modern cell biology were discussed at the Congress of the Spanish Society for Cell Biology (SSCB), held in Malaga (Spain) in December 2011, where both Spanish and foreign scientists exposed an discussed the most significant advances attained in their laboratories. This

book

includes

the

most

important

communications, in article form with an easy format, constituting a summary of "Hot topics in Cell Biology". It does not contain all of the issues that could be treated under that subject, but only part of what was presented at the conference. The authors have not been forced to adopt a specific style; instead, they have been given freedom to express themselves how they wanted, taking only some formal matters in consideration. The editors are very grateful to all authors who have participated and hope that they find their ideas faithfully reflected. We would also like to thank María del Mar Bueno for her creativity and for putting her efforts into the edition of this book. We are particularly thankful to the SSCB and its Executive

Board

for

their

assistance

and

encouragement and to the Spanish and Andalusian Governments for their financial support. José Becerra and Leonor Santos-Ruiz, Editors

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Contributors María R. Aburto Institute for Biomedical Research “Alberto Sols”, CSIC-UAM, CIBERER, Madrid, Spain.

Jordi Alberch Dept of Cellular Biology, Immunology and Neuroscience. Medical School. IDIBAPS. CIBERNED. University of Barcelona. Casanova 143, 08036 Barcelona. Spain.

María Alieva Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

Miguel A. Alonso Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

José A. Andrades Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, University of Málaga. CIBER-BBN. 29071 Málaga, Spain.

Inés M. Antón Centro Nacional de Biotecnología (CNB-CSIC). 28049 Madrid, Spain; CIBERNED,

Centro

Investigación

Biomédica

Red

Enfermedades

Neurodegenerativas, Spain.

Juan F. Aranda Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Laura Asin Instituto de Nanociencia de Aragón (INA) (Universidad de Zaragoza) Zaragoza, Spain.

František Baluška IZMB, University of Bonn, Kirschallee 1, 53115 Bonn, Germany

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Inmaculada Bañón Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Domingo F. Barber Department of Immunology & Oncology, Centro Nacional de Biotecnología (CNBCSIC), Darwin 3, Cantoblanco 28049, Madrid, Spain

José Becerra BIONAND-UMA. Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, University of Malaga. CIBER-BBN. Málaga, Spain.

Judit Berman Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain

Miguel Bernabé-Rubio Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Ilenia Bernascone Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Jerónimo Blanco Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

Mª Angela Burrell Departamento de Histología y Anatomía Patológica, Universidad de Navarra. Pamplona, Spain

Josep M. Canals Dept of Cellular Biology, Immunology and Neuroscience. Medical School. IDIBAPS. CIBERNED. University of Barcelona. Casanova 143, 08036 Barcelona. Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

iii

Amparo Cano Departamento de Bioquímica, UAM. Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM. Instituto de Investigación Sanitaria IdiPAZ. c/Arzobispo Morcillo, 2. 28029 Madrid. Spain

Ana I. Caño-Delgado Molecular Genetics Department, Centre for Research in Agricultural Genomics (CRAG), Bellaterra, Barcelona, Spain.

Teresa Capell Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain

Marta Carretero Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT, Centro de Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER), Madrid, Spain

Paul Christou Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain. Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 08018, Barcelona, Spain

Joan X. Comella Cell Signaling and Apoptosis Group, Vall d'Hebron-Institut de Recerca. Network Biomedical Research Center for Neurodegenerative Diseases (CIBERNED) Barcelona, Spain.

Antonella Consiglio Institute for Biomedicine (IBUB), University of Barcelona, Barcelona, Spain. Department of Biomedical Science and Biotechnology, University of Brescia, Brescia, Italy

Isabel Correas Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

José C. Dávila Depto. Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, CIBERNED, Spain.

Lidia Delgado Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.

Gustavo Egea Departamento de Biologia Celular, Inmunología y Neurociencias, Facultad de Medicina, Universidad de Barcelona-IDIBAPS, 08036 Barcelona, Spain

Isabel Fabregat Bellvitge Biomedical Research Institute (IDIBELL). Hospitalet de Llobregat, Barcelona, Spain

Gemma Farre Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain

Isabel Mª Fernández-Golbano Instituto de Biología Molecular de Barcelona (IBMB-CSIC), C/ Baldiri Reixac 15, 08028 Barcelona, Spain.

Rodrigo Fernández-Pacheco Laboratorio Microscopias Avanzadas (LMA) (Universidad de Zaragoza) Zaragoza, Spain.

Olaia Fernández-Vila Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

Jesus M. De La Fuente Instituto de Nanociencia de Aragón (INA) (Universidad de Zaragoza) Zaragoza, Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Manuel Gálvez-Santisteban Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

José M. García-Verdugo. Department of Comparative Neurobiology. Cavanilles Institute. University of Valencia. CIBERNED. Spain.

Mª Isabel Geli Instituto de Biología Molecular de Barcelona (IBMB-CSIC), C/ Baldiri Reixac 15, 08028 Barcelona, Spain.

Silvia Ginés Dept of Cellular Biology, Immunology and Neuroscience. Medical School. IDIBAPS. CIBERNED. University of Barcelona. Casanova 143, 08036 Barcelona. Spain.

María J. Gómez-Lechón Unidad de Hepatología Experimental. IIS Hospital La Fe, Avda. Campanar 21, 46009 Valencia. CIBEREHD. Spain.

Mary-Paz González-García Molecular Genetics Department, Centre for Research in Agricultural Genomics (CRAG), Bellaterra, Barcelona, Spain.

Gerardo F. Goya Departamento de Física de la Materia Condensada,Universidad de Zaragoza, Zaragoza, Spain. Instituto de Nanociencia de Aragón (INA) (Universidad de Zaragoza) Zaragoza, Spain.

Sara Guerrero-Aspizua Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT. Department of Bioengineering, Universidad Carlos III (UC3M), Centro de Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER), Madrid, Spain

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Antonia Gutiérrez Dept. of Cell Biology, Genetics and Physiology, Faculty of Sciences, University of Malaga. Network Biomedical Research Center for Neurodegenerative Diseases (CIBERNED), Spain.

Nieves Hernández Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.

Manuel R. Ibarra Departamento de Física de la Materia Condensada,Universidad de Zaragoza, Zaragoza, Spain. Instituto de Nanociencia de Aragón (INA) (Universidad de Zaragoza) Zaragoza, Spain. Laboratorio Microscopias Avanzadas (LMA) (Universidad de Zaragoza) Zaragoza, Sapain.

Fatima-Zahra Idrissi Instituto de Biología Molecular de Barcelona (IBMB-CSIC), C/ Baldiri Reixac 15, 08028 Barcelona, Spain.

Alba Juanes-García Universidad Autónoma de Madrid, Facultad de Medicina and Instituto de Investigación Sanitaria- Princesa, c/Diego de León 62, 28006 Madrid, Spain

Rebecca Klippstein CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO), Americo Vespucio s/n. Parque Cientifico y Tecnologico Cartuja 93, E-41092, Seville, Spain. Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Sanchez Pizjuan, 4. E-41009, Seville, Spain

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

vii

Soledad López-Enríquez CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO), Americo Vespucio s/n. Parque Cientifico y Tecnologico Cartuja 93, E-41092, Seville, Spain. Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Sanchez Pizjuan, 4. E-41009, Seville, Spain

Carmen López-Iglesias Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.

Guillermo López-Lluch Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, CIBERER, Instituto Carlos III, Carretera de Utrera Km. 1, 41013 Sevilla, Spain.

Marta Magariños Institute for Biomedical Research “Alberto Sols”, CSIC-UAM, CIBERER, Madrid, Spain.

María M. Malagón Departamento de Biología Celular, Fisiología e Inmunología, IMIBIC, Universidad de Córdoba, 14014 Córdoba, Spain

Beatriz Marcos-Ramiro Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Manuel Marí-Beffa Departamento de Biología Celular, Genética y Fisiología Facultad de Ciencias. Universidad de Málaga. CIBER-BBN. 29071-Málaga, Spain.

Clara Marquina Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC- Universidad de Zaragoza, Zaragoza, Spain. Departamento de Física de la Materia Condensada,Universidad de Zaragoza, Zaragoza, Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

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Fernando Martín-Belmonte Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Gema Martínez Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.

Raquel Mejías Department of Immunology & Oncology, Centro Nacional de Biotecnología (CNBCSIC), Darwin 3, Cantoblanco 28049, Madrid, Spain.

Jaime Millán Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Gema Moreno-Bueno Departamento de Bioquímica, UAM. Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM. Instituto de Investigación Sanitaria IdiPAZ. c/Arzobispo Morcillo, 2. 28029 Madrid. Spain

Yolanda Muela Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.

Alejandro Pérez-Luque IFAPA, Centro Alameda del Obispo, Área de Mejora y Biotecnología, PO Box 3092, 14004 Córdoba, Spain.

Esther Pérez-Navarro Dept of Cellular Biology, Immunology and Neuroscience. Medical School. IDIBAPS. CIBERNED. University of Barcelona. Casanova 143, 08036 Barcelona. Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

David Pozo CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO), Americo Vespucio s/n. Parque Cientifico y Tecnologico Cartuja 93, E-41092, Seville, Spain. Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Sanchez Pizjuan, 4. E-41009, Seville, Spain. BIONAND-Andalusian Center for Nanomedicine and Biotechnology. Av. Severo Ochoa, 34, Parque Tecnologico de Andalucia, E-29590 Malaga, Spain.

Felipe Prósper Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain

Ángel Raya Control of Stem Cell Potency Group, Institute for Bioengineering of Catalonia (IBEC),

Center

for

Networked

Biomedical

Research

Bioengineering,

Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Natalia Reglero-Real Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Jaime Renau-Piqueras Sección de Biología y Patología Celular, Centro de Investigación, Hospital La Fe, Valencia, Spain.

Marcela del Río Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT, Madrid. Department of Bioengineering, Universidad Carlos III (UC3M), Madrid. Centro de Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER), Madrid, Spain.

María-Carmen Risueño Plant Development and Nuclear Architecture. Biological Research Center, CIBCSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Dolores Rodríguez Centro

Hispano-Luso

Investigaciones

Agrarias

(CIALE), University

Salamanca, Spain.

Rafael Rodríguez Chief Technology Officer and co-founder of Wimasis. Quantitative Image Analysis Automation. Av. Gran Capitan, 46 3 8.Cordoba, Spain. Wimasis GmbH. Karlstrasse 55. D-80333 Munich, Germany

Julio Rodríguez-Bagó Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain.

Alejo E. Rodríguez-Fraticelli Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Nuria Rubio Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain

Ana Ruiz-Sáenz Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

Adriana Sánchez-Danés Institute for Biomedicine (IBUB), University of Barcelona, Barcelona, Spain

Leonor Santos-Ruiz BIONAND-UMA. Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, University of Malaga. CIBER-BBN. Málaga, Spain.

Mª Salomé Sirerol-Piquer Department of Comparative Neurobiology. Cavanilles Institute. University of Valencia. CIBERNED. Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Teng Teng Department of Cancer and Cell Biology. Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, University of Cincinnati, OH 45220, USA

Pilar S. Testillano Plant Development and Nuclear Architecture. Biological Research Center, CIBCSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

George Thomas Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, University of Cincinnati, OH 45220, USA. Catalan Institute of Oncology, Bellvitge Biomedical Research Institute, IDIBELL, Gran Via de l'Hospitalet, 199, 08908 Hospitalet de Llobregat, Barcelona, Spain.

Isabel Varela-Nieto Instituto Investigaciones Biomedicas “Alberto Sols” CSIC-UAM, CIBERER, IdiPaz, Madrid, Spain

Silvia Vergarajáuregui Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Miguel Vicente-Manzanares Universidad Autónoma de Madrid, Facultad de Medicina and Instituto de Investigación Sanitaria- Princesa, c/Diego de León 62, 28006 Madrid, Spain

Josep Vilarrasa-Blasi Molecular Genetics Department, Centre for Research in Agricultural Genomics (CRAG), Bellaterra, Barcelona, Spain.

Rick Visser BIONAND-UMA. Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, University of Malaga. CIBER-BBN. Málaga, Spain.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

xii

Javier Vitorica Dept Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville (IBIS). Network Biomedical Research Center for Neurodegenerative Diseases (CIBERNED). Sevilla, Spain.

Agustin G Zapata Department of Cell Biology. Faculty of Biology. Complutense University. 28040 Madrid, Spain

Zhu Changfu Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain

Uxue Zorrilla-L贸pez Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain

HOT TOPICS IN CELL BIOLOGY - Edited by Jos茅 Becerra and Leonor Santos-Ruiz

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1. INTRODUCTION An overview of cell biology in the 21st century Rick Visser and José Becerra BIONAND-UMA. Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, UMA. CIBER-BBN, Málaga, Spain. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, (CIBER-BBN), 29071 Málaga, Spain

It soon will be 350 years ago since Robert Hooke wrote (and drew) his outstanding Micrographia, in which the term cellula was used for the first time. Although Hooke’s cell was actually just the description of a purely morphologic unit, referring to the polyhedral shapes that he observed in the thin slices of cork he studied with his primitive microscope, his discovery, together with those made by the contemporary Antonie van Leeuwenhoek, resulted to be one of the first steps towards the setting up of what we today know as Cell Biology. However, almost another 200 years had to pass for these little cellulae to acquire their true value, when Jacob Schleiden and Theodor Schwann postulated the first principles of the Cell Theory, defining the cell as the fundamental unit of structure and function in all living things. The knowledge about the cell and its functioning that we have gained during the 20th century is surely far behind the imagination Hooke and his contemporaries may have had. Nevertheless, the great complexity of the mechanisms that underlie cell physiology and the relations a cell establishes with its environment and with other cells determines that, instead of reaching full understanding of the cell’s biology, new hot topics arise continuously within the discipline. In this chapter we will briefly review some of these current issues. In the last decade, one of these topics has certainly been everything there is around epigenetics. Although the term is already more than 50 years old, its definition has experienced a significant change during the last years. Nowadays, epigenetics refers to the study of heritable changes of gene function that cannot be explained by changes in the DNA sequence, and has profoundly altered our concept of inheritance [1]. Although the molecular basis of heritable epigenetics has been studied in a variety of organisms, transgenerational so-called epimutations have been mainly observed in plants [2]. However, non-mutational changes that are transmitted from one cell to its daughter cells during one or more division cycles have also been described in animals, and more and more examples are brought to light thanks to the development of new molecular biology techniques, such as ChIP- or bisulfite-sequencing, or DNA adenine methyltransferase identification (DamID). Epigenetic changes mainly originate by two types of mechanisms: DNA methylation and chromatin remodelling through post-translational modifications of histones. The fact that these epigenetic changes and their effects on phenotype are potentially affected by ageing and/or environmental stimuli somehow brings some Lamarckian ideas back from exile. However, many of the mechanisms by which epigenetic gene silencing occur and is transmitted between cell generations remain unknown, and epigenetics will still give a lot to talk about the next years.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Epigenetics is not the only newly discovered way of regulating gene expression. With the development of sophisticated sequencing techniques that have allowed revealing the entire genome of many species, researchers were able to identify great regions of the human genome that were initially thought to be non-functional. Nevertheless, the evolutionary conservation across the mammalian genomes of much more sequence than can be explained by the protein-coding regions already indicated that many of these socalled non-coding regions might have a certain function in the cell. By now it is known that these regions are in fact transcribed to non-coding RNAs (ncRNAs), which have an increasingly important role in gene regulation and biological processes fundamental to both normal development and disease [3]. Among the different types of ncRNAs, the group of the microRNAs (or miRNAs) is the most widely studied so far. These miRNAs, from which there are estimated to be over 1000 in the human genome and which might regulate up to 60% of it, are involved in a large number of essential biological functions that are critical to normal development. Furthermore, the deregulation of these same miRNAs has been implicated in numerous disease states, including cancer and cardiomyopathies [4]. miRNAs are mainly known to cause mRNA degradation or to inhibit their translation by binding to their 3’ untranslated regions. However, there are evidences suggesting that miRNAs might be involved in positive regulation of mRNA translation as well. In animals, one specific miRNAs usually targets a diverse set of mRNAs and, therefore, has the capacity to regulate multiple genes and to redirect or reprogram biological pathways. Hence, the importance of miRNAs and other ncRNAs has become clearer during the last years, and miRNA-based therapies are currently under investigation, as well as miRNA expression patterns are being tested as part of clinical decision algorithms in clinical trials [5]. Besides miRNAs, other small ncRNAs (small interfering RNAs or siRNAs and Piwiinteracting RNAs or piRNAs) are also known to be involved in gene regulation [6]. Based on the mechanisms by which these small RNAs control gene expression, a whole new technical field has been developed for post-transcriptional gene silencing: RNA interference (RNAi). These techniques have become very powerful tools in reverse genetics for in vivo and in vitro systems, and will not only allow scientist to better understand gene function by achieving easy down-knocking of gene expression , but are also likely to be valuable for therapeutic applications [7]. In fact, the first evidences supporting a therapeutic potential of RNAi were published in 2003 by Song and co-workers, which demonstrated protection against liver fibrosis using siRNAs targeted to a cell death receptor’s mRNA in a mouse model of autoimmune hepatitis [8]. Since then, many therapeutic programs based on RNAi for the treatment of several human diseases, including age-related macular degeneration, hepatitis B and C, AIDS, asthma or different types of cancer, have entered phase I or II clinical trials [7]. But not only animal cell biology benefits from the discovery of RNAi. Since miRNAs have been widely described in plants, RNAi-based biotechnological approaches have been and are being currently used to produce plants with lower levels of natural toxins or allergens, or to obtain plants with an increased resistance to viral diseases of agronomic relevance [9]. During the last years, not only the research focused on directly studying gene regulation has experienced significant, sometimes paradigm-shifting, advances. We have also gained important new knowledge about cellular processes such as programmed cell death or autophagy. For a long time, apoptosis was thought to be the sole form of programmed cell death during development, homeostasis and disease. In opposition, necrosis has been considered an unregulated (and, thus, uncontrollable) process. Recent evidences have now revealed that some forms of necrosis actively involve defined signalling pathways that contribute to

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

cell death [10]. This type of regulated necrosis, termed necroptosis, requires the kinase activity of receptor-interacting protein-1 (RIPK1) and RIPK3, and seems to be negatively regulated by the apoptotic inducer caspase-8, since genetic deletion of RIPK3 rescues caspase-8-deficient mice from embryonic lethality. Necroptosis is now recognized to be a cellular defence mechanism against viral infections and as being critically involved in ischemia-reperfusion damage, and future studies will have to determine which roles this type of regulated necrosis play in cell death processes that are thought to shape embryonic development but are unaffected when apoptosis is blocked. Autophagy is a generic term for all the pathways, which include macro-, micro- and chaperone-mediated autophagy, by which cytoplasmic materials are delivered to the lysosome in animal cells or to the vacuole in plant cells and yeasts [11]. Of these three types, macroautophagy is the major and most extensively studied type of autophagy. Under conditions of stress, autophagy serves to generate much needed nutrients for the cell. However, when it is overactivated, the destruction of organelles can lead to cell death, being autophagy, at times, an alternative to apoptosis to eliminate unwanted or damaged cells. Recently, complex relations between certain diseases and autophagic mechanisms have been described. For example, autophagy seems to have a tumor-supressing role, especially in the liver where, when autophagy-related genes (ATG) are deleted, spontaneous benign tumorogenesis is observed in mice. Nevertheless, it has also been shown that basal autophagy is used by certain tumor cells as a survival mechanism to protect against ischemia and apoptotic signals [12]. Besides its relation with cancer, the deregulation of autophagic mechanisms has also been related with several other diseases, such as Parkinson, Alzheimer or cystic fibrosis. Furthermore, the fact that many regimes that promote longevity, such as caloric restriction, TOR suppression or sirtuin activation, also induce autophagy raise the question of how much might autophagy contribute to prolonging life span. Finally, the importance of autophagic mechanisms during development is currently an active field of research (as an example, see the chapter written by Aburto MR et al. in this book, where the role of autophagy in otic neurogenesis is discussed). Although many of these and other questions are being answered as research continues, many others still remain, and the physiological role of autophagy in certain key organs (bone, skin, blood vessels…) is still unknown. Despite the great amount of knowledge gained during the last decade about the above mentioned and other important topics, if members of the scientific community were asked to choose the most relevant finding of these times, probably many of them would mention the induced pluripotent stem cells (iPSCs). Since Takahashi and Yamanaka showed, in 2006, that fibroblasts can be reprogrammed to become embryonic stem cell-like cells by ectopically co-expressing just four transcription factors (SOX2, OCT4, KLF4 and c-MYC), their worldwide praised discovery not only changed our point of view on cell differentiation and plasticity, but also opened new doors to regenerative medicine, especially after a great amount of follow-up studies demonstrated that human fibroblasts and other human cell types (pancreatic β cells, neural stem cells, mature B cells, stomach and liver cells, melanocytes, adipose stem cells and keratinocytes, among others) can also be successfully reprogrammed [13]. However, after the initial burst of optimism, stem cell biologists have now taken a step back to better analyse whether iPSCs and embryonic stem cells (ECSs) are really identical or not. Although both pluripotent cell types should be, in theory, functionally equivalent, the truth is that they show some genetic and epigenetic differences that reflect their different histories. Nevertheless, it should also be noticed that, per se, the heterogeneity within each population of cells is greater than initially thought [14]. In fact, after the reprogramming process, only a small fraction of “good quality” iPSCs can be obtained, and the selection of

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

these fully reprogrammed, not aberrant cells remains a challenge [13]. The differences between iPSCs and ESCs, and the inherent heterogeneity among iPSCs populations clearly affect the utility of these cells in both research and therapeutics although, given the evident advantages of iPSCs over ESCs, this does not reduce the potential of iPSCs [14]. At short term, iPSCs might become especially useful for drug screening applications or for providing human models for studying certain diseases that lack adequate animal models to date. For these purposes, iPSCs from representative patients have to be obtained and, afterwards, differentiated to disease-relevant cell types. These type of approaches are currently being used by several research groups to study human diseases such as type I diabetes, Duchenne muscular dystrophy, the fragile X syndrome, amyotrophic lateral sclerosis or Parkinson’s disease. In particular, an illustrative example of modeling Parkinson’s disease using iPSCs can be found in this book, in the chapter written by Sánchez-Danés A et al. Besides the above mentioned, some other issues need to be solved regarding iPSCs derivation and use. Even though the reprogramming of cells with DNA-free techniques has already been achieved, so avoiding the problems associated with the use of viral vectors, the generation of iPSCs is still a low efficiency process and the frequency of mutations and genomic aberrations remains too high. In addition, cell reprogramming to iPSCs is known to inhibit tumour-suppressing genes and to activate oncogenes. These facts indicate that the reprogramming procedures used are far from perfect and, therefore, improved, more uniform protocols and better control analysis need to be developed in order to obtain standardized cell lines to be used in both basic and applied studies; this would not only give developmental and cell biology a very powerful tool, but would also bring the field a great step closer to personalized medicine. A more extensive analysis of the potentiality of iPSCs can be found later in this book (see the chapter written by Zapata AG). Of course, the list of research topics mentioned here does only cover a minuscule percentage of what is going on in cell biology nowadays. To specify them all would be an impossible task. However, they could be considered as representative of what type of researches cell biologists are mainly focused on and, in the next chapters of this book, the reader can find these and many other examples of burning issues in cell biology. Taking into account that this 21st century can still be considered brand new, we might question ourselves about the near future: is cell biology heading in the right direction? The answer to this question might be “Yes, but…” “Yes” because the number of scientific publications per year has not stopped growing since the beginning of the century (from 613.803 articles indexed in PubMed in 2000 to over one million in 2011, what makes a total of almost eight and a half million articles published in the past eleven years); a significant percentage of these publications are related to cell biology topics. “But…” because of this very same reason; the amount of data that is being collected is so big that filtering and integration have become almost impossible. In our frenzy to collect data, we need to learn to ask the right questions and how to extract useful information from that data. Cell biology should tend towards tissues and organisms, integrating biological processes to find out really how cellular components form complexes, how these assemble into organelles and how organelles form cells which, in turn, build organs and organisms. This is, however, one of the biggest challenges we might face, since this requires developing new experimental and analytical methods to trespass across the traditional borders between fields [15]. Furthermore, due to our technical limitations, cell biology has focused mainly on a relatively small number of cell types (most often unpolarized, cultured cell lines), overlooking the enormous amount of different cell types with specialized functions that can be found in vivo. Developing better ways of performing in vivo cell biology, which would allow us to study

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

the real cellular behaviours in the context of tissues and organs, would also be an important step towards complete understanding of how life works. However, besides systems biology, a mechanistic understanding of biology is also necessary. We need to know the biochemical and physical principles underlying the biological events. Unfortunately, social and economic pressures are forcing research in the opposite direction, being applied science generally favoured over basic studies. This is not likely to change in the near future and will surely determine the goals cell biology might reach in the coming decades. Anyhow since we, human beings, have not evolved very much during the past 350 years, as happened to Hooke, these goals will probably also be far behind our own imagination.

Acknowledgements Supported by grants from the Spanish Government BIO2009-13903-C02-01; Red de Terapia Celular, RD06/0010/0014), and the Andalusian Government (P07-CVI-2781). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

References 1. Russo, V. E. A., Martienssen, R. A. & Riggs, A. D. (eds) Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor Laboratory Press, Woodbury, 1996. 2. Bird A. Perceptions of epigenetics. Nature. 2007;447(7143):396-8. 3. Van Wynsberghe PM, Chan SP, Slack FJ, Pasquinelli AE. Analysis of microRNA expression and function. Methods Cell Biol. 2011;106:219-52. 4. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet. 2011;12(12):861-74. 5. Nana-Sinkam SP, Croce CM. Non-coding RNAs in cancer initiation and progression and as novel biomarkers. Mol Oncol. 2011;5(6):483-91. 6. Sasidharan R, Gerstein M. Genomics: protein fossils live on as RNA. Nature. 2008;453(7196):729-31. 7. Sifuentes-Romero I, Milton SL, García-Gasca A. Post-transcriptional gene silencing by RNA interference in non-mammalian vertebrate systems: where do we stand? Mutat Res. 2011;728(3):158-71. 8. Song E, Lee S, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis, Nat. Med. 2003;9:347–351. 9. Simón-Mateo C, García JA. Antiviral strategies in plants based on RNA silencing. Biochim Biophys Acta. 2011;1809(11-12):722-31. 10. Vanlangenakker N, Vanden Berghe T, Vandenabeele P. Many stimuli pull the necrotic trigger, an overview. Cell Death Differ. 2012;19(1):75-86. 11. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147(4):728-41. 12. Bialik S, Kimchi A. Autophagy and tumor suppression: recent advances in understanding the link between autophagic cell death pathways and tumor development. Adv Exp Med Biol. 2008;615:177-200. 13. Bilic J, Izpisua Belmonte JC. Concise review: Induced pluripotent stem cells versus embryonic stem cells: close enough or yet too far apart? Stem Cells. 2012;30(1):33-41. 14. Robinton DA, Daley GQ. The promise of induced pluripotent stem cells in research and therapy. Nature. 2012;481(7381):295-305.

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15. Akhtar A, Fuchs E, Mitchison T, Shaw RJ, St Johnston D, Strasser A, Taylor S, Walczak C, Zerial M. A decade of molecular cell biology: achievements and challenges. Nat Rev Mol Cell Biol. 2011;12(10):669-74.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

1.1. Dynamics of cell compartments

Dynamics of cell compartments and pathological implications Inés M. Antón∞ and Jaime Renau-Piqueras* ∞ Centro Nacional de Biotecnología (CNB-CSIC). 28049 Madrid, Spain; CIBERNED,

Centro

Investigación

Biomédica

Red

Enfermedades

Neurodegenerativas, Spain * Sección de Biología y Patología Celular, Centro de Investigación, Hospital La Fe, Valencia, Spain Movement is intrinsic to life. Biologists have established that most forms of directed nanoscopic, microscopic and, ultimately, macroscopic movements are powered by molecular motors from the dynein, myosin and kinesin superfamilies. These motor proteins literally walk, step by step, along polymeric filaments, carrying out essential tasks such as organelle transport.

Dynamics refers to “forces that cause motions of bodies”; in cell biology, it can apply to intracellular organelle movement, to changes in organelle shape or number, or to molecular composition when dynamics of proteins (e.g., receptors) or lipids are considered. As a general term, dynamics of cell compartments is intimately linked to intracellular trafficking. Since this process will be discussed specifically in another section of this book (Section 1.3. The intracellular trafficking), here we will focus mainly on updated concepts related to organelle shape and redistribution and cell polarization, as well as actin and tubulin cytoskeleton reorganization. Correct intracellular displacement of organelles is necessary for cell viability and function. Dynamics is crucial to cell biology, controlling essential processes such as cytokinesis and cell division, cell polarization, cell shape change and reorganization during morphogenesis, as

well as

cytoskeletal mechanics. Abnormal dynamics

of cell

compartments is therefore a major cause of the most common severe human pathologies including cancer, cardiovascular disease and neurodegenerative disorders. Morphological characteristics of organelles are important for their function in the cell. These characteristics include not only their shape, number and size, but also their distribution within the cell. The dynamics of processes that lead to changes in any of these characteristics (organelle fission, fusion, autophagy, transport) significantly influence cell function. Organelle homeostasis in eukaryotic cells is precisely regulated (2). Organelles like mitochondria, vacuoles and peroxisomes can be formed by fission of pre-existing ones,

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

leading to an increase in organelle number. The opposite, organelle fusion, is also possible for mitochondria and some types of vacuoles, and promotes a decrease in organelle number. Other cell structures like peroxisomes, vacuoles and the Golgi complex can also be formed de novo, for instance from the endoplasmic reticulum (ER), while autophagy contributes to removal of entire organelles from the cell. During cell division, organelles are divided between mother and daughter cells, leading to a decrease in organelle number in the mother cell and an increase in the daughter cell. Similarly, chromosome motility and segregation during mitosis is orchestrated by dynamic organelles termed kinetochores that itself support protein kinetics (3). Some organelles (such as the nucleus in mammalian cells) can disassemble and reassemble during the cell cycle.

The nuclear envelope is a physical barrier that

encapsulates, organizes and protects the genome and contains nuclear pore complexes able to regulate the passage of molecules to and from the nucleus (4). Recent imaging approaches that focus on single molecules have provided unexpected insight into this crucial step in information flow. Other organelles, like the ER, are in permanent modification. The ER is a multifunctional organelle formed by functionally and morphologically distinguishable domains: the planar nuclear envelope and the peripheral cytoplasmic ER (a lattice of sheetlike cisternae interconnected with tubules) that change constantly in shape and relative content.

These dynamic modifications depend on the interplay among many forces,

including the interaction between the ER and microtubules, membrane fusion and fission events, and the contribution of membrane-shaping proteins, in turn pivotal for the appropriate execution of the varied functions of this organelle (5). Better understanding of these dynamic processes requires the development of quantitative approaches.

Recent methods aimed to improve quantification of organelle

abundance, morphology and the kinetics of the processes that cause changes in these properties, include mainly biochemical and microscopy techniques (2).

Biochemical

approaches to study fusion or degradation of organelles are generally based on activation of a protein following cleavage or scission of a reporter protein, which generates a lower molecular weight product detectable by Western blot analysis.

In the past 25 years,

however, most developments in quantitative analysis of organelle abundance and dynamics have developed in parallel to advanced fluorescence microscopy milestones: enormous progress has been made thanks to the introduction of new, improved fluorescent probes and proteins (e.g., photoactivatable proteins), better microscopes (confocal laser scanning microscope and automated microscopes), as well as the development of image analysis software. Program improvement, in combination with hardware development, are expected fields to make quantitative analysis of cell architecture routinely applicable in cell and systems biology. Live cell imaging has undoubtedly boosted the tremendous advancement seen by cell biology in recent decades. We will also comment on some examples of the pathological effects derived from abnormal dynamics of certain cell compartments. Mitochondria are essential organelles for energy production in the cell, whose morphology varies between elongated interconnected mitochondrial networks (by fusion) and fragmented disconnected arrays (by fission), which allow the transmission of signaling messengers and exchange of metabolites within the cell.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

Two of the most energy-demanding tissues are muscle and nerves. Besides undergoing usual reshaping, neuronal mitochondria must also move along neurites to localize at sites of high-energy use, such as synapses. Neurons are particularly sensitive and vulnerable to any abnormalities in mitochondrial dynamics, due to their large energy demand and long extended processes.

It is therefore not surprising that there is increasing evidence

supporting a strong link between mitochondria and neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's disease (6).

A specific example are mutant

SOD1 (Cu,Zn superoxide dismutase) motor neurons, responsible for familial amyotrophic lateral sclerosis (FALS). These neurons show impaired mitochondrial fusion and retrograde axonal transport, with reduced frequency and velocity of movement.

Both fusion and

transport defects are associated with decreased mitochondrial size and density as well as defective mitochondrial membrane potential.

In vitro, mislocalization of mitochondria at

synapses among motor neurons correlates with abnormal synaptic number, structure, and function, suggesting that impaired mitochondrial dynamics contributes to the selective degeneration of motor neurons (7). New evidence has recently emerged that shows how remodeling of the mitochondrial network is altered in several cardiac pathologies that affect cardiac development, the response to ischemia-reperfusion injury, and heart failure (8).

Small, disorganized

mitochondria are observed in dilated cardiomyopathies and in experimental models of ischemic myocardia in which the mitochondrial network is fragmented, whereas giant mitochondria are found in patients with idiopathic dilated cardiomyopathy or in hypoxia models. Both establishment and maintenance of cell polarity require the actin and microtubule cytoskeletons (9) and depend on spatio-temporally controlled dynamic reorganization of multiple cell compartments. Next two articles provided perceptive mechanistic views on the generation of cell polarity in distinct cell processes, including generation of front-back polarity by the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) during cell migration (Juanes-GarcĂa and Miguel Vicente-Manzanares, in this book), and generation of apicalbasal polarity during epithelial development and lumen formation (MartĂn Belmonte et al., in this book). Epithelial cells are highly polarized, with an apical pole usually facing the lumen, and a basal pole in contact with the underlying basal membrane. Epithelial tissue homeostasis depends on cell-cell and cell-matrix interactions. Increasing evidence demonstrates that loss of polarity, by altered expression, location or activity of cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages of tumor progression and invasiveness (10). The recent identification of pivotal roles for these proteins in epithelial function and proliferation also points to their association with early stages of tumorigenesis. In summary, great expectations have been placed on understanding the mechanisms that control dynamics of cell compartment, in the hope they will provide new therapeutic targets for treating cancer, neurodegenerative disorders and cardiovascular diseases.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

References 1. von Delius M, Leigh DA. Walking molecules. 2011. Chem Soc Rev 40(7):3656-76. 2. van Zutphen T, van der Klei IJ. Quantitative analysis of organelle abundance, morphology and dynamics. 2011. Curr Opin Biotechnol 22(1):127-132. 3. Dorn JF, Maddox PS. Kinetochore dynamics: how protein dynamics affect chromosome segregation. 2011. Curr Opin Cell Biol. Dec 29. http://dx.doi.org/10.1016/j.ceb.2011.12.003 4. Grünwald D, Singer RH, Rout M. Nuclear export dynamics of RNA-protein complexes. 2011. Nature 475(7356):333-41 5. Pendin D, McNew JA, Daga A. Balancing ER dynamics: shaping, bending, severing, and mending membranes. 2011. Curr Opin Cell Biol 23(4):435-42. 6. Han XJ, Tomizawa K, Fujimura A, Ohmori I, Nishiki T, Matsushita M, Matsui H. Regulation of mitochondrial dynamics and neurodegenerative diseases. 2011. Acta Med Okayama 65(1):1-10. 7. Magrané J, Sahawneh MA, Przedborski S, Estévez ÁG, Manfredi G. Mitochondrial dynamics and bioenergetic dysfunction is associated with synaptic alterations in mutant SOD1 motor neurons. 2012. J Neurosci 32(1):229-42. 8. Kuzmicic J, Del Campo A, López-Crisosto C, Morales PE, Pennanen C, Bravo-Sagua R, Hechenleitner J, Zepeda R, Castro PF, Verdejo HE, Parra V, Chiong M, Lavandero S. Mitochondrial dynamics: a potential new therapeutic target for heart failure. 2011. Rev Esp Cardiol 64(10):916-23. 9. de Forges H, Bouissou A, Perez F. Interplay between microtubule dynamics and intracellular organization. 2012. Int J Biochem Cell Biol 44(2):266-74. 10. Martin-Belmonte F, Perez-Moreno M. Epithelial cell polarity, stem cells and cancer. 2011. Nat Rev Cancer 12(1):23-38.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Non-Muscle Myosin II Integrates Cell Adhesion and Migration by Controlling the Compartmentalization of Adhesive Signaling Alba Juanes-García and Miguel Vicente-Manzanares* Universidad Autónoma de Madrid, Facultad de Medicina and Instituto de Investigación Sanitaria- Princesa, c/Diego de León 62, 28006-Madrid * Corresponding author: Dr. Miguel Vicente-Manzanares, Universidad Autónoma de Madrid, Facultad de Medicina, U.D. Hospital de la Princesa, c/Diego de León 62, 28006-Madrid. Tel: 34-91-520.23.70; E-mail: miguel.vicente@uam.es

Abstract Front-back polarization requires exquisite compartmentalization of the signals that control actin-based protrusion and adhesive signaling. In this study, we review recent evidence of the role of non-muscle myosin II (NMII) as a master regulator of the polarization of front-back signals. We underscore the cooperative role of the two myosin II isoforms, A and B, in the generation and stabilization, respectively, of actin bundles and focal adhesions in

polarized

mesenchymal

cells.

Furthermore,

NMII

controls

adhesive

signaling

compartmentalization at the leading edge. We suggest the existence of mechano-sensitive signaling mechanisms that respond to alterations in the balance of plasma membranecytoskeleton tension to spatially promote, or block, protrusion, during cell migration.

Introduction Motile cells display asymmetry as they migrate (1). Most cells display a single leading edge enriched in dendritic actin in the direction of migration (2). Dendritic actin is branched actin generated by the concerted action of formins at the barbed end and the Arp2/3 at the pointed end (3). Both the formins and the Arp2/3 complex are under the control of small Rho GTPases, e.g. RhoA and Rac (4). The protrusive leading edge that contains dendritic actin is often termed lamellipodium (5). It is ~1µm-wide and very dynamic, undergoing intense actin polymerization and depolymerization. The lamellipodium enables cell movement through its attachment to the substratum through small, dynamic adhesions (6). Opposite to the leading edge is the cell rear, which is a non-protrusive end that contains large stable adhesions and actin bundles. In some cells, forward motion is achieved through the coordinated extension of the front and contraction of the rear, but whether this is a general mechanism is not well established (7-9). The development and maintenance of migratory front-back polarity has been studied in some detail in the last years, and several key regulators have emerged. Non-muscle myosin

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

(NMII) is an actin binding protein implicated in force generation and cytoskeletal remodeling. Recent evidence from several groups indicates its central role in cellular reshaping and polarization, adhesion dynamics and cell migration (10-13). The molecular basis of these functions relies on its actin cross-linking and contractile activities. NMII is a endpoint of extracellular signals as multiple pathways converge on its activation or inactivation (14). NMII is comprised of two heavy chains (MHCII), two light essential light chains (ELC) and two regulatory light chains (RLC) that regulate NMII activity through its phosphorylation. There are three NMII isoforms determined by three different genes encoding the heavy chain: MHCII-A, II-B and II-C (15). NMII-A and NMII-B are widely expressed, whereas NMIIC is restricted to specific tissues and may be up-regulated in some tumors (16). They all share a similar structure and co-regulate different cellular phenomena. However, their function is not redundant, although sometimes they cooperatively regulate different stages of the same process. This research was aimed to understand the possible synergies between NMII-A and NMII-B during cell migration, and how NMII determines the cellular signaling that enables protrusion at the front and shuts it down at the rear of migrating cells.

Cooperative and hierarchical roles of NMII-A and NMIIB in actin bundling and adhesion dynamics Previous evidence from our group had established the mandatory role of NMII in formation of the rear of migrating mesenchymal cells (13, 17). These studies clearly showed that NMII-B-deficient cells displayed no clear front and rear, exhibited multiple protrusions and were unable to migrate. Conversely, NMII-A-deficient cells displayed non-retractile long rears devoid of actin bundles. These cells were highly protrusive and, when examined shortterm, they apparently migrated actively. However, lack of retraction impeded long-term migration. Based on these precedents, we sought out to determine the specific role of each NMII isoform in the formation of well-defined cell rears of polarized mesenchymal cells. To do this, we observed cells as they became polarized from very early time-points. We noted that most cells that later became polarized and migratory displayed an “actomyosin protobundle”. The proto-bundle was an F-actin-containing patch also decorated with NMII-A, NMII-B and phosphorylated (active) RLC. We observed that the proto-bundle was a physical precursor of the cell rear, as the rear often emerged in the region of the cell where the protobundle resided, evolving into large actomyosin bundles as polarity develops and the cell migrates. To demonstrate the role of the NMII isoforms in proto-bundle formation, we analyzed the effect of isoform-specific depletions. We found that depletion of NMII-A abrogated formation of the proto-bundle, whereas elimination of NMII-B allowed the protobundle to assemble; but it was highly unstable and dynamic, forming and disassembling rapidly during early spreading. From these experiments, we concluded that NMII-A was required for the initial assembly and NMII-B for the stabilization of the proto-bundle and its subsequent evolution into the cell rear. NMII-B, by itself, was not able to form the protobundle; this suggested a hierarchical relationship between the NMII isoforms in terms of assembly of the proto-bundle and its evolution into the trailing edge. Using two-color, high

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

spatiotemporal resolution time-lapse confocal microscopy and single-molecule expression vectors (18), we observed that NMII-A became immobilized first and promoted the formation of bundled actin- and myosin-containing structures, whereas NMII-B incorporated at later time points, and only to structures pre-formed by NMII-A (19). NMII is also implicated in the evolution of nascent adhesions into elongated mature adhesions (20). Nascent adhesions form underneath the lamellipodium in a NMIIindependent manner during forward motion (6, 21). Previous work from the group had also described the role of NMII-A in initial adhesion maturation, by catalyzing the formation of thin actin bundles that act as a physical scaffolds, or templates, for adhesion maturation (6).

However, a fraction of these elongated adhesions and actin bundles still disassemble, suggesting that NMII-A is not enough to completely stabilize these structures (19). Based on these precedents, we tested the hypothesis that NMII-B was required for the stabilization of NMII-A-initiated adhesion maturation. We confirmed that NMII-A was required for the

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initiation of adhesion maturation, as NMII-A-depleted cells lacked elongated (mature) adhesions. Conversely, NMII-B-deficient cells displayed some elongated adhesions, but those adhesions were small and less numerous compared to control cells (19). Furthermore, using two-color time-lapse confocal microscopy, we noted that adhesions associated to NMII-A+NMII-B-decorated actin bundles did not disassemble, whereas those associated to bundles containing NMII-A alone disassembled comparably to those observed in wild type cells (19). From these experiments, we concluded that: 1) NMII-A plays a initiating role during the beginning of actin bundling and adhesion maturation, but it is not able to stabilize these structures; 2) NMII-B is not able to initiate these processes, but it is essential for the stabilization of actin bundling and adhesions; 3) NMII-A provides a physical actin scaffold for NMII-B to bind, stabilize and enhance actin bundling and promote adhesion maturation (Fig. 1).

NMII confines protrusion to the leading edge by compartmentalizing adhesive signaling and Rac activation How protrusion is confined to the leading edge is still a matter of speculation. The most likely scenario is the confinement of a rate-limiting component (either signals or mechanical forces, or both) to the leading edge that activates protrusion at the front. Given the crucial role of NMII in forming the rear of the cell (13, 17), we hypothesized that NMII was implicated in excluding protrusive signals locally, impeding protrusion in this area and thereby enabling protrusion in a different region of the cell, which then becomes the front. The physical force that pushes the membrane at the leading edge is driven by actin polymerization, which is nucleated by the Arp2/3 complex under the control of Rac. The morphology of the cell rear, lacking protrusions, suggests that Rac activation is lower in this cellular region, which was further confirmed by FRET (22). In order to address whether NMII-driven rear formation down-regulates Rac in one pole of the cell, i.e. the rear, we used constitutively activated Rac mutants, an activator of Rac (Tiam1) and a photo-activated Rac construct (23). Using these tools, we observed that isotropic activation of Rac causes cell depolarization and formation of protrusions around the entire periphery of the cell. When Rac was locally photo-activated at the rear, it also induced protrusion, even in areas where NMII-B had generated large bundles. This data suggests that inhibition of protrusion at the rear occurs upstream of Rac. We therefore studied the upstream signaling that leads to Rac activation. We found that two GEFs that activate Rac, βPIX and DOCK 180, are excluded from the rear, where NMII has generated large actin bundles. Similarly, the adhesive signaling that recruits these Rac activators to the plasma membrane, including phosphoS273-paxillin-GIT for βPIX and phospho-Y31/Y118-paxillin-p130CAS-CrkII for DOCK180 (24-26), was attenuated in the regions of the plasma membrane closer to NMII-decorated large actin bundles. These data indicate that the asymmetric localized activation of Rac that maintains front-back polarization is based on NMII-dependent polarized localization of its activators (Fig. 2). How NMII distributes these signals asymmetrically remains unsolved. A

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hypothesis is that NMII shunts tension (which is required for switching on protrusive signaling pathways) from the plasma membrane to the actomyosin bundles, locally limiting their activation. This hypothesis suggests the existence of a mechano-sensitive signaling relay that is activated only when the plasma membrane-cytoskeletal tension is optimized by NMII. Future studies will be aimed at describing the specific molecular nature of the cytoskeletal-plasma membrane force relationship and the existence of one or multiple mechanosensitive scaffolds and kinases.

Acknowledgements The authors thank Laura Hernández Téllez for the artistic impression used for Figure 1. MV-M is supported through a Ramón y Cajal contract (RYC-2010-06094) from the Spanish Ministry of Science and Education (MICINN), Plan Nacional I+D (SAF2011-24953) and a Marie Curie CIG award (PCIG09-GA-2011-293719).

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

References 1. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. 2003. Science. 302(5651):1704-9. 2. Bagorda A, Parent CA. Eukaryotic chemotaxis at a glance. 2008. J Cell Sci. 121(Pt 16):2621-4. 3. Campellone KG, Welch MD. A nucleator arms race: cellular control of actin assembly. 2010. Nat Rev Mol Cell Biol. 11(4):237-51. 4. Hall A. Rho GTPases and the control of cell behaviour. 2005. Biochem Soc Trans. 33(Pt 5):891-5. 5. Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, Danuser G. Two distinct actin networks drive the protrusion of migrating cells. 2004. Science. 305(5691):1782-6. 6. Choi CK, Vicente-Manzanares M, Zareno J, Whitmore LA, Mogilner A, Horwitz AR. Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. 2008. Nat Cell Biol. 10(9):1039-50. 7. Poincloux R, Collin O, Lizarraga F, Romao M, Debray M, Piel M, et al. Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel. 2011. Proc Natl Acad Sci U S A. 108(5):1943-8. 8. Solecki DJ, Trivedi N, Govek EE, Kerekes RA, Gleason SS, Hatten ME. Myosin II motors and F-actin dynamics drive the coordinated movement of the centrosome and soma during CNS glial-guided neuronal migration. 2009. Neuron. 63(1):63-80. 9. Martini FJ, Valdeolmillos M. Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons. 2010. J Neurosci. 30(25):8660-70. 10. Cai Y, Biais N, Giannone G, Tanase M, Jiang G, Hofman JM, et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. 2006. Biophys J. 91(10):3907-20. 11. Lo CM, Buxton DB, Chua GC, Dembo M, Adelstein RS, Wang YL. Nonmuscle myosin IIb is involved in the guidance of fibroblast migration. 2004. Mol Biol Cell. 15(3):982-9. 12. Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS, Yamada KM. Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. 2007. Nat Cell Biol. 9(3):299309. 13. Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. 2007. J Cell Biol. 176(5):573-80. 14. Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR. Non-muscle myosin II takes centre stage in cell adhesion and migration. 2009. Nat Rev Mol Cell Biol. 10(11):778-90. 15. Wang A, Ma X, Conti MA, Adelstein RS. Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains. 2011. Biochem Soc Trans. 39(5):1131-5. 16. Jana SS, Kawamoto S, Adelstein RS. A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line. 2006. J Biol Chem. 281(34):24662-70. 17. Vicente-Manzanares M, Koach MA, Whitmore L, Lamers ML, Horwitz AF. Segregation and activation of myosin IIB creates a rear in migrating cells. 2008. J Cell Biol. 183(3):54354. 18. Watanabe N, Mitchison TJ. Single-molecule speckle analysis of actin filament turnover in lamellipodia. 2002. Science. 295(5557):1083-6. 19. Vicente-Manzanares M, Newell-Litwa K, Bachir AI, Whitmore LA, Horwitz AR. Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells. 2011. J Cell Biol. 193(2):381-96.

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20. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, et al. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. 2004. Nat Cell Biol. 6(2):154-61. 21. Alexandrova AY, Arnold K, Schaub S, Vasiliev JM, Meister JJ, Bershadsky AD, et al. Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow. 2008. PLoS One. 3(9):e3234. 22. Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, Hahn KM. Localized Rac activation dynamics visualized in living cells. 2000. Science. 290(5490):3337. 23. Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, et al. A genetically encoded photoactivatable Rac controls the motility of living cells. 2009. Nature . 461(7260):104-8. 24. Nayal A, Webb DJ, Brown CM, Schaefer EM, Vicente-Manzanares M, Horwitz AR. Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics. 2006. J Cell Biol. 173(4):587-9. 25. Kiyokawa E, Hashimoto Y, Kobayashi S, Sugimura H, Kurata T, Matsuda M. Activation of Rac1 by a Crk SH3-binding protein, DOCK180. 1998. Genes Dev. 12(21):3331-6. 26. Kiyokawa E, Hashimoto Y, Kurata T, Sugimura H, Matsuda M. Evidence that DOCK180 up-regulates signals from the CrkII-p130(Cas) complex. 1998. J Biol Chem. 273(38):2447984.

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A 3D-functional-screening identifies new components of the machinery for epithelial lumen morphogenesis Fernando Martín-Belmonte, Manuel GálvezSantisteban, Alejo E. Rodriguez-Fraticelli, Silvia Vergarajáuregui, Inmaculada Bañón, and Ilenia Bernascone Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Abstract Lumen formation is a key event in epithelial morphogenesis. During morphogenesis, gene expression is modified to accommodate the specific needs of individual cell types. However, how cell polarity is controlled at a transcriptional level during epithelial morphogenesis in vertebrates is poorly understood. Here, we performed a two-step screening based on a transcriptomic analysis, followed by a functional screening with RNAi to identify new proteins required for lumen formation using MDCK 3D cell cultures. We identified 15 new candidate regulators of epithelial polarity and lumen formation, which are frequently downregulated in epithelial cancers, suggesting an importance in the maintenance of a differentiated epithelial phenotype in vivo.

Introduction Epithelia represent some of the most important tissues in animal biology. Epithelial tissues are formed by layers of cells that cover organ surfaces such as the surface of the skin, the digestive tract, and the intricate network of kidney tubules. During development, epithelial cells develop apico-basal polarity, a specific type of constitutive cell polarity in which cells organize their architecture towards an apical (luminal) surface. The epithelial plasma membrane is divided into two surfaces: apical and basolateral, separated by cellular junctions (Figure 1). The establishment of apical-basolateral polarity depends on the asymmetric segregation of membrane protein and lipids. To create this asymmetry, membrane proteins are delivered to specific regions of the plasma membrane (Figure 1) (1,2). One critical step in development is the formation of the central lumen. Disruption of lumen formation may result in altered polarity and cancer and therefore, understanding these modifications is a major challenge for the future (3).

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Epithelial cells create lumens following a wide variety of tubulogenesis mechanisms. Despite this diversity, emerging data from different model systems have concluded that in most systems, cells follow a series of similar events to create tubes that include apical membrane biogenesis, vectorial transport of apical vesicles to the plasma membrane, secretion and regulated expansion (Figure 2) (4,5). Recent data on 3D models has demonstrated a molecular chain linking membrane-trafficking machinery with delivery of the Par3â&#x20AC;&#x201C;aPKCâ&#x20AC;&#x201C;Cdc42 complex to the nascent apical surface during lumen formation. This trafficking machinery included the Rab GTPases Rab11a and Rab8a, the exocyst complex, and the motor protein myo5B (Figure 2) (6,7), which emphasize the complex spatiotemporal orchestration required to construct a new membrane. In addition, certain phosphoinositide species such as PtdIns(3,4,5)P3 and PtdIns(4,5)P2, specify membrane identity and are also required for apical trafficking and lumen formation (8,9). Remarkably, the Rabs cascade, membrane phosphinositides and the exocysts

also regulates mammalian ciliogenesis, and an homologous yeast pathway regulates budding, suggesting it is an ancient polarity-generating module (10). Despite these recent advances, most of the key players essential for lumen formation, and the molecular machinery controlled by them during epithelial morphogenesis remain to be characterized.

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During development, polarized traffic, and certain signaling pathways are modified to accommodate the specific needs of individual cell types. Studies performed mainly in epithelial tissues of Drosophila melanogaster have shown that transcriptional promotion and repression control the function of multiple genes involved in apical polarity and luminal development, including membrane trafficking pathways, polarity complexes, etc (11,12). However, how cell polarity is controlled at a transcriptional level during epithelial morphogenesis is poorly understood. In our laboratory, we performed a two-step screening based on a transcriptomic analysis, and a posterior functional screening to identify new proteins required for lumen formation using MDCK 3D cell cultures. Interestingly, most of the genes identified in the screening are also downregulated upon oncogenic transformation in important epithelial cancers, such as renal and breast.

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Results An RNAi-screening reveals 14 novel regulators of epithelial morphogenesis To identify new genes upregulated during epithelial morphogenesis, and potential candidates to regulate cell polarity and lumen formation, we performed a two-step functional screening using MDCK cells. The first step was a genome wide analysis to identify genes upregulated in 3D-morphogenesis. A microarray-based differential expression analysis was conducted using the Affymetrix Canine Genome 2.0 platform (Figure 3). MDCK type II cells were grown at confluence in 2D or 3D conditions in Matrigel© and RNA was isolated at 36h and submitted for microarray analysis. The data revealed a set of 1597 probes upregulated in 3D and 1304 probes that were downregulated. To complement the microarray analysis and prioritize the functional analysis, the results obtained in this screening were verified by quantitative PCR (qPCR). A set of 99 upregulated genes of interest was selected using these bioinformatic tools and then validated by qPCR (n=4). Finally a stealth®-siRNA library was custom designed to target 47 candidate genes, including internal positive controls, based on previous data (13,14). To test the efficiency of the siRNA duplexes, MDCK cells transfected with siRNA were cultured in 2D and 3D conditions, and RNA extracts were analyzed by RT-PCR. For functional analysis, transfected MDCK cells were grown for 3 days in Matrigel to form cysts, and lumen formation efficiency was quantified by confocal analysis using as a readout the localization of podocalyxin (gp135), the integrity of the actin cytoskeleton and tight junctions (TJs) (Fig. 3), and membrane phosphoinositides (not shown). We found a set of 16 genes required for lumen formation, of which 14 had not been previously related with epithelial morphogenesis in MDCK cells, and two other were our internal positive controls.

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After applying bioinformatic pathway analysis tools, some of these genes were found to be connected in common functional mechanisms. Interestingly, most of the potential novel regulators of lumen formation identified in the screening were for the most part uncharacterized proteins, and their functions and mechanism in epithelial morphogenesis are still to be elucidated. To analyze a possible relevance of these novel genes in vivo, we analyzed human cancer gene expression using Oncomine, an oncogenic gene-expression analysis tool. We analyzed whether genes that produced a phenotype in 3D were being downregulated in epithelial cancers (p<0.05), suggesting an importance in the maintenance of a differentiated epithelial phenotype in vivo (not shown). Renal, breast and skin tissues have the highest scores, with 10, 8 and 7 downregulated candidate genes, respectively.

Discussion Transcriptional and functional screenings have demonstrated to be powerful tools to identify novel factors in many biological processes. For instance, some functional screenings using RNAi techniques have been previously performed to identify regulators of epithelial morphogenesis. However, these studies were based in screenings targeting specific subset of genes, such as apical proteins or activators for Rho-family GTPases (15,16), instead of a genome-wide screenings as the one performed here. Therefore, we show here how a wider screening is a useful tool to identify specific regulators of epithelial morphogenesis that were never shown before related to this process. Interestingly, most of the potential novel regulators of lumen formation identified in the screening were for the most part uncharacterized proteins, and their functions and mechanism in epithelial morphogenesis are still to be elucidated. In addition, most of the genes identified to be upregulated during morphogenesis have been shown to be downregulated in many epithelial cancers, indicating they might be acting as tumor suppressors and thus providing potential clinical relevance.

References: 1. Bagnat, M., I.D. Cheung, K.E. Mostov, and D.Y. Stainier. 2007. Genetic control of single lumen formation in the zebrafish gut. Nat Cell Biol. 9:954-960. 2- Brady, D.C., J.K. Alan, J.P. Madigan, A.S. Fanning, and A.D. Cox. 2009. The transforming Rho family GTPase Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis. Mol Cell Biol. 29:1035-1049. 3. Bryant, D.M., A. Datta, A.E. Rodriguez-Fraticelli, J. Peranen, F. Martin-Belmonte, and K.E. Mostov. 2010. A molecular network for de novo generation of the apical surface and lumen. Nat Cell Biol. 12:1035-1045. 4. Bryant, D.M., and K.E. Mostov. 2008. From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol. 9:887-901.

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5. Datta, A., D.M. Bryant, and K.E. Mostov. 2011. Molecular regulation of lumen morphogenesis. Curr Biol. 21:R126-136. 6. Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat Cell Biol. 8:963-970. 7. Kerman, B.E., A.M. Cheshire, M.M. Myat, and D.J. Andrew. 2008. Ribbon modulates apical membrane during tube elongation through Crumbs and Moesin. Dev Biol. 320:278288. 8. Lubarsky, B., and M.A. Krasnow. 2003. Tube morphogenesis: making and shaping biological tubes. Cell. 112:19-28. 9. Martin-Belmonte, F., A. Gassama, A. Datta, W. Yu, U. Rescher, V. Gerke, and K. Mostov. 2007. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell. 128:383-397. 10. Mostov, K., T. Su, and M. ter Beest. 2003. Polarized epithelial membrane traffic: conservation and plasticity. Nat Cell Biol. 5:287-293. 11. Myat, M.M., and D.J. Andrew. 2002. Epithelial tube morphology is determined by the polarized growth and delivery of apical membrane. Cell. 111:879-891. 12. Quyn, A.J., P.L. Appleton, F.A. Carey, R.J. Steele, N. Barker, H. Clevers, R.A. Ridgway, O.J. Sansom, and I.S. Nathke. 2010. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell. 6:175-181. 13. Rodriguez-Boulan, E., A. Musch, and A. Le Bivic. 2004. Epithelial trafficking: new routes to familiar places. Curr Opin Cell Biol. 16:436-442. 14. Rodriguez-Fraticelli, A.E., M. Galvez-Santisteban, and F. Martin-Belmonte. 2011. Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis. Curr Opin Cell Biol. 23:638-646. 15. Roland, J.T., D.M. Bryant, A. Datta, A. Itzen, K.E. Mostov, and J.R. Goldenring. 2011. 16. Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization. Proc Natl Acad Sci U S A. 108:2789-2794. 17. Torkko, J.M., A. Manninen, S. Schuck, and K. Simons. 2008. Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis. J Cell Sci. 121:11931203.

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1.2. The intracellular trafficking

Some basic ideas about the intracellular membrane trafficking Gustavo Egea∞ María M. Malagón* ∞ Departamento de Biologia Celular, Inmunología y Neurociencias, Facultad de Medicina, Universidad de Barcelona-IDIBAPS, 08036 Barcelona. * Departamento de Biología Celular, Fisiología e Inmunología, IMIBIC, Universidad de Córdoba, 14014 Córdoba.

Within the eukaryotic, proteins and lipids need to be transported from the location of their synthesis to the location of their destiny where they perform their functions. There are various intracellular compartments that play different roles in synthesis, maturation and function of proteins and lipids. Thus, a secreted protein (a hormone for instance) is synthesized in the lumen of the endoplasmic reticulum (ER), then packaged in transport intermediates or transport carriers and transported to the Golgi apparatus (where it is usually modified and mature) to be finally transported to the plasma membrane in secretory vesicles, which fuse with the cell membrane (exocytosis) to be released to the extracellular medium. Transport intermediates are membrane carriers with a vesicular or tubular shape that contain cargo and move between intracellular compartments. The cargo is first sorted and concentrated from the donor compartment (ER, endoplasmic reticulum-Golgi intermediate compartment/ERGIC, Golgi cisternae, trans-Golgi-network/TGN, the plasma membrane, and early, late and recycling endosomes). After budding off the donor compartment, transport carriers are directed along the cytoskeleton to the acceptor compartment (the same compartments indicated above, including the lysosomes) where they fuse and the cargo is released into the lumen of the acceptor compartment (1). There are two transport models in membrane trafficking: the vesicular and the maturation models (2,3). In the former, the intracellular compartments are physically distinct, static and communicate between them through cycles of fusion and fission of small transport carriers (vesicles); in the latter, cargoes do not really move from one compartment to the other, but the own compartment with the content. Although some of the molecular machinery involved in fission and fusion of transport carriers are known and characterized, the early steps of the required membrane deformation necessary to sustain both processes is poorly understood. In this respect, some proteins (such as ArfGAPs and coat proteins) and certain lipids (such as diacylglycerol and phosphatidic acid, among others) result necessary. The first step in intracellular transport to move a cargo (lipid or protein) from one side to another is to sort it into the transport carrier. It has been reported that receptors in the acceptor membrane

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interact with protein cargo to capture and concentrate it in a nascent bud (4). The mechanisms of lipid sorting are much less characterized, but it seems that the generation of membrane curvature could be a process by which certain lipids could be sorted in flat or curved membranes (5,6). Other intriguing aspect of intracellular transport is the maintenance of the molecular identity of the intracellular compartments despite the continuous and intense membrane remodeling that occurs following the fusion and fission cycles of transport carriers. Intracellular compartments are easily identified by their morphology under the electron microscopy. However, they need a specific set of membrane and luminal proteins (and lipids as well) for carrying out their respective functions. This is the case of glycosylation enzymes for the Golgi apparatus or of glycohydrolases for lysosomes. There are other set of proteins that help to maintain such identity such as the small GTPases Arf and Rab proteins (7-9). Besides protein markers, some lipids are differently enriched in intracellular

compartments.

This

the

case

cholesterol,

sphingomyelin

and

phosphoinosite family members (10). How the cell maintains this lipid heterogeneity and protein concentration in the membrane is nowadays an intense field of study in membrane trafficking, and the molecular mechanism will differ depending on the transport model that is postulated, which implies a different membrane composition for transport vesicles (vesicular model) or some kind of molecular or physical barrier to the passive lipid diffusion (maturation model). Once the transport carrier is formed and contains cargo (lipids and proteins) it should be transported and correctly addressed to the acceptor compartment, through processes known as targeting and tethering. This involves interactions between proteins present in the transport carrier (coat subunits as clathrin and COP proteins, Rab proteins, vSNAREs) and proteins present in the surface of the acceptor membrane (tethering complexes, t-SNAREs). Not only molecular interactors seems to be required for providing specificity to the transport, but also physical parameters such as membrane tension, which together with the cytoskeleton, plays a significant role in determining other factors such as the membrane curvature (for example, the highly fenestrated lateral portions of the Golgi cisternae) and local lipid packing (for example, the lipid raft). The postulate that is current and extensively investigated is that each (endo)membrane could have a characteristic degree of lipid packing depending on its physico-chemical characteristics, thereby contributing to the identity of the compartment or restricting a set of proteins (for example, ArfGAPs) or both, which altogether specify membrane trafficking. In this respect, several short oral presentations

focused

on the

role of lipids

such as

phospholipids,

glycerophsopholipids and phosphoinositides in the generation of transport carriers have been presented. An important element of the cell that contributes to the structural integrity of intracellular compartments and to the transport of transport intermediates is the cytoskeleton and its molecular motors (11). Both microtubules and actin filaments are necessary for correct Golgi positioning, architecture and trafficking (12). Recent evidences show that the Golgi functions as an actin- and microtubule-nucleating organelle, and in this sense, the new insights to the functional understanding of acetylated microtubules in trafficking and cell migration and division has been also presented in our session.

Other important role of cytoskeletal

elements is to generate force to induce membrane deformations and/or fission events in the

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formation of transport carriers. This has been particularly relevant in the case of the actin cytoskeleton, focused mainly on myosins (the actin motors), and the actin-based polymerization machinery. In this line of evidence, the results shown by Idrissi et al. (next article) represent a significant step forward in the understanding of the role played by the sequential assembly of molecular scaffolds (including actin-based machinery) in the formation of an endocytic transport intermediate. Our current knowledge of the membrane trafficking field is the result of biochemical, molecular and genetic approaches, but in the last decade the incorporation of new light imaging techniques such as GFP-based technology and video-microscopy in living cells has resulted essential for getting novel and significant insights in the field (13). A good example of this technology applied to the endocytic pathway is given in Peñalva et al. (14), who utilizes Aspergillus as a new emerging working model for the study of membrane trafficking events. In conclusion, intracellular trafficking is an active research area in Cell and Molecular Biology, in which new technologies have opened new fields to be explored and have contributed to address new concepts and revealed novel insights into the molecular mechanisms that govern the traffic of transport carriers and maintain the structural and molecular identity of intracellular compartments.

References 1. Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. 2004. Cell 116(2):153-66. 2. Glick BS, Nakano A. Membrane traffic within the Golgi apparatus. 2009. Annu Rev Cell Dev Biol. 25:113-32. 3. Nakano A, Luini A. Passage through the Golgi. 2010. Curr Opin Cell Biol. 22(4):471-8. 4. Traub LM. Tickets to ride: selecting cargo for clathrin-regulated internalization. 2009. Nat Rev Mol Cell Biol. 10(9):583-96. 5. van Meer G, Voelker DR, Feigenson GW. Membrane lipids: where they are and how they behave. 2008. Nat Rev Mol Cell Biol. 9(2):112-24. 6. Callan-Jones A, Sorre B, Bassereau P. Curvature-driven lipid sorting in biomembranes. 2011. Cold Spring Harb Perspect Biol. 3(2). 7. Stenmark H. Rab GTPases as coordinators of vesicle traffic. 2009. Nat Rev Mol Cell Biol. 10(8):513-25. 8. Donaldson JG, Jackson CL. ARF family G proteins and their regulators: roles in membrane transport, development and disease. 2011. Nat Rev Mol Cell Biol. 12(6):362-75. 9. Itzen A, Goody RS. GTPases involved in vesicular trafficking: structures and mechanisms. 2011. Semin Cell Dev Biol. 22(1):48-56. 10. Behnia R, Munro S. Organelle identity and the signposts for membrane traffic. 2005. Nature 438(7068):597-604. 11. Ross JL, Ali MY, Warshaw DM. Cargo transport: molecular motors navigate a complex cytoskeleton. 2008. Curr Opin Cell Biol. 20(1):41-7. 12. Egea G, Rios R. The role of the cytoskeleton in the structure and function of the Golgi apparatus. In: The Golgi apparatus-State of the Art 110 years after Camilo Golgi’s discovery. AA Mironov and M Pavelka (Eds). SpringerWien, 2008.

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13. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA. Fluorescent proteins and their applications in imaging living cells and tissues. 2010. Physiol Rev. 90(3):1103-63. 14. PeĂąalva MA, Galindo A, Abenza JF, Pinar M, Calcagno-Pizarelli AM, Arst HN, Pantazopoulou A. Searching for gold beyond mitosis: Mining intracellular membrane traffic in Aspergillus nidulans. Cell Logist. 2012 Jan 1;2(1):2-14.

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Introducing the temporal dimension to the ultrastructural analysis of membrane budding in S. cerevisiae Fatima-Zahra Idrissi, Isabel María Fernández-Golbano and María Isabel Geli∞ Instituto de Biología Molecular de Barcelona (IBMB-CSIC), C/ Baldiri Reixac 15, 08028 Barcelona, Spain. ∞ Corresponding autor. E-mail mgfbmc@ibmb.csic.es.

Budding of transport intermediates from cellular membranes is an energetically unfavorable process that can be effected by a variety of molecular machines. Protein coats, membrane sculpting domains such as ENTHs and BARs, enzymes that translocate or modify lipids, actin nucleating promoting factors (NPFs) and/or molecular motors have been proposed to participate in membrane deformation and/or vesicle scission [1, 2]. However, how these molecular machines are organized to effectively bend the lipid bilayer and promote membrane fission in vivo is far from being understood. The formation of clathrin and actin-dependent endocytic vesicles from the PM is one of the best-characterized budding events, which possibly uses the concerted action of all currently postulated membrane deforming mechanisms [3]. Thus, it is an ideal experimental system to unveil the molecular mechanisms driving membrane bending and vesicle scission. In the yeast S. cerevisiae analysis of endocytic budding started in the early 90´s with the isolation of mutants that block the process. The genetic approaches leaded to the identification of more than 50 genes involved in endocytic budding from the plasma membrane and showed that the machinery is conserved from yeast to mammals [4]. Subsequent development of live-cell fluorescence microscopy techniques to analyze the dynamics of the endocytic proteins at sites where an endocytic vesicles is formed, clarified the sequence of recruitment of the different factors involved and served to initially define functional modules (Fig. 1). Analogous experiments in mammals also demonstrated that the choreography of proteins involved in clathrin and actin-dependent endocytosis is basically conserved ([5] and references therein) [6]. In yeast, the fluorescence microscopy showed that the recruitment of clathrin shortly follows the arrival of the early module, which includes the scaffolding protein Ede1/Eps15 and the F-BAR protein Syp1/FCHO1. Up to 90 seconds later, the intermediate coat module formed by the yeast epsins Ent1/2 and the HIP1r homologue Sla2 is recruited to the patch, immediately followed by the late coat components, which include the clathrin adaptor Sla1 and the Arp2/3-dependent NPFs, Las17/WASP and Pan1. Actin polymerization is initiated a few seconds later, upon recruitment of Bzz1/syndapin and Vrp1/WIP, two proteins that modulate the activity of the NPFs. After an initial stage of actin-dependent restrained motility or corralled movement, the endocytic coat initiates a slow movement into the cytosol.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Initiation of the inward movement coincides with the recruitment of the third NPF, the myosin-I Myo5, and with initiation of massive actin polymerization. Arrival of the amphiphysins Rvs161/167 precedes scission of the vesicles, which travel rapidly into the cytosol while still attached to actin. The myosin-I ATPase and NPF activities are essential to power the slow inward movement of the coat whereas Las17 and Pan1 act earlier in the process (reviewed in [5]) (Fig 1).

The fluorescence live-cell imaging experiments established the basis for the molecular models explaining clathrin and actin-dependent endocytosis [7-9]. However, the resolution offered by the most sophisticated versions of this technique can hardly resolve changes in the morphology of the lipid bilayer coupled to the dynamics of endocytic proteins and therefore, it is insufficient to unveil how endocytic proteins are organized to deform the membrane. Thus, for example, the fluorescence microscopy could not resolve if the slow inward movement of the endocytic coat corresponded to the invagination of the plasma membrane or to the slow movement of an already detached vesicle. In this framework, our group started to develop the technology to analyze endocytic budding in yeast at the ultrastructural level. To unequivocally define the primary endocytic

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

profiles in yeast, the chromosomal copies of several genes encoding components of the endocytic coat were HA-tagged and plasma membrane profiles specifically labeled with immunogolds against these proteins were searched on ulthrathin sections of chemically fixed cells by electron microscopy. Interestingly, immunogolds labeling Clathrin, Sla1 and Pan1 all appeared associated with the tip of tubular invaginations of about 50 nm in diameter and up to 180 nm in length (Fig. 2A), suggesting that the slow inward movement of the

endocytic coat, observed in the fluorescence microscopy experiments, might correspond to the growth of a tubular profile, rather than to the movement of a detached vesicle. To test this hypothesis, a number of endocytic proteins sequentially recruited during the slow inward movement were also HA-taggedt (i. e. Myo5, Abp1, Rvs167) and the length of the invaginations labeled for each particular protein were statistically analyzed using a General Lineal Model. Consistent with our hypothesis, we found that proteins recruited early during the process (i. e Sla1, Pan1 or clathrin) were more represented in the shortest invaginations, whereas those recruited late (i. e Rvs167), were found more frequently in the longer profiles [10] (Fig. 2B). This observation demonstrated that the length of the endocytic invagination could be used as a parameter to define their age, thereby allowing introduction of the temporal dimension to the ultrastructural analysis of membrane budding.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

To investigate how the localization of different endocytic proteins along the tubular invaginations evolved as the profiles matured, we next defined a number of parameters to describe the position of immunogolds relative to the endocytic invaginations. In addition to the length of the labelled invagination (IL), the distance from the gold particle to the basal plasma membrane (GDPM), the distance from the gold particle to the invagination tip (GDIT = GDPM - IL) and the relative position of the gold particle with respect to the invagination (GRP = GDPM / IL) were analyzed (Fig. 3). GRPs close to 1 described golds localized at the invagination tip (IT) and GRPs near 0 defined gold particles situated at the base of the invagination (PM), close to the negatively curved area (Fig. 3) [10]. After collecting images of a minimum of 120 gold particles decorating different endocytic proteins

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

(Clathrin, Sla1, Pan1, actin, Abp1, Las17, Myo5, Bbc1 and Rvs167) the parameters defining the position of each particular protein were statistically compared for short (IL ≤ 50 nm), intermediate (50 < IL ≤ 100 nm) and long (> 100 nm) invaginations using a Kruskal Wallis Test. Further, similarities and difference between the relative positions of different proteins could be described using a General Lineal Model. Besides unequivocally identifying the primary endocytic profiles in yeast, our quantitative immunoelectron microscopy approach served to initially define the dynamics of nine endocytic proteins with a resolution down to 8 nm [10], much lower than that offered by the conventional fluorescence microscopy techniques. Such resolution provided major insights into the architecture of the endocytic complexes coupled to the lipid bilayer and redefined some of the functional modules described by the fluorescence microscopy experiments. For example, our approach demonstrated that the endocytic coat in yeast forms a hemisphere of about 30 nm, which covers the tip of the invaginations and travels into the cytosol as the profile elongate. Further, and in contrast to what it was believed at the time, we could demonstrate that the endocytic NPFs Myo5 and Las17 work at different positions along the endocytic invagination and therefore, they probably have distinct functions during endocytic budding [10]. At present, we are using this technique to build up a comprehensive map of the endocytic machinery with unprecedented spatio-temporal resolution. Further, we are analysing the morphology of the endocytic invaginations in several yeast mutants with the purpose of dissecting the process into mechanistically distinct phases. Altogether, we believe that we will provide important information about the mechanism of clathrin and actin dependent endocytic budding at an intermediate resolution between the fluorescence microscopy and the X-Ray crystallography, which will very much contribute to refine the current models explaining the process.

References 1. Farsad, K. and P. De Camilli, Mechanisms of membrane deformation. Curr Opin Cell Biol, 2003. 15(4): p. 372-81. 2. Praefcke, G.J. and H.T. McMahon, The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol, 2004. 5(2): p. 133-47. 3. McMahon, H., Harvey McMahon: ahead of the curve on membrane dynamics. J Cell Biol, 2011. 193(4): p. 598-9. 4. Engqvist-Goldstein, A.E. and D.G. Drubin, Actin assembly and endocytosis: from yeast to mammals. Annu Rev Cell Dev Biol, 2003. 19: p. 287-332. 5. Weinberg, J. and D.G. Drubin, Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol, 2012. 22(1): p. 1-13. 6. Taylor, M.J., D. Perrais, and C.J. Merrifield, A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biol, 2011. 9(3): p. e1000604. 7. Kaksonen, M., C.P. Toret, and D.G. Drubin, Harnessing actin dynamics for clathrinmediated endocytosis. Nat Rev Mol Cell Biol, 2006. 7(6): p. 404-14. 8. Galletta, B.J., O.L. Mooren, and J.A. Cooper, Actin dynamics and endocytosis in yeast and mammals. Curr Opin Biotechnol, 2010. 21(5): p. 604-10.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

9. Boettner, D.R., R.J. Chi, and S.K. Lemmon, Lessons from yeast for clathrin-mediated endocytosis. Nat Cell Biol, 2012. 14(1): p. 2-10. 10. Idrissi, F.Z., et al., Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane. J Cell Biol, 2008. 180(6): p. 1219-32. 11. Grotsch, H., et al., Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites. Embo J, 2010. 29(17): p. 2899-914.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

1.3. Cell signalling

Cell signalling and communication Isabel Fabregat Bellvitge Biomedical Research Institute (IDIBELL). Hospitalet de Llobregat, Barcelona, Spain. During the past two decades, we have observed great advances in our understanding of cell signalling. These include the appreciation of how phosphorylation, ubiquitination and acetylation events drive protein activities, as well as protein-protein interactions. Technological advances, included live-cell image techniques, high-throughput genomic sequencing, proteomics, RNA interference or the use of genetically encoded fluorescent proteins have allowed the analysis of cell signalling in space and time (1). Recent discoveries have pointed out to the realization that protein/enzyme compartmentalization determines signalling specificity. In this sense, the advance in our knowledge about the function of cell membrane compartmentalization in specialized microdomains or lipid rafts is the research line of Miguel Angel Alonso Lebrero, from the Center of Molecular Biology Severo Ochoa (CBMSO), Madrid, which has been working during the last years in the identification of components of the protein machinery that operates in the lipid rafts to characterize its mechanism of action, using lymphocytes T as a model. MAL is the original member of the MAL family of proteins, which includes a small number of proteins with overall structural homology. The alignment of sequences of the MAL family allowed a novel domain, referred to as MARVEL, to be identified, which is involved in intracellular transport and membrane apposition events. The MAL protein was the first integral membrane component of the machinery for the raft-mediated route of direct transport to the apical surface in epithelial cells to be characterized. Miguel Angelâ&#x20AC;&#x2122;s group demonstrated a novel role for MAL as an element of the machinery for a clathrin-dependent, raft-mediated route of endocytosis from the apical membrane (2). More recently, they have shown that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signalling and immunological synapse assembly (3). MAL2, a second member of the MAL family, was identified as an essential element of the machinery for the transcytotic route. Different experiments revealed a functional mechanism whereby Cdc42, the formin INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation (4). Indeed, the characterization and analysis of the function of unedited members of the MAL family of proteins, and how MAL proteins promote transport processes in epithelial cells and T lymphocytes, is one of the current interests in this lab. In summary, Dr. Alonso Lebreroâ&#x20AC;&#x2122;s work is contributing to a deeper understanding of

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

the function of the intracellular routes of transport and to the functional analysis of the growing family of MARVEL-domain-containing proteins. Signal transduction was defined as the coordinated relay of messages derived from the extracellular factors to intracellular effectors. The information received on the cell surface is processed across the plasma membrane and transduced to intracellular targets. Many evidences in the recent literature (1) support that the activators, effectors, enzymes and substrates that respond to cellular signals come together when and where they need to and cell-cell communication plays a relevant role in this process.

References 1. Scott JD, Pawson T. Cell signalling is space and time: where proteins come together and when they’re apart. 2009. Science 326:1220-4. 2. Martín-Belmonte F, Martínez-Menárquez JA, Aranda JF, Ballesta J, de Marco MC, Alonso MA. MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells. 2003. J Cell Biol 163(1):155-64. 3. Antón O, Batista A, Millán J, Andrés-Delgado L, Puertollano R, Correas I, Alonso MA. An essential role for the MAL protein in targeting Lck to the plasma membrane of human T lymphocytes. 2008. J Exp Med 205(13):3201-13. 4. Madrid R, Aranda JF, Rodríguez-Fraticelli AE, Ventimiglia L, Andrés-Delgado L, Shehata M, Fanayan S, Shahheydari H, Gómez S, Jiménez A, Martín-Belmonte F, Byme JA, Alonso MA. The formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2. 2010. Dev Cell 18(5):814-827.

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MYADM, a member of the MAL protein family, regulates Rac1 targeting to membrane rafts and cell migration Juan F. Aranda, Natalia Reglero-Real, Beatriz MarcosRamiro, Ana Ruiz-Sáenz, Miguel Bernabé-Rubio, Isabel Correas, Jaime Millán, Miguel A. Alonso Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain. -

Correspondence

should

(maalonso@cbm.uam.es),

Centro

be de

addressed Biología

Universidad Autonoma, 28049-Madrid, Spain.

to:

Molecular

Alonso

“Severo

Ochoa”,

Tel.: 34-911964401; Fax: 34-

911964420.

Abstract The MAL family of proteins has been proposed as being machinery for raft lipid organization. The MAL family members characterized so far contain four transmembrane domains, are expressed in a restricted range of tissues, and are involved in specialized raftmediated functions. Here we discussv that MYADM, a member of the MAL family with eight transmembrane segments and ubiquitous expression, is involved in the organization of raft lipids to make raft membranes competent for recruitment of Rac1 with the subsequent repercussion in cell spreading and migration.

Membrane domains generated by lipid-lipid and lipidprotein interactions: lipid and membrane rafts Lipid rafts are defined as membrane micro- or nano-domains enriched in cholesterol, glycosphingolipids and other saturated lipids, as well as with specific types of proteins. The current model of raft structure proposes that sphingolipids, which contain a sphingosine chain and a long, largely saturated, fatty acyl chain, are packed, leaving voids between the hydrocarbon chains caused by the bulky headgroups, and that are filled with cholesterol [1]. According to this model, this cholesterol-dependent liquid-ordered structure co-exists with liquid-disordered domains in cell membranes. Partitioning of proteins into lipid rafts in cells has primarily been assessed based on their association with detergent-resistant membrane (DRM) fractions, which are generally assume to be enriched in raft membranes [2]. However, DRM association reflects only a preference for rafts by proteins or lipids and does not prove that raft association precedes experimental manipulation [1]. Therefore, alternative

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

methods should be employed to assign a protein as raft associated. One of method that is becoming increasingly popular to visualize rafts in cells is the measure of membrane order by

staining

with

fluorescent

probes,

such

Laurdan

(6-dodecanoyl-2-

dimethylaminonaphthalene), whose range of wavelength emission is different in ordered and disordered membranes [3]. Proteins anchored to the extracellular leaflet of the plasma membrane by a glycosylphosphatidylinositol moiety and cytoplasmic proteins modified with saturated acyl chains such as palmitoyl moieties have been identified as DRM-associated proteins. Most transmembrane proteins are excluded from DRMs, although certain receptors are specifically recruited, at least partially, following ligand binding or cell activation [1]. Lipid rafts are, therefore, envisioned as functional platforms that compartmentalize biological membranes to confine specific proteins to regulate a large variety of cellular functions, including signalling, protein sorting and cell polarity [4]. Despite the apparent involvement of rafts in a large repertoire of cellular functions, the very existence of rafts in intact cells has been a matter of debate because of their submicroscopic size (probably less than 50 nm) and because of concerns over methods for studying rafts [5,6]. In revisiting the raft model, proteinâ&#x20AC;&#x201C;protein and proteinâ&#x20AC;&#x201C;lipid interactions, in addition to the lipid-lipid interactions of the classical model, have been gaining recognition as important factors in the dynamics of raft microdomains. Considering the importance of proteins in the raft organization, lipid rafts were renamed membrane rafts [7]. Specific protein machinery (e.g., caveolins, MAL proteins, flotillins) modulates stability, size and/or structure of membrane domains, creating specific subsets of micro- or nano-domains.

The MAL family of proteins The tetra-spanning MARVEL (MAL and related proteins for vesicle trafficking and membrane link) membrane domain [8] is present in 28 human integral proteins grouped into different families, including occludin proteins, synaptophysins and MAL proteins. MAL and MAL2, the best documented proteins of the MAL family (Fig. 1), and BENE, plasmolipin (PLLP) and CMTM8 contain four transmembrane segments, are expressed in a restricted range of tissues and are involved in specialized membrane trafficking processes [9-12]. The other three members, myeloid differentiation-associated marker (MYADM), MYADM-like1 (MYADML1) and MYADM-like2 (MYADML2), contain additional transmembrane segments, form an independent branch within the MAL family and their function is totally unknown. A distinctive feature of all the MAL proteins examined so far is their association with DRMs in all the cell types in which they are expressed.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

MAL was the first integral membrane protein to be shown as an essential component of the machinery for the direct route of apical transport, which was previously postulated to be mediated by membrane rafts. Apical transport is blocked in MDCK cells with reduced levels of MAL and this block is closely correlated with the inability of apical cargo to associate with DRMs during biosynthetic transport [9,13]. In T cells, MAL was shown to be crucial for the transport of tyrosine kinase Lck to the plasma membrane and for normal assembly of the supramolecular activation cluster (SMAC) at the immunological synapse [14,15]. In T cells depleted of endogenous MAL, Lck is retained intracellularly and this defect correlates with the inability of Lck to partition into DRMs. This and several other observations prompted us to propose a model in which MAL organizes raft lipids to make them competent to recruit cargo molecules (e.g.: HA, Lck) for polarized transport (Fig. 2).

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

In addition to MAL, other members of the MAL family have been characterized in our laboratory. MAL2 is expressed in hepatocytes and other polarized epithelia and is essential for the indirect route of apical transport [11]. This pathway consists of the targeting of cargo molecules to the basolateral membrane to be subsequently internalized and directed to the apical membrane by transcellular transport in a process called transcytosis. The BENE protein was characterized in epithelial ECV304 cells. In these cells, BENE associates with caveolin-1 and appears to control cholesterol transport [10]. Since condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the function of raft membranes for cell migration.

MYADM regulates cell migration by mediating Rac1 targeting to ordered membranes To search for MAL-family proteins with ubiquitous expression and plasma membrane localization, we have analyzed the expression and distribution of the three MYADM proteins. Our

analyses

MYADM

expression

PCR

and

MYADM

distribution

immunofluorescence microscopy revealed that only MYADM had a widespread expression

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

range and a plasma membrane distribution. Morphological analysis in epithelial cervical HeLa and prostate PC3 cells showed that MYADM knockdown (KD) cells were more rounded than control cells and presented a reduced spreading area. In addition, MYADM KD cells moved more slowly and followed a more tortuous itinerary than control cells. The change in cell shape and directionality were quantified by measuring the elliptical factor (length/breadth) and the index of directionality (the net distance divided by the total distance travelled by the cell), respectively. The cellular functions affected in MYADM KD cells (shape, spreading, and migration) are all functions regulated by Rac, a member of the Rho GTPase family of proteins. To be functional, in addition to GTP loading, Rac requires correct targeting to plasma membrane rafts for efficient signaling. As the levels neither of active GTP-loaded Rac1 nor of RhoA or Cdc42 were significantly altered in MYADM KD cells, we investigated whether MYADM regulates membrane condensation and, subsequently, the targeting of these GTPases to condensed membranes. MYADM-enriched membrane regions were found highly ordered as analyzed by Laurdan fluorescence. It is of note that membrane order was significantly reduced in MYADM KD cells to levels similar to those obtained by treating the cells with methyl-Ă&#x;-cyclodextrin a cholesterol-sequestering agent known to disrupt membrane rafts. These results indicate that MYADM regulates cell membrane condensation. Importantly, Rac1, but not RhoA and Cdc42, was found confined in condensed membranes in HeLa cells. Consistent with this role of MYADM, the absence of MYADM expression prevented Rac1 from segregating into DRMs compared with control cells. MYADM, therefore, mediates the targeting of Rac1 to condensed, raft membranes.

Conclusions We think plausible that the deficient targeting of Rac1 is a major cause of the defects of cell shape and motility observed in MYADM KD cells. Therefore, similar to the model of MAL function presented in Figure 2, whereby the primary function of MAL is to organize raft lipids to make rafts competent for cargo recruitment, a primary function of MYADM is the organization of raft membranes to make them competent for Rac1 recruitment with the subsequent repercussion in cell spreading and migration (Fig. 3). This model is consistent with previous reports showing that Rac1 is targeted to membrane ordered domains upon adhesion and that integrity of cholesterol-enriched membranes is essential for Rac1 localization and cell migration [16,17]

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

Acknowledgments This work was supported by grants BFU2009-07886 and CONSOLIDER COAT CSD2009-00016 to MAA, SAF2011-22624 to JM, and BFU2011-22859 to IC all of them from the Ministerio de EconomĂa y Competitividad and grant S2010/BMD-2305 from the Comunidad de Madrid to IC.

References 1. Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. 2010. Science 327:46-50. 2. Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. 1992. Cell 68:533-544. 3. Gaus K, Gratton E, Kable EPW, Jones AS, Gelissen I, Kritharides L et al. Visualizing lipid structure and raft domains in living cells with two-photon microscopy. 2003. Proc Nat Acad Sci 100:15554-15559.

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4. Kim T, Fiedler K, Madison DL, Krueger WH, Pfeiffer SE. Cloning and characterization of MVP17: a developmentally regulated myelin protein in oligodendrocytes. 1995. J Neurosci Res 142:413-422. 5. Shaw AS. Lipid rafts: now you see them, now you don't. 2006. Nat Immunol 7:1139-1142. 6. Munro S. Lipid rafts: elusive or illusive? 2003. Cell 115:377-388. 7. Pike LJ. Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. 2006. J Lipid Res 47:1597-1598. 8. Sanchez-Pulido L, Martin-Belmonte F, Valencia A, Alonso MA. MARVEL: a conserved domain involved in membrane apposition events. 2002. Trends Biochem Sci 27:599-601. 9. Puertollano R, Martin-Belmonte F, Millan J, de Marco MC, Albar JP, Kremer L et al. The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells. 1999. J Cell Biol 145:141151. 10. de Marco MC, Kremer L, Albar JP, Martinez-Menarguez JA, Ballesta J, Garcia-Lopez MA et al. BENE, a novel raft-associated protein of the MAL proteolipid family, interacts with caveolin-1 in human endothelial-like ECV304 cells. 2001. J Biol Chem 276:23009-23017. 11. de Marco MC, Martin-Belmonte F, Kremer L, Albar JP, Correas I, Vaerman JP et al. MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells. 2002. J Cell Biol 159:37-44. 12. Bosse F, Hasse B, Pippirs U, Greiner-Petter R, Muller HW. Proteolipid plasmolipin: localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin. 2003. J Neurochem 86:508-518. 13. Martín-Belmonte F, Puertollano R, Millán J, Alonso MA. The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and Fischer rat thyroid cell lines. 2000. Mol Biol Cell 11:20332045. 14. Antón O, Batista A, Millán J, Andrés-Delgado L, Puertollano R, Correas I et al. An essential role for the MAL protein in targeting Lck to the plasma membrane of human T lymphocytes. 2008. J Exp Med 205:3201-3213. 15. Anton OM, Andres-Delgado L, Reglero-Real N, Batista A, Alonso MA. MAL protein controls protein sorting at the supramolecular activation cluster of human T lymphocytes. 2011. J Immunol 186:6345-6356. 16. del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, Anderson RG, Schwartz MA. Integrins regulate Rac targeting by internalization of membrane domains. 2004. Science 303:839-842. 17. Palazzo AF, Eng CH, Schlaepfer DD, Marcantonio EE, Gundersen GG. Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. 2004. Science 303:836-839.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

1.4. Autophagy, apoptosis and cell homeostasis

An overview of cell homeostasis Isabel Varela-Nieto∞ and Mª Angela Burrell* ∞ Instituto Investigaciones Biomedicas “Alberto Sols” CSIC-UAM, CIBERER, IdiPaz, Madrid. E-mail: ivarela@iib.uam.es. * Departamento de Histología y Anatomía Patológica, Universidad de Navarra. Pamplona. E-mail: mburrell@unav.es. During development the sequential generation of the cell types that will form the adult organism requires the coordination of cell proliferation with cell differentiation programs, the strict regulation of cell survival and the metabolic homeostasis of cellular precursors. A network of intracellular signals operates to coordinate the transcriptional response to the extracellular input. Understanding the molecular clues that direct development is fundamental to understand adult physiology and pathological disorders. Furthermore, it is crucial for the design of novel treatments based on gene and cellular therapies. Adult cell homeostasis also depends on the careful balance among biological processes as cell death, proliferation and differentiation in a tissue-specific manner. The importance of autophagy and apoptosis in adult cell homeostasis and disease is starting to be understood. Apoptosis can be defined as the set of biochemical reactions that occur in a cell leading to its death in a quiet and orderly way. The apoptotic cell undergoes a series of morphological changes that define it as such. The plasma membrane is altered, cell volume is significantly reduced, the cytoplasm condenses and the chromatin becomes dense and collapses. The apoptotic cell is phagocytized in an ATP-dependent manner by macrophages or neighboring cells, avoiding the local inflammatory response that causes necrosis. Autophagy is a self-degradation process that regulates protein and organelle removal in a variety of tissues during development. Autophagy plays a dual role as a physiological cell death mechanism and as a survival key process during critical times of embryogenesis. Through cytoplasmic remodeling autophagy participates in cellular death, proliferation and differentiation. The activation of the autophagic machinery could promote either cell death or survival depending on the cellular context; autophagy is thus an extraordinary tool for the developing organs and for tissue remodeling. Defects in autophagy may be related to a variety of pathologies, in this context, exciting new findings on the etiology of cancer and neurodegenerative diseases are emerging. Recent excellent reviews covering these aspects are those by Mizushima and Komatsu [1] and by Codogno et al. [2]. To understand how autophagy, cell death and disease are linked it is first necessary to answer many basic questions. The molecular interactions between autophagy and apoptosis are very complex, both processes may be triggered by common upstream signals and share common machinery. There are numerous examples of the mutual regulation of apoptosis

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

and autophagy, each process is likely to regulate and modify the activity of the other. Autophagy regulators can control not only whether the cell dies or not, but also the way cells die. Conversely, apoptosis could control autophagy, although less is known about the involved mechanisms. Mitochondria have a crucial role in this crosstalk. p62 and beclin, among others, seem to be key regulators at the core of autophagy-apoptosis interactions. Gump and Thorburn [3] point out the outstanding issues that need to be unraveled in the coming years in order to apply this information in a clinical context.

References 1. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. 2011. Cell 147(4):728-41. 2. Codogno P, Mehrpour M, Proikas-Cezanne T. Canonical and non-canonical autophagy: variations on a common theme of self-eating? 2011. Nat Rev Mol Cell Biol 13(1):7-12. 3. Gump JM, Thorburn A. Autophagy and apoptosis: what is the connection? 2011. Trends Cell Biol 21(7):387-392.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

Differential regulation of autophagy, proliferation and cell survival during otic neurogenesis María R. Aburto1,2, Marta Magariños1,2,3 & Isabel VarelaNieto1,2,4* 1 2

Institute for Biomedical Research “Alberto Sols”, CSIC-UAM, Madrid, Spain. Unit 761, Centro de Investigación Biomédica en Red de Enfermedades Raras

(CIBERER), Instituto de Salud Carlos III, Madrid, Spain. 3

Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain.

IdiPAZ, Madrid, Spain

* Author for the correspondence ivarela@iib.uam.es

Early otic vertebrates

development

and

neurogenesis

The vertebrate inner ear contains multiple cell types that derive from a simple ectodermal placode [1]. The otic placode invaginates to form the otic cup, which pinches off in birds and mice, or cavitates in fish to produce the otic vesicle or otocyst [2, 3]. The otic epithelium also generates the neurons for the acoustic-vestibular ganglion (AVG) that will eventually innervate the hair cells of the inner ear [4, 5]. Neuronal progenitors are specified within the neurogenic otic epithelium, where they lose connectivity and delaminate into the adjacent mesenchyme to form the AVG, which later develops into the acoustic and vestibular ganglia that connect the sensory epithelia to the brain through the VIII cranial nerve. Otic neuronal progenitors pass through different cellular stages characterized by a distinct combination of molecular markers before became competent neurons (Fig. 1). The first visible event in otic neurogenesis is the migration of the epithelial neuroblasts from the proneural region of the otic cup towards the mesenchymal space loosing the connectivity with the otic epithelium. Ganglionar neuroblasts undergo a series of divisions that expand and maintain the population. Later on, as neurogenesis proceeds, these neuroblasts will exit the cell cycle and differentiate into mature neurons, extending their axons to innervate the sensory patches [3]. Extrinsic and intrinsic factors are involved in regulating and coordinating different aspects of this process, among them insulin-like growth factor (IGF-I) is fundamental for early neurogenesis. [2, 6].

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Regulation of cell proliferation, cell death and neurogenesis by IGF-I: PI3K-AKT and RAF-MEK-ERK pathways Insulin-like growth factor I (IGF-I) is a member of the insulin family of peptides that modulates

cell

survival,

epithelial

morphogenesis,

neurogenesis

and

late

neural

differentiation in the inner ear [3, 7]. IGF-I deficiency is associated to profound neurosensorial deafness in mice and men and is fundamental for the regulation of cochlear development, growth, and differentiation [8, 9]. During development, IGF-I is necessary for cell survival and proliferation in otic vesicle explants, and also for survival and differentiation of otic neuroblasts in the chicken embryo. Accordingly, blockade of endogenous IGF-I activity in otic vesicle explants, inhibits the formation of the AVG and increases cell death. IGF-I acts through the high affinity tyrosine kinase type 1 IGF receptor to activate multiple signal transduction pathways including the

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phosphatidylinositol 3-kinase (Pl3K) pathway and the mitogen-activated protein kinase cascade (MAPK, the RAF-MEK-ERK pathway) [10] (Fig. 2).

The PI3K-AKT signaling pathway is crucial for cell survival. AKT is a nodal molecule within this complex signaling network that phosphorylates a large number of proteins that are either stimulated or, more frequently, inhibited. AKT-modulated pathways regulate metabolism, protein synthesis, cell cycle, cell survival and cell death [11, 12]. IGF-I strongly promotes the proliferation of otic neuroblasts a process that is totally inhibited by treatment with either LY294002 or AKTi VIII inhibitors of the PI3K-AKT pathway. In contrast, postmitotic neuroblasts are not affected by these inhibitors nor respond to IGF-I, which suggests that their survival depends principally on other factors, such as neurotrophins. Therefore, the existing data indicate that otic neurogenesis dependence on the activation of the PI3K-AKT pathway by IGF-I takes place in a specific temporal window during neurogenesis and is marked by the neural differentiation state [7].

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

IGF-I also activates the signaling cascade RAF-MEK-ERK that is essential for cell proliferation in the inner ear [13, 14]. The A, B and C-RAF kinase family are serine/threonine kinases that play a central role in normal and pathologic cellular processes, including cell regeneration, cell senescence and cancer. B and C-RAF are essential for regulating the balance between cell death and cell proliferation in the otic neuroepithelial population and also for otic neuron differentiation and axonal growth at the AVG [14]. By using the specific B- and C-RAF inhibitor Sorafenib and the C-RAF inhibitor GW5074, we have been able to describe that C-RAF promotes antiapoptotic signals by a MEK-ERK-independent pathway, whereas B-RAF regulates epithelial neuroblast proliferation.

Autophagy is essential for otic neurogenesis Autophagy is a catabolic process that supplies energy during development and in response to nutrient stress by carrying out lysosomal degradation of cell contents. Autophagy also plays a housekeeping role preventing the accumulation of proteins and clearing damaged organelles and even cells [15]. Accordingly, autophagy in vertebrates has key roles in development [16], cancer and neuronal degeneration [17]. Autophagy begins with the formation of a phagophore that expands to form a double-membrane autophagosome. This engulfs intra-cellular cargo, such as protein aggregates, and organelles. The autophagosome then fuses with a lysosome and acid hydrolases degrade its contents, the Atg proteins participate at different stages of this process. Autophagy can be studied by using specific chemical inhibitors as 3-methyladenine (3-MA) or rapamicyn. Gene expression studies in the E18.5 cochlea showed the expression of key autophagy genes as Beclin-1, Lc3b (microtubule-associated protein light chain 3b), and Atg genes including Atg4b and d [6; Gene Expression Omnibus accession number GSE11821]. Recently, we have shown that the autophagic machinery is expressed in early stages of inner ear development and that it is an active process during early inner ear development [18]. Inhibition of autophagy in cultured otic vesicles with 3-MA caused the accumulation of apoptotic cells that cannot be eaten up by macrophages. The addition of an external source of ATP recovered the normal phenotype of the otic vesicle. Taken together these data indicate that autophagy provides the metabolic energy needed for the structure to process apoptotic-dying cells. Moreover, autophagy inhibition impaired early AVG formation and, in later stages of differentiation, axonal guidance. In summary, early otic neurogenesis requires

the

strict

regulation

biological

processes that are coordinated by the concerted action of extrinsic and intrinsic factors. Otic epithelial cells have to escape apoptosis, survive, nurture themselves by autophagy, proliferate, migrate and differentiate to generate the neurons of the AVG that will eventually innervate the hair cells of the inner ear. To this end, IGF-I acts differentially trough the PI3K-AKT and the RAF-MEK-ERK signaling pathways.

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Acknowledgements This work was partially supported by SAF2008 and SAF2011 grants to IVN and by a JAE-CSIC contract to Maria R Aburto. We thank Javier Perez and the IIB Image Unit for their technical support. We warmly thank our colleagues from the Neurobiology of Hearing Group and Dr. Patricia Boya (CIB-CSIC, Madrid) for the critical reading of the manuscript and the generous sharing of results and reactives.

References 1. Driver EC, Kelley MW. Specification of cell fate in the mammalian cochlea. 2009. Birth Defects Res C Embryo Today 87(3):212-21. 2. Sanchez-Calderon H, Milo M, Leon Y, Varela-Nieto I. A network of growth and transcription factors controls neuronal differentation and survival in the developing ear. 2007. Int J Dev Biol 51:557-570. 3. Magariños M, Contreras-Rodríguez J, Aburto MR, Varela-Nieto I. Early development of the vertebrate inner ear. 2012. Anat Rec (In Press). 4. Fekete DM, Wu DK.. Revisiting cell fate specification in the inner ear. 2002. Curr Opin Neurobiol 12:35-42. 5. Yang T, Kersigo J, Jahan I, Pan N, Fritzsch B. The molecular basis of making spiral ganglion neurons and connecting them to hair cells of the organ of Corti. 2011. Hear Res 278:21-33. 6. Sánchez-Calderón H, Rodríguez-de la Rosa L, Milo M, Pichel JG, Holley M, Varela-Nieto I. RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors. 2010. PLoS ONE 5:e8699. 7. Aburto MR, Magariños M, Leon, Y, Varela-Nieto I, Sanchez-Calderon H. AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors. 2012. PLoS ONE 7:e30790. 8. Walenkamp MJE, Wit JM. Genetic disorders in the growth hormone - insulin-like growth factor-I axis. 2006. Horm Res 66:221-230. 9. Murillo-Cuesta S, Rodríguez-de la Rosa L, Cediel R, Lassaletta L, Varela-Nieto, I. The role of insulin-like growth factor-I in the physiopathology of hearing. 2011. Front Mol Neurosci 4:11. 10. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. 2006. Nat Rev Mol Cell Biol 7:85-96. 11. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. 2007. Cell 29;129(7):1261-74. 12. Sale EM, Sale GJ. Protein kinase B: signalling roles and therapeutic targeting. 2008. Cell Mol Life Sci 65(1):113-27. 13. Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. 2004. Nat Rev Mol Cell Biol 5:875-885. 14. Magariños M, Aburto MR, Sánchez-Calderón H, Muñoz-Agudo C, Rapp UR, VarelaNieto I.. RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development. 2010. PLoS ONE 5:e14435. 15. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, et al. . Regulation of mammalian autophagy in physiology and pathophysiology. 2010. Physiol Rev 90:1383-1435. 16. Montero JA, Hurlé JM. Sculpturing digit shape by cell death. 2010. Apoptosis 15: 365375.

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17. Wong E, Cuervo AM. Autophagy gone awry in neurodegenerative diseases. 2010. Nat Neurosci 13:805-811. 18. Aburto MR, Hurlé JM, Varela-Nieto I and Magariños M. Autophagy during Vertebrate Development. Cells. In the press. 2012.

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1.5. Cell biology of aging

Overview of cell senescence and longevity Guillermo LĂłpez-Lluch Centro Andaluz de BiologĂa del Desarrollo, Universidad Pablo de Olavide, CIBERER, Instituto Carlos III, Carretera de Utrera Km. 1, 41013 Sevilla, Spain. After a considerable number of studies, we can consider that ageing is a complex process involving near all the cells and tissues of the organism. Several theories have been proposed to explain the whole process but none of them has been able to fit all the factors involved in aging. However, one of the common denominators of this process is that cells accumulate damaged substances and structures inside and outside them that impair their activity and contribute to the physiological decay of the whole organism. In this accumulation, the activity of metabolic factors, antioxidant and reparation mechanism and recycling procedures such as autophagy, mitophagy and the proteasome-dependent protein degradation are involved. Further, mechanisms involved in cell cycle control and epigenetic factors involved in gene regulation also contribute in an important degree with the decrease of the capability of cells to maintain their activity during aging. During the last years, it has been demonstrated that the metabolic activity is one of the main sources of cell damaging factors such as reactive oxygen and nitrogen species being mitochondrial activity one of the key processes involved in production of these radicals. For this reason, the accumulation of damaged mitochondria into cells ends in a deleterious cycle where higher radicals produced by damaged mitochondria increase damage in mitochondria and in the rest of the cell. Relating to the regulation of metabolism and its effect on aging, calorie restriction (CR) is the only non-genomic intervention able to increase lifespan in any of the organisms studied to date. Some years ago we demonstrated that CR induces a more balanced activity in mitochondria reducing reactive oxygen species (ROS) production and reducing damage (1). Further, oxidative damage is also reduced by the activation of coenzyme Q-dependent antioxidant reductases such as cytochrome b5-reductase and NQO1 (2). Caloric restriction not only reduces metabolic effect but also improves the activity of recycling mechanisms related to autophagy and protective mechanisms such as antioxidant systems and damagerepair mechanisms (3). On the other hand, resveratrol, a polyphenol found in grapes and berries, has been shown to mimic CR effect and modulate mitochondrial activity (4). Resveratrol also controls mitochondrial activity by increasing respiratory efficiency and reducing oxidative damage. Although the effect of resveratrol on sirtuin activity and the role of these deacetylases in longevity are under controversy (5), it seems clear that they show essential roles on the control of mitochondrial activity and its importance in aging (6). Most of the seven different sirtuins found in mammals show metabolism-related regulatory roles mainly of them involved

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in the regulation of cell metabolism to a higher oxidative respiration. They are also involved in the regulation of endogenous antioxidant systems coupled to the oxidative metabolism. Autophagic mechanisms are also involved in longevity. In fact, inhibition of this process by genetic mechanisms induces degenerative changes resembling those related to aging. Further, many pharmacological compounds able to increase longevity often stimulate autophagy indicating that the elimination of accumulated damaged structures in cells is importantly involved in aging (3). On the other hand, the mechanisms involved in cell cycle regulation have been recently related to cell senescence. These mechanisms are regulated after cellular damage and direct the cells death by apoptosis or senescence. Very recently one of these regulators, the lnk4/Arf locus involving p16lnka, p19Arf and p15lnk4b tumor suppressors have been related to the mechanisms involved in the progression of aging. The expression of these factors progressively increases during aging and many genome-wide association studies have linked them to a number of aging-associated diseases and also frailty in humans. Recent studies indicate that the activity of this locus is associated with a global anti-aging effect probably by favoring quiescence and preventing unnecessary proliferation of cells (7-8). In all these mechanisms it seems that the activity of AMP activated protein kinase (AMPK), sirtuins (SIRT) and target of rapamycin (TOR) pathways are involved. In fact, caloric restriction, resveratrol, sirtuins and exercise, all of the mechanisms that have demonstrated prolongevity effects or at least positive effects on health-span, seem to produce their effect by affecting these pathways. Interestingly, all these pathways have been related to metabolic control, regenerative processes, protective mechanisms and control of cell cycle. It seems clear that metabolism plays an important role in the progression of aging and longevity and further research involving metabolic processes and regulation of cell growth and senescence will permit us to clarify the regulatory factors involving in these processes. Taken together, the latter publications in aging indicate that this process is a continuous phenomenon where the equilibrium between cell damage and cell renovation is one of the main factors. This equilibrium is maintained during early times by higher ratios of damage elimination and balance cell growth/apoptosis. When years accumulate, the capacity of stem cells to proliferate and renovate tissues and the intracellular recycling mechanisms slow and permit the accumulation of cell damage or decrease the renew of cells in tissues and organs. Later results indicate that senescence of cells can be a protective mechanism against cell growth progression when cell is damaged avoiding cancer. Further, new research involving epigenetic regulatory processes such as DNA and histone metilation but also microRNA-dependent regulatory processes will highlight some other important aspects to identify the importance of genetic and epigenetic processes in aging and the relationship of aging and metabolism (9-10).

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References 1. López-Lluch G, Hunt N, Jones B, Zhu M, Jamieson H, Hilmer S, Cascajo MV, Allard J, Ingram DK, Navas P, de Cabo R. Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency. 2006. Proc Natl Acad Sci U S A. 103(6):1768-73. 2. De Cabo R, Cabello R, Rios M, López-Lluch G, Ingram DK, Lane MA, Navas P. Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver. 2004. Exp Gerontol. 39(3):297-304. 3. Rubinsztein DC, Mariño G, Kroemer G. Autophagy and aging. 2011. Cell. 146(5):682-95. 4: Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA. Resveratrol improves health and survival of mice on a high-calorie diet. 2006. Nature. 444(7117):337-42. 5. Burnett C, Valentini S, Cabreiro F, Goss M, Somogyvári M, Piper MD, Hoddinott M, Sutphin GL, Leko V, McElwee JJ, Vazquez-Manrique RP, Orfila AM, Ackerman D, Au C, Vinti G, Riesen M, Howard K, Neri C, Bedalov A, Kaeberlein M, Soti C, Partridge L, Gems D. Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila. 2011. Nature. 477(7365):482-5. 6. Santa-Cruz Calvo S, Navas P, López-Lluch, G. Sirtuin-dependent metabolic control and its role in the aging process. In: Bioenergetics. (Kevin Clark ed). In Tech., 2012. pp; 95-120. 7. Serrano M. Cancer: final act of senescence. 2011. Nature. 479(7374):481-2. 8. Matheu A, Maraver A, Collado M, Garcia-Cao I, Cañamero M, Borras C, Flores JM, Klatt P, Viña J, Serrano M. Anti-aging activity of the Ink4/Arf locus. 2009. Aging Cell. 8(2):152-61. 9. Simboeck E, Ribeiro JD, Teichmann S, Di Croce L. Epigenetics and senescence: learning from the INK4-ARF locus. 2011. Biochem Pharmacol. 82(10):1361-70. 10. Vlaming H, van Leeuwen F. Crosstalk between aging and the epigenome. 2012. Epigenomics. 4(1):5-7.

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Mitochondrial homeostasis and healthy aging Guillermo López-Lluch Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, CIBERER, Instituto Carlos III, Carretera de Utrera Km. 1, 41013 Sevilla, Spain.

Introduction Aging is accompanied by the accumulation of damaged molecules in cells mainly due to the injury produced by external and internal factors. Among these factors, reactive oxygen species (ROS) produced by cell metabolism are considered essential damaging factors. Although under discussion, mitochondria-dependent functions seem to be in a central position to explain aging. Several studies have demonstrated that aging-delaying compounds or nutrition or exercise-based procedures are able to affect the activity and biogenesis-turnover of mitochondria, especially in post-mitotic tissues. It is then important to highlight the importance of mitochondria to explain the aging process in different models or organisms. However it is important to determine if mitochondrial activity malfunction is the cause or is simply another mark of the pass of the time.

The free radical theory of aging revisited It is known that mitochondrial dysfunction leads to increased levels of ROS and that ROS are important factors in development of mitochondrial dysfunction (1). This vicious cycle is the basis of the ‘free radical theory of aging’ proposed by Harman (2). Harman postulated that biomolecules are damaged by ROS, which would over time result in the functional impairment that defines aging. He proposed that the most likely source of such radicals would be ‘‘the interaction of the respiratory enzymes involved in the direct utilization

of molecular oxygen’’. Every single aerobic cell produces low levels of ROS and shows a certain level of oxidative stress under normal conditions. This means that even when antioxidant mechanisms are ancient and evolutionarily developed; they are not completely efficient. Oxidation of lipids, proteins and DNA increases with age in different species and correlates with higher levels of mitochondrial O2●- and H2O 2 production. Harman’s theory was later refined by himself and others to propose that mitochondria, besides being the main cellular source of ROS (3), are also the most relevant free radical target in aging (4-6). Most work in the field during the 80s and 90s focused on extensive characterization of mitochondrial dysfunction associated with aging, including reduced bioenergetic capacity, increased ROS production and mtDNA oxidative damage and mutation accumulation. Reduction of antioxidant defences results in high ROS levels inducing shorter lifespan in yeast, flies and mice (7-9) and confirming the role of ROS in aging. Then, if lifespan

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extension is caused by a reduction of ROS levels, treatment with antioxidant agents should increase lifespan in organisms. However this has not been clearly demonstrated so far. The accumulation of mutations and errors induced by ROS in mitochondria affects the mitochondrial function and can explain to some extent aging process (10) although this association is not straightforward (11). Damaging in DNA may impair mitochondrial DNA replication and/or induce mitochondrial DNA degradation, both of which would result in a decline in levels of mitochondrial DNA, mRNA, and protein. Reduced levels of mitochondrial proteins would result in decreased ATP synthesis and an eventual decline in cellular function (12). Further, the disruption of the dynamic process of fusion and fission of mitochondria has been also related to the process of cellular senescence and the development of age-related diseases (13). On the other hand, energy produced in cells is used to maintain many functions including the maintenance of cell homeostasis, renewal, reparation and removal of damaged structures. From the discovery of the effect of the mutation of age-1 gene in C. elegans, and its pro-longevity effect most of the prolongevity genes discovered to date are involved in modifications of cell metabolism. Caloric restriction (CR), the only non-genetic mechanism discover to date able to decrease aging progression, explains how a better balance of metabolism is responsible of lengthening life (14). However, CR not only modulates the efficiency of the metabolism but also activates mechanisms of maintenance and reparation of cells and tissues. In all these mechanisms, mitochondrial activity plays a key role. It has been proposed that CR shows low probability to affect human longevity because our low metabolic rate. However, CR does affect and increase life-span in short-living animals with a high metabolism rate: worms, flights, mice, etc.

Mitochondrial activity during development influences aging Metabolism is a key factor in development at both embryonic and post-embryonic phases. A recent paradigm indicates that development in uterus and adaptations during early life are part of the environmental factors affecting aging. Then, metabolic changes produced during pregnancy must condition early life in humans that can favour or impair life expectancy. A large body of epidemiological data indicate that an adverse early environment produces metabolic diseases in adult offspring and shortens lifespan. Moreover, epidemiological studies carried out in humans have demonstrated that both, under- and over-nutrition severely affect health in the offspring inducing a high level of prevalence of obesity aggravated with a high incidence of diabetes (15). Very recently it has been shown that paternal diet affects the epigenetic marks in spermatozoa and induces environmental reprogramming of the metabolic gene expression of the offspring (16). Further, these metabolic changes have been also involved in the process of neurological disorders including neuropathies that severely affect life-span (17). It has been also shown that protein-restricted diet during pregnancy alters the future regulation of metabolism in cardiac

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muscle of the offspring at the epigenetic level (18). It is then clear that during gestation a metabolic reprogramming happens and that it affects the health and development of mature offspring. Then, it is important to know the epigenetic mechanisms occurring during gestation that affect mitochondrial biogenesis and functions affecting aging. Although ROS levels coming from mitochondria have been considered as major factors in cells damage they are also important components in cell signalling and development. ROS production by mitochondria can condition the optimum development during gestation and the onset of diseases during maturity or aging. During the last years a body of results indicate that the transition between aerobic glycolysis to respiration induces ROS production that affects differentiation of stem cells of the mesenchimal lineage (19-21). These results indicate that a balanced activity of mitochondria is essential for early developmental processes that can further affect lifespan. The repercussions of embryonic development and early life environment on metabolism are currently two of the most promising fields of research in aging. For this reason, we consider that the knowledge of mitochondria functions and its regulation during embryonic development and early-life, and its association with cellular modifications/damage could highlight the mechanisms involved in the conditioning of the organism to develop certain diseases during adulthood and aging. The knowledge of the epigenetic and regulatory processes involved in the reprogramming process of mitochondria would support a better knowledge and would lead to the identification of therapeutic targets to extend the healthy years of life.

Molecular mechanisms involved in the lengthening of aging To explain how caloric restriction, exercise or other interventions that modify metabolic homeostasis and mitochondrial activity produce protective effects on aging we have to focus on the main factors involved in aging. However, it is necessary to take into consideration that to date there is not a theory that is generally accepted to explain aging. As it has been above indicated, the accumulation of damage along life and the incapacity to remove damaged components of cells and tissues is directly related to the malfunction of cells and the deterioration of physiological functions that aggravates during aging. In this aspect, the turnover of mitochondria seems to be a key factor since experiments carried out in mouse demonstrate that exercise is able to reduce the incidence of mitochondrial mutations responsible of the accumulation of dysfunctional mitochondria. Further, several papers indicate that the exercise-dependent induction of mitochondrial biogenesis in muscle is able to protect muscle against mitochondrial damages (22,23). Further, CR and the polyphenol that acts in several aspects as CR-mimetic also affect mitochondrial biogenesis and turnover (14,24). Then, it is important to determine the factors involved in mitochondrial biogenesis to understand how this process can be related to aging. These factors can be clustered in three main groups: ubiquitous transcription factors (SP1, YY1, CREB, MEF-2/E-Box),

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nuclear respiratory factors (NRF- 1, -2, MT1- to -4) and co-activators (PGC-1α, 1B and PRC) (25). Other factors are also involved in the metabolic adaptation to fasting and energy requirements such as peroxisome proliferator activated receptor (PPAR) that together with PGC-1α increase mitochondrial biogenesis. Very interestingly, the most recent results indicate that PGC-1α is required for preventing the decline in mitochondrial activity but also to induce the expression of antioxidant enzymes (26). Among the molecular components involved in mitochondrial turnover PGC-1α is the common denominator. The biogenesis of new mitochondria is stimulated by the PGC-1α-NRF1 pathway. PGC-1α is the first stimulator of mitochondrial biogenesis whereas NRF1 is an intermediate transcription factor which stimulates the synthesis of transcription factor A mitochondrial (TFAM). In mitochondria TFAM activates the duplication of mitochondrial DNA molecules. One of the reasons by which mitochondria activity decreases during aging is because this pathway is impaired (10,27). The response of the organism to environmental agents characterized by a low dose stimulation of beneficial effects whereas high doses produce toxic and also deleterious effects is called hormesis (28). As in the case of ischemia, dietary restriction or low doses of phytochemicals, or exercise also induce mild stress in the organism inducing stressdependent signals that activate kinases, deacetylases and transcription factors such as Nrf2 or NF-κB that increase the amount of protective and antioxidant proteins and the activity of reparation systems of all kind of biomolecules including DNA and proteins (30).

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This process makes metabolic sensors such as sirtuins (31) or AMP-activated protein kinase (AMPK) the most interesting key components to understand how metabolic homeostasis can be involved in aging. In fact, PGC1Îą (10), AMPK (32) and sirtuins (31) are currently being studied in deep to dissect their role in aging and to design therapeutic strategies or pharmacological products able to induce mitochondrial turnover by inducing biogenesis and autophagic processes at the same time but also the activity of endogenous antioxidant and reparation systems (Figure 1). In conclusion, similarly to the case of an old but well maintained car, if we are able to maintain the activity of the engine at the same time that repair exhausted pieces or damages, our organism is able to maintain a high activity until reaching our maximum longevity life. In agreement with other authors, I consider that working on aging research must be focus on the goal of adding live to the years whereas adding years to life is a utopian goal for the moment.

References 1. Cassarino DS, Bennett JP, Jr. An evaluation of the role of mitochondria in neurodegenerative diseases: mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration. 1999. Brain Res Rev. 29:1-25. 2. Harman D. Aging: a theory based on free radical and radiation chemistry. 1956. J Gerontol. 11:298-300. 3. Sesti F, Liu S, Cai SQ. Oxidation of potassium channels by ROS: a general mechanism of aging and neurodegeneration? 2010. Trends Cell Biol. 20:45-51. 4. Fleming JE, Reveillaud I, Niedzwiecki A. Role of oxidative stress in Drosophila aging. 1992. Mutation Res. 275:267-79. 5. Ku HH, Brunk UT, Sohal RS. Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species. 1993. Free Rad Biol Med. 15:621-7. 6. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. 1996. Science. 273:59-63. 7. Elchuri S, Oberley TD, Qi W, Eisenstein RS, Jackson Roberts L, Van Remmen H, Epstein CJ, Huang TT. CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. 2005. Oncogene. 24:367-80. 8. Phillips JP, Campbell SD, Michaud D, Charbonneau M, Hilliker AJ. Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. 1989. Proc. Natl Acad Sci USA. 86:2761-5. 9. Wawryn J, Krzepilko A, Myszka A, Bilinski T. Deficiency in superoxide dismutases shortens life span of yeast cells. 1999. Acta Biochim Pol. 46:249-53. 10. Lopez-Lluch G, Irusta PM, Navas P, de Cabo R. Mitochondrial biogenesis and healthy aging. 2008. Exp Gerontol. 43:813-9. 11. Vermulst M, Bielas JH, Kujoth GC, Ladiges WC, Rabinovitch PS, Prolla TA, Loeb LA.Mitochondrial point mutations do not limit the natural lifespan of mice. 2007. Nat Genet. 2007;39:540-3. 12. Hebert SL, Lanza IR, Nair KS. Mitochondrial DNA alterations and reduced mitochondrial function in aging. 2010. Mech Ageing Develop.131:451-62. 13. Bonda DJ, Wang X, Perry G, Smith MA, Zhu X. Mitochondrial dynamics in Alzheimer's disease: opportunities for future treatment strategies. 2010. Drugs Aging. 27:181-92.

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14. Lopez-Lluch G, Hunt N, Jones B, et al. Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency. 2006. Proc. Natl Acad Sci USA. 103:1768-73. 15. Sullivan EL, Grove KL. Metabolic imprinting in obesity. 2010. Forum Nutr. 63:186-94. 16. Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Meissner A, Weng Z, Hofmann HA, Friedman N, Rando OJ. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. 2010. Cell. 143:1084-96. 17. Levin BE. The obesity epidemic: metabolic imprinting on genetically susceptible neural circuits. 2000. Obesity Res. 8:342-7. 18. Slater-Jefferies JL, Lillycrop KA, Townsend PA, Torrens C, Hoile SP, Hanson MA, Burdge GC. Feeding a protein-restricted diet during pregnancy induces altered epigenetic regulation of peroxisomal proliferator-activated receptor-a in the heart of the offspring. 2010. J Develop Ori Health Dis. 2(4):250-255. 19. Crespo FL, Sobrado VR, Gomez L, Cervera AM, McCreath KJ. Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose. 2010. Stem cells. 28:1132-42. 20. Owusu-Ansah E, Banerjee U. Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. 2009. Nature. 461:537-41. 21. Ji AR, Ku SY, Cho MS, Kim YY, Kim YJ, Oh SK, Kim SH, Moon SY, Choi YM. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. 2010. Exp Mol Med. 42:175-86. 22. Argiles JM, Busquets S, Lopez-Soriano FJ, Figueras M. Fisiologia de la sarcopenia. Similitudes y diferencias con la caquexia neoplasica. 2006. Nutr Hosp. 21(Supl 3):38-45. 23. Clark DJ, Patten C, Reid KF, Carabello RJ, Phillips EM, Fielding RA. Muscle performance and physical function are associated with voluntary rate of neuromuscular activation in older adults. 2011. J Gerontol A Biol Sci Med Sci. 66:115-21. 24. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA. Resveratrol improves health and survival of mice on a high-calorie diet. 2006. Nature. 444(7117):337-42. 25. Visvanathan R, Chapman I. Preventig sarcopaenia in older people. 2010. Maturitas. 66:383-8. 26. Frontera WR, Meredith CN, O’Reilly KP, Knuttgen HG, Evans WJ. Strength conditioning in older men: skeletal muscle hypertrophy and improved function. 1988. J Appl Physiol. 64:1038-44. 27. Fiatarone MA, Marks EC, Ryan ND, Meredith CN, Lipsitz LA, Evans WJ. Hingh-intesity strenght training in nonagenarians. Effects on skeletal muscle. 1990. JAMA. 263:3029-34. 28. Fiatarone MA, O’Neill EF, Ryan ND, Clements KM, Solares GR, Nelson ME, Roberts SB, Kehayias JJ, Lipsitz LA, Evans WJ. Exercise training and nutricional supplementation for physical frailty in very elderly people. 1994. N Engl J Med. 330:1769-75. 29. Lexell J, Downhm DY, Larsson Y, Bruhn E, Morsing B. Heavy-resistance training in older Scandinavian men and women: short- and long-term effects on arm and leg muscles. 1995. Scan J Med Sci Sports. 5:329-41. 30. Carmeli E, Reznick AZ, Coleman R, Carmeli V. Muscle strength and mas of lower extremities in relation to functional abilities in ederly adults. 2000. Gerontology. 46:249-57. 31. Santa-Cruz Calvo S, Navas P, López-Lluch, G. Sirtuin-dependent metabolic control and its role in the aging process. In: Bioenergetics. (Kevin Clark ed). In Tech., 2012. pp; 95-120. 32. Salminen A, Kaarniranta K. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network. 2012. Ageing Res Rev. 11:230.241.

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1.6. Plant cell biology

Cell biology of plant development and adaptation Pilar S. Testillano∞ and Dolores Rodríguez* ∞ Plant Development and Nuclear Architecture. Biological Research Center, CIBCSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. testillano@cib.csic.es *Centro Hispano-Luso de Investigaciones Agrarias (CIALE), University of Salamanca, Spain. mdr@usal.es

Plant biology research has been historically conducted using traditional genetic approaches to solve relevant challenges in agriculture. The application of molecular biology approaches has produced, over the last decades, an increasing knowledge on the genes controlling plant growth and development, as well as plant response and adaptation to changing and adverse environmental conditions, a considerable number of genes being identified. More recently, plant research interests have been focused on the understanding of how a multicellular organism can grow and develop from a single cell and how the appropriate cell types are specified in the right place in an organism, the plant cell arising as an interesting model to explore common basic processes in plant and animal cells like totipotency, differentiation, stress response, or cell reprogramming. To focus on these challenges, cell biology approaches have became essential tools. The exploitation of fluorescent proteins has heralded a new age in the in vivo analysis of subcellular events, and has overcome many of the limitations that are associated with the investigation of cellular and molecular processes in plant cells. Modern bioimaging and cell biology techniques have significantly contributed to the wealth of recent exciting data on the broad field of plant cell biology. In this context, the present chapter on “Cell Biology of Plant Adaptation and Development” includes four significant papers of representative outstanding topics of the field. Taking the root as a model system, new information has been recently obtained on the role of polar auxin transport as electric signalling in root movements (tropisms); new evidences are discussed to support the exciting model of the synaptic-like secretory polar auxin transport which is behind of defined root sensory-motoric circuits. Other plant hormones, the brassinosteroids (BRs) are known as growth-promoting steroid hormones that mediate both developmental and environmental signals. Recent work has focused on investigating the role of BRs in controlling root growth by a genetic, molecular and cellular approach; the new data has revealed that BR signaling is required to promote cell growth, this control affecting the balance between cell cycle progression and cell differentiation. In vitro plant systems are very convenient models to explore the mechanisms controlling plant

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cell reprogramming and embryogenic competence acquisition by stress treatments. Nuclear reprogramming requires the removal of the cell type-specific epigenetic patterns imposed on chromatin during cellular differentiation. The analysis of the stress-induced pollen reprogramming to embryogenesis has provided new evidences showing that changes in DNA methylation, accompanied by the reorganization of the nuclear domains including Cajal bodies, are involved in the regulation of cell reprogramming, differentiation, proliferation and programmed cell death. Finally, a potent combinatorial genetic transformation strategy has been successfully applied to recreate the carotenoid biosynthetic pathway in carotenoiddeficient maize plants, resulting in the production of plants stacking further nutritional components into corn. Despite their benefits, the application of the potentials of this strategy from the laboratory to the market place is limited by sociopolitical constraints. The results and discussions reviewed in the four papers of this chapter permit to gain a deeper insight into the basic principles of stem cell specification, cell fate and differentiation, cell reprogramming, pattern formation, and their signalling pathways during plant development and adaptation to environmental stresses, as well as to the new biotechnology strategies to increase nutritional content of agro-food crops.

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Root Behaviour: From Sensory Perceptions to Motoric Actions František Baluška IZMB, University of Bonn, Kirschallee 1, 53115 Bonn, Germany Email: baluska@uni-bonn.de

Plant Roots Perform Exploratory Movements Plant roots perform complex movements (tropisms) in order to navigate successfully in the soil. In order to provide plants with nutrition, they need to search for water- and nutritionrich soil patches and avoid dangerous dry or toxic patches. This exploratory root behaviour is crucial for healthy plant growth and it constitutes a major key to successful agriculture. Sensory events are located at the very tip of the root, in the root cap, whereas the motoric (bending) processes are located basally, in the elongation region. In-between the root cap and the elongation zone, lies the apical meristem and the adjoining transition zone. This transition zone plays crucial role in the initiation of root tropisms. Moreover, similarly to the root cap, the transition zone has sensory modalities, especially for light, gravity and mechanostimulation. As the sensory-motoric circuit in gravitropism is very rapid and effective, both chemical and electric cell-cell communications are likely to be involved. Our current understanding of the sensory-motoric circuits of roots is limited. Recent data implicate synaptic-like secretory polar auxin transport behind of these root sensory-motoric circuits. In roots, the most active electric signalling has been reported for the transition zone (1), which is also the most active one with respect to polar auxin transport and endocytic vesicle recycling (2).

Sensory-Motoric Circuits Behind Worm-Like Crawling of Plants As Charles and Francis Darwin revealed in their book ‘The Power of Movements in Plants’ (3) already in 1880, plants have sensory-motoric circuits which underly their tropisms. Roots, show active and adaptive behaviour (3,4). Although this new view of plants was not accepted by leading botanists of that early time (4), it proved to be correct one in the last decade (4-9). Darwins implicated moveable transmitters mediating integration of sensory and motoric events. Subsequently, auxin was discovered as one of these transmitters moving from cell-to-cell to integrate sensory-motoric circuits. Currently dominating concept considers auxin transporters of the PIN family to transport auxin only across the plasma membrane (10,11). However, there are numerous data incompatible with this concept. First of all, there is no tight correlation, which one would expect if the current model would be

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correct, between the number of PIN transporters at the plasma membrane and the auxin transport (for root apices see 12-16). On the other hand, there is tight correlation between the rate of PIN vesicular recycling and auxin transport (for root apices see 17-19). Obviously, at least in cells of the transition zone, besides the classical transport mode across the plasma membrane, significant portion of the auxin transport is accomplished via the neurotransmitter-like secretion of auxin (20,21). Growing roots have two bending zones, one is located in the transition zone whereas the second, more basal one, is located in the central part of elongation region (22-24). If roots are placed in the horizontal plane, both these motoric zone accomplish root bending in a coordinated manner, allowing roots to perform worm-like crawling movements (24), resembling lower animals as proposed by Charles and Francis Darwin in 1880 (3). During this root crawling behavior, root tip repeatedly touches the substrate, trying to penetrate down the gravity vector. If this is not possible within few hours, then bending of the elongation zone lifts up the whole root tip which then repeats, via the apical transition zone, again the next touch-down (24). This co-ordination of two motoric zones in their activities implicates not only chemical but also rapid electrical (1) cell-cell communication between, as well as functional sensory-motoric integration (2-4,24). Our recent studies have revealed important role of light in control of the crawling root behaviour (25-27).

References 1. Masi E et al (2009) Spatio-temporal dynamics of the electrical network activity in the root apex. Proc Natl Acad Sci USA 106: 4048-4053 2. Baluška F, Mancuso S, Volkmann D, Barlow PW (2010) Root apex transition zone: a signalling – response nexus in the root. Trends Plant Sci 15: 402-408 3. Darwin C (1880) The Power of Movements in Plants. John Murray, London 4. Baluška F et al (2009) The 'root-brain' hypothesis of Charles and Francis Darwin: Revival after more than 125 years. Plant Signal Behav 4: 1121-1127 5. Monshausen GB, Gilroy S (2009) The exploring root: root growth responses to local environmental conditions. Curr Opin Plant Biol 12: 766-772 6. Hodges A (2009) Root decisions. Plant Cell Environ 32: 628-640 7. Trewavas A (2009) What is plant behavior? Plant Cell Environ 32: 606-616 8. Gruntman M, Novoplansky A (2004) Physiologically mediated self/non-self discrimination in roots. Proc Natl Acad Sci USA 101: 3863-3867 9. Baluška F ed (2009) Plant-Environment Interactions: From Sensory Plant Biology to Active Plant Behavior. Springer Verlag 10. Benjamins R, Scheres B (2008) Auxin: the looping star in plant development. Annu Rev Plant Biol 59: 443-465 11. Petrásek J, Friml J (2009) Auxin transport routes in plant development. Development 136: 2675-2688 12. Mancuso S et al (2005) Non-invasive and continuous recordings of auxin fluxes in intact root apex with a carbon-nanotube-modified and self-referencing microelectrode. Anal Biochem 341: 344-351 13. Schlicht M et al (2006) Auxin immunolocalization implicates vesicular neurotransmitterlike mode of polar auxin transport in root apices. Plant Signal Behav 1: 122-133

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14. Mancuso S et al (2007) Phospholipase Dζ2 drives vesicular secretion of auxin for its polar cell-cell transport in the transition zone of the root apex. Plant Signal Behav 2: 240-244 15. Shen H et al (2008) Aluminium toxicity targets PIN2 in Arabidopsis root apices: Effects on PIN2 endocytosis, vesicular recycling, and polar auxin transport. Chinese Sci Bull 53: 2480-2487 16. Baluška F et al (2008) Vesicular secretion of auxin: Evidences and implications. Plant Signal Behav 3: 254-256 17. Li G, Xue HW (2007) Arabidopsis PLDzeta2 regulates vesicle trafficking and is required for auxin response. Plant Cell 19: 281-295 18. Yang X et al (2008) Membrane steroid binding protein 1 (MSBP1) stimulates tropism by regulating vesicle trafficking and auxin redistribution. Mol Plant 1: 1077-1087 19. Wang Y et al (2009) The role of Arabidopsis 5PTase13 in root gravitropism through modulation of vesicle trafficking. Cell Res 19: 1191-1204 20. Baluška F et al (2003) Polar transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion? Trends Cell Biol 13: 282-285 21. Baluška F et al (2005) Plant synapses: actin-based adhesion domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 22. Wolverton C et al (2000) Two distinct regions of response drive differential growth in Vigna root electrotropism. Plant Cell Environ 23: 1275-1280 23. Wolverton C et al (2002) The kinetics of root gravitropism: dual motors and sensors. J Plant Growth Regul 21: 102-112 24. Baluška F et al (2009a) Intracellular domains and polarity in root apices: from synaptic domains to plant neurobiology. Nova Acta Leopold 96: 103-122 25. Yokawa K, Kagenishi T, Kawano T, Mancuso S, Baluška F (2011) Illumination of Arabidopsis roots induces immediate burst of ROS production. Plant Signal Behav 6: 14571461 26. Burbach C, Markus K, Yin Z, Schlicht M, Baluška F (2012). Photophobic behaviour of maize roots. Plant Signal Behav 7: 876-880 27. Wan Y-L, Jasik J, Wang L, Hao H, Volkmann D, Menzel D, Mancuso S, Baluška F, Lin JX (2012) The signal transducer NPH3 integrates the Phototropin1 photosensor with PIN2based polar auxin transport in Arabidopsis root phototropism. Plant Cell; 24: 551-565

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Plant steroid hormones control cell cycle and differentiation in the Arabidopsis root Mary-Paz González-García, Josep Vilarrasa-Blasi and Ana I. Caño-Delgado. Molecular Genetics Department, Centre for Research in Agricultural Genomics (CRAG), Bellaterra, Barcelona, Spain. Address: maripaz.gonzalez@cragenomica.es; ana.cano@cragenomica.es Dept. Genética Molecular Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB Edificio CRAG- Campus UAB Bellaterra (Cerdanyola del Vallés) 08193 Barcelona, España. Tel: +34 93 563 66 00 ext. 3210 Fax: +34 93 563 66 01 www.cragenomica.es The molecular and genetic mechanisms that control organogenesis are largely unknown. Plant and animal growth differ from one another at important features in their cells. Plant cells have a cell wall, cannot migrate, and the number of organs is not fixed during embryogenesis stage as in animals. Thus, plants need to regulate their growth in a very precise manner in order to survive to a constantly changing environment. Brassinosteroids (BRs) are growth-promoting steroid hormones that mediate both developmental and environmental signals during growth, as well as responses to stress factors in the plant [1, 2]. The described mechanism for plant steroid perception at the cell surface of the plasma membrane is different from that of animal cells, where steroids are perceived by a nuclear receptor family of transcription factors [3]. BRs are bound to members of the BRI1-like family of Leucine-Rich Repeat-Receptor Like Kinases (LRR-RLK) [4, 5]. Binding of the BR to the BRI1 receptor at the plasma membrane activates downstream signaling events which involve BES1 and BZR1 transcription factors causing changes in gene expression needed for the control of BR-mediated responses in the nuclei [6-8]. Previous work in several plant species has shown that BRs promote both cellular and organ growth of the aerial vegetative organs [9]. In Arabidopsis thaliana (Arabidopsis), BR loss-of-function mutants have round-shaped, dark-green leaves with short petioles [10-14], while the BRs gain-of-function mutants have bigger elongated leaves with longer petioles [6, 7].

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The role of BRs in the root development has not been studied in great detail. This organ has an indeterminate growth and provides an excellent model for understanding organ formation and maintenance in plants [15-17]. The simplicity of its radial and longitudinal patterns and its stereotyped organization cell layers are key features for the study of developmental processes. Our work focused on investigating the role of BRs in controlling root growth, which is the consequence of an increase in cell size, cell number, or both. Previous physiological and genetic studies showed that BRs control cell size in the root by promoting cell elongation [12, 18, 19]. However, our work has revealed that the positive effects of BRs on root cell elongation are not sufficient to explain the short-root phenotypes observed in both gain- and loss-of-function BR mutants. These results led to us to further analyze the role of BRs in modulating meristem cell number, which depends on cell division and cell differentiation (Figure 1). By counting the

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number of epidermis cells in the meristem, in the loss-of-function bri1-116 mutant and the gain-of-function bes1-D mutant, a reduced number of meristematic cells was found. In addition, using different concentrations of Brassinolide (BL), the most active BRs compound, our study demonstrated that the reduction in the root meristem is dose-dependent. All these results show us that BRs control primary root growth by regulating the number of meristem cells in the root apex. To unravel whether regulation of meristem cell number by BRs is linked to cell division or cell differentiation, we analyzed the expression of specific cell cycle markers. KNOLLE, a cytokinesis-specific syntaxin, was used as a marker of the cell division plane [20] and

CYCB1;1 reporter was used for labelling cells in the G2-M stage of the cell cycle [21]. In bri1-116 both markers showed a significant reduction in their expression [22], and in addition cell-cycle inhibitor ICK2/KRP2 [23] displayed an increased expression in the same background. Furthermore, overexpression of CYCD3;1 [24] in a null bri1 allele reverted the short meristem size phenotype back to wild type, collectively indicating that BRs play a role in maintaining normal cell cycle progression in the root meristem [22]. Recent studies have demonstrated that growth defects can be recovered expressing BRI1 in the epidermis of shoot and roots [25,26] and BR-mediated growth can be modulated in the primary root by differential regulation of PIN2 and PIN4 but not of PIN1, PIN3 and PIN7 [27]. To further understand how BRs regulate root growth, we investigated the role of BRs in the stem cell activities in the meristem. The stem cell niche consists of the quiescent centre (QC), a group of four cells that hardly ever divide, and the surrounding stem cell initials [2830]. The correct maintenance of these cells is necessary for sustained root growth and development. Morphological analyses have shown that this involves at least two cellular processes: maintenance of the QC quiescence and suppression of the stem cell differentiation [17, 28, 31]. The effect of BRs in the QC maintenance was investigated by analysing the expression of several QC markers in different BRs mutant background. Physiological and genetic evidence supported the idea that BRs play a role in regulating QC identity. We found that the expression of the pWOX5:GFP, pAGL42:GFP, pSCR:GFP, QC25:CFP and QC142:GUS was increased in a BL concentration-dependent manner [22]. In addition, genetic evidence showed that bri1 null mutant roots exhibited a reduced expression of pWOX5:GFP marker. Conversely, the BRI1-GFP roots showed an increased number of cells expressing the WOX5 marker, an effect that was even stronger in a bes1-D background, indicating that BRs are essential for proper function and maintenance of the QC. These results suggest that BRs promote the division of the QC cells and the newly formed cell appear to retain QC identity as they expressed pSCR:GFP [22]. Following the hierarchy of celullar process in the Arabidopsis root the effect of BRs in cell differentiation was investigated. It is well known that BRs are required for xylem differentiation, since application of BL accelerated the timing of tracheary elements differentiation in Zinnia mesophyll cell cultures [32, 33]. In the plant shoot, BRs mutants of Arabidopsis and rice exhibit vasculature differentiation defects as reduced xylem differentiation compared to the wild type [12, 26, 34-36]. In the root, it was shown that

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increased BRs levels promote the appearance of root hairs near the root tip and columella stem cell differentiation, indicating that BRs force a premature cell cycle exit prompting cell differentiation in the primary root [22]. Taken together, these results led us to conclude that BR signalling is required to promote cell growth not only by modulating cell size, but also by regulating the cell number. This control is exerted by a fine balance

between cell cycle progression and cell

differentiation, which results in the final control of root size in the plant and affects overall plant development (Fig 1).

References 1. Bajguz, A. and Hayat, S. 2009. Effects of brassinosteroids on the plant responses to environmental stresses. Plant physiology and biochemistry : PPB / Société française de physiologie végétale 47, 1-8 2. Clouse, S.D. 2011. Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development. The Plant cell 23, 1219-30 3. Thummel, C.S. and Chory, J. 2002. Steroid signaling in plants and insects--common themes, different pathways. Genes & development 16, 3113-29 4. Shiu, S. and Bleecker, A. 2001. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proceedings of the National Academy of Sciences of the United States of America 98, 10763-8 5. Kinoshita, T., Cano-Delgado, A., Seto, H., Hiranuma, S., Fujioka, S., Yoshida, S., and Chory, J. 2005. Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433, 167-71 6. Yin, Y., Wang, Z.-Y., Mora-García, S., Li, J., Yoshida, S., Asami, T., and Chory, J. 2002. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109, 181-91 7. Wang, Z.-Y., Nakano, T., Gendron, J., He, J., Chen, M., Vafeados, D., Yang, Y., Fujioka, S., Yoshida, S., Asami, T., and Chory, J. 2002. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Developmental Cell 2, 505-13 8. Kim, T.-W., Guan, S., Sun, Y., Deng, Z., Tang, W., Shang, J.-X., Sun, Y., Burlingame, A.L., and Wang, Z.-Y. 2009. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nature Cell Biology 11, 1254-60 9. Clouse, S.D. 1996. Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. Plant J 10, 1-8 10. Clouse, S.D., Langford, M., and McMorris, T.C. 1996. A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol 111, 671-8 11. Li, J. and Chory, J. 1997. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, 929-38 12. Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A., Altmann, T., Redei, G.P., Nagy, F., Schell, J., and Koncz, C. 1996. Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85, 171-82

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13. Choe, S., Dilkes, B.P., Fujioka, S., Takatsuto, S., Sakurai, A., and Feldmann, K.A. 1998. The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis. The Plant cell 10, 231-43 14. Li, J., Nam, K.H., Vafeados, D., and Chory, J. 2001. BIN2, a new brassinosteroidinsensitive locus in Arabidopsis. Plant Physiol 127, 14-22 15. Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K., and Scheres, B. 1993. Cellular organisation of the Arabidopsis thaliana root. Development 119, 71-84 16. Wildwater, M., Campilho, A., Perez-Perez, J.M., Heidstra, R., Blilou, I., Korthout, H., Chatterjee, J., Mariconti, L., Gruissem, W., and Scheres, B. 2005. The RETINOBLASTOMARELATED gene regulates stem cell maintenance in Arabidopsis roots. Cell 123, 1337-49 17. Sarkar, A.K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, K., Scheres, B., Heidstra, R., and Laux, T. 2007. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 446, 811-4 18. Müssig, C., Shin, G.-H., and Altmann, T. 2003. Brassinosteroids promote root growth in Arabidopsis. Plant physiology 133, 1261-71 19. Mouchel, C.F., Osmont, K.S., and Hardtke, C.S. 2006. BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth. Nature 443, 458-61 20. Volker, A., Stierhof, Y.D., and Jurgens, G. 2001. Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis. Journal of cell science 114, 3001-12 21. Colon-Carmona, A., You, R., Haimovitch-Gal, T., and Doerner, P. 1999. Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J 20, 503-8 22. González-García, M.P., Vilarrasa-Blasi, J., Zhiponova, M., Divol, F., Mora-García, S., Russinova, E., and Caño-Delgado, A.I. 2011. Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots. Development (Cambridge, England) 138, 849-59 23. De Veylder, L., Beeckman, T., Beemster, G.T.S., Krols, L., Terras, P., Landrieu, I., Van der Schueren, E., Maes, S., Naudts, M., and Inze, D. 2001. Functional analysis of cyclindependent kinase inhibitors of Arabidopsis. The Plant cell 13, 1653-67 24. Riou-Khamlichi, C., Huntley, R., Jacqmard, A., and Murray, J.A. 1999. Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283, 1541-4 25. Hacham, Y., Holland, N., Butterfield, C., Ubeda-Tomas, S., Bennett, M.J., Chory, J., and Savaldi-Goldstein, S. 2011. Brassinosteroid perception in the epidermis controls root meristem size. Development (Cambridge, England) 138, 839-48 26. Savaldi-Goldstein, S., Peto, C., and Chory, J. 2007. The epidermis both drives and restricts plant shoot growth. Nature 446, 199-202 27. Hacham, Y., Sela, A., Friedlander, L., and Savaldi-Goldstein, S. 2012. BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers. Plant signaling & behavior 7, 68-70 28. van den Berg, C., Willemsen, V., Hendriks, G., Weisbeek, P., and Scheres, B. 1997. Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390, 287-9 29. Scheres, B. 2007. Stem-cell niches: nursery rhymes across kingdoms. Nature reviews Molecular cell biology 8, 345-54 30. Dolan, L. 2009. Meristems: the root of stem cell regulation. Current biology : CB 19, R459-60 31. Ortega-Martinez, O., Pernas, M., Carol, R.J., and Dolan, L. 2007. Ethylene modulates stem cell division in the Arabidopsis thaliana root. Science 317, 507-10

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32. Iwasaki, T. and Shibaoka, H. 1991. Brassinosteroids Act as Regulators of TrachearyElement Differentiation in Isolated Zinnia Mesophyll-Cells. Plant and Cell Physiology 32, 1007-14 33. Clouse, S.D. and Zurek, D. 1991. Molecular Analysis of Brassinolide Action in PlantGrowth and Development. Acs Symposium Series 474, 122-40 34. A Kauschmann, A.J., C Koncz, M Szekeres, L Willmitzer, and T Altmann 1996. Genetic evidence for an essential role of brassinosteroids in plant development. Plant Journal 9, 70113 35. Caño-Delgado, A., Yin, Y., Yu, C., Vafeados, D., Mora-García, S., Cheng, J.-C., Nam, K.H., Li, J., and Chory, J. 2004. BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development (Cambridge, England) 131, 5341-51 36. Nakamura, A., Fujioka, S., Sunohara, H., Kamiya, N., Hong, Z., Inukai, Y., Miura, K., Takatsuto, S., Yoshida, S., Ueguchi-Tanaka, M., Hasegawa, Y., Kitano, H., and Matsuoka, M. 2006. The role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice. Plant Physiol 140, 580-90

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Nuclear reprogramming of plant differentiating cells: remodelling of nuclear domains and epigenetic regulation Pilar S. Testillano and María-Carmen Risueño Plant Development and Nuclear Architecture. Biological Research Center, CIBCSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. E-mail: testillano@cib.csic.es Mechanisms underlaying nuclear reprogramming are thought to involve genome-wide changes in chromatin structure and gene expression, and require the factors that contribute to regulate the flexibility of genome (1, 2). The architecture of the cell nucleus, highly dynamic and organized in distinct functional domains, reflects these changes of gene expression and modifies its organization. In this sense, chromatin regulation plays a critical role in determining cell fate of totipotent cells, the organization of chromatin domains providing an additional platform for a regulatory level controlling genetic information. (3). The architecture of the well organized nuclear functional domains – condensed chromatin, interchromatin region, nuclear bodies and nucleolus – changing in response to DNA replication, RNA transcription, processing and transport. Microspore embryogenesis constitutes an intriguing system in which a cell is reprogrammed from its genetically controlled gametophytic programme towards an embryogenic pathway. After a stress treatment, in vitro cultured microspores are reprogrammed and change their developmental pathway, the process constituting an useful system of cell reprogramming in isolated cells, which also represents an important tool in plant breeding to obtain double-haploid plants (4).

Bioimaging and in situ molecular identification techniques to track gene and protein expression during microspore reprogramming Confocal Laser Scanning Microscopy technology and bio-imaging are powerful tools for three-dimensional and colocalization molecular analysis of the plant cell reprogramming. Strategies with fluorescent-labelled probes for in situ hybridization and immunofluorescence have provided unique images of the spatial and temporal pattern of the expression of genes and proteins, and of the sub-cellular rearrangements that accompany the microspore embryogenesis (5). Using this strategy, various signalling and stress proteins have been shown to be differentially expressed in reprogrammed microspores and young embryos (5); specific endosperm and embryo genes were expressed in microspore-derived embryos at different stages (6).

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Chromatin pattern and nuclear domains dynamics parallel the change of developmental program and cell fate The know-how of the dynamics of the nuclear domains during the activation of the reprogramming process will give new insights into the mechanisms that regulate it. Several reports have revealed defined changes in nuclear domains that accompany the stressinduced pollen reprogramming to embryogenesis (7, 8). They have characterized specific rearrangements of the nuclear domains during the switch of plant differentiating cells to proliferation, by using in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling (MAPKs, ERKs) and stress (HSPs) proteins (9-13). These works showed that the chromatin pattern is also affected by the microspore reprogramming and embryogenesis induction, constituting a good marker of developmental fate; in microspores committed to embryogenesis, all nuclei show a similar decondensed chromatin pattern, allowing for an abundant interchromatin region (7, 12).

Cajal body: a marker for microspore reprogramming The structures present in the interchromatin domain, the nuclear domain between chromosome territories, have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). Recent results revealed characteristic changes in size, shape and distribution of the different interchromatin structures, specifically the Cajal bodies, as a consequence of the reprogramming of the microspore, allowing to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events and suggesting that RNA associated structures could be a regulatory mechanism in the cell reprogramming process (14). Cajal bodies are specifically involved on the storage and maturation of both snRNPs and snoRNPs, as well as other splicing factors, necessary for mRNA and pre-rRNA processing, but not directly on the transcription. Several evidences showed that the number of Cajal bodies increase after microspore reprogramming and during the early stages of microspore embryogenic development, and suggested that Cajal bodies may have a role in the transcriptionally active, proliferative stages that characterise early microspore development, aFter reprogramming (8, 14).

Epigenetic marks reprogramming

reorganize

with

microspore

Nuclear reprogramming requires the removal of the cell type-specific epigenetic pattern imposed on chromatin during cellular differentiation and division (15). Chromatin regulation plays a critical role in determining cell fate of totipotent cells, the organization of chromatin

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

domains providing an additional platform for a regulatory level controlling genetic information. Epigenetic modifications, like DNA methylation and histone modifications, are key factors regulating DNA accessibility of factors responsible of activate or repress gene expression. However, the relationship between DNA methylation and large-scale organization of nuclear architecture is still poorly understood. New evidences showed that changes in DNA methylation accompany the reorganization of the nuclear domains during plant cell differentiation, proliferation and programmed cell death (16). Some data indicated that genomic DNA methylation increases and is associated with heterochromatinization during cell differentiation, whereas DNA methylation and histone H4 acetylation decreases with the cell reprogramming, embryogenic switch and proliferation (17, 18), as well as during other plant developmental processes like floral determination of buds (19).

Concluding remark The successful use of reprogrammed cells requires the maintenance of genomic stability and cell survival to ensure long-term function. The information summarized here will provide a wider understanding of the mechanisms controlling plant cell reprogramming and embryogenic competence acquisition by stress treatments. This data will be useful to design and optimize protocols with higher efficiency in cell reprogramming in vitro systems in agrofoodstuffs and forest industry, besides to set the bases for transformation strategies.

Acknowledgements Work supported by project granted by the Spanish Ministry of Science and Innovation, MICINN, BFU2011-23752.

References 1. Arnholdt-Schmitt B. Stress-induced cell reprogramming. A role for global genome regulation? 2004. Plant Physiol 136 (1):2579-2586. 2. Chinnusamy V, Zhu JK. Epigenetic regulation of stress responses in plants. 2009. Curr Opin Plant Biol 12 (2):133-139. 3. Sang Y, Wu M-F, Wagner D. The stem cell-chromatin connection. 2009. Seminars in Cell & Developmental Biology 20:1143-1148. 4. Maluszynski M, Kasha KJ, Forster BP, Szarejko I. (eds.) Doubled Haploid Production in Crop Plants. 2003. Kluwer Academic Publishers. Dordrecht, Boston, London. 5. Testillano PS, Risueño MC. Tracking gene and protein expression during microspore embryogenesis by Confocal Laser Scanning Microscopy. 2009. In: Advances in Haploid Production in Higher Plants (Eds: Touraev A, Forster BP, Mohan Jain S.) Springer Science and Bussines Media B.V. UK. pp. 339-347. 6. Massonneau A, Coronado MJ, Audran A, Bagniewska-Zadworna A, Mol R, Testillano PS, Goralski G, Dumas C, Risueño MC, Matthys-Rochon E. Multicellular structures that develop during in vitro maize pollen embryogenesis express both endosperm- and embryo-specific genes: which is which? 2005. Eur. J. Cell Biol. 84: 663-675.

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7. Testillano PS, Coronado, MJ, Seguí JM, Domenech J, González-Melendi P, Raska I, Risueño MC. Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis. 2000. J. Struct. Biol. 129:223-232.. 8. Seguí-Simarro JM, Bárány I, Suárez R, Fadón B, Testillano PS, Risueño MC. Nuclear bodies domain changes with microspore reprogramming to embryogenesis. 2006. Eur. J. Histochem. 50:35-44. 9. Coronado MJ, González-Melendi P, Seguí JM, Ramírez C, Barany I, Testillano PS, Risueño MC. MAPKs entry into the nucleus at specific interchromatin domains in plant differentiation and proliferation processes. 2002. J Struct. Biol. 140:200-213. 10. Ramírez C, Testillano PS, Pintos B, Moreno MA, Bueno MA, Risueño MC. Changes in pectins and MAPKs related to cell development during early microspore embryogenesis in Quercus suber L. 2004. Eur. J. Cell Biol. 83:213-225. 11. Silva MS, Fortes AM, Testillano PS, Risueño MC, Pais S. Differential expression and cellular localization of ERKs during organogenic nodule formation from internodes of Humulus lupulus var. Nugget. 2004.Eur J Cell Biol. 83:425-433. 12. Testillano PS, González-Melendi P, Coronado MJ, Seguí JM, Moreno MA, Risueño MC. Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains. 2005. Cytogenet. Genome Res. 109:166-174. 13. Seguí-Simarro JM, Testillano PS, Jouannic S, Henry Y, Risueño, M.C. MAP kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus. 2005. Histochem.Cell Biol. 123:541-551. 14. Seguí-Simarro JM, Corral-Martínez P, Corredor E, Raska I, Testillano PS, Risueño MC. A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L. 2011. J Plant Physiol. 168:746-757. 15. Marión RM, Blasco MA. Telomere rejuvenation during nuclear reprogramming. 2010. Curr Opin Genet Dev 20(2):190-196. 16. Solís MT. Reprogramación del polen a embriogénesis inducida por estrés: identidad celular, muerte celular programada y papel de la metilación del DNA. 2012. Tesis Doctoral. Universidad Complutense de Madrid. 17. Solís MT, Risueño MC, Testillano PS. Genomic DNA methylation levels and distribution pattern change during plant cell differentiation and cell reprogramming. 2012. Submitted. 18. Testillano PS, Solís MT, Rodríguez-Serrano M, Risueño MC. DNA methylation and expression of MET1 are regulated during pollen development and pollen reprogramming to embryogenesis. 2010. Proceedings of XVII Congress of the Federation of European Societies of Plant Biology FESPB, Valencia, 4-9 July 2010. 19. Meijón M, Valledor L, Santamaría E, Testillano PS, Risueño MC, Rodríguez R, Feito I, Cañal MJ. Epigenetic characterization of the vegetative and floral stages of azalea buds: dynamics of DNA methylation and histone H4 acetylation. 2009. J. Plant Physiol. 166:16241636.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Multi-gene engineering for reconstruction and extension of complex plant biosynthetic pathways and sociopolitical constraints limiting the transition from the laboratory to the market place Gemma Farré1, Uxue Zorrilla-Lopez1, Teresa Capell1, Judit Berman1, Changfu Zhu1, Paul Christou1,2,* 1 Department of Plant Production and Forestry Science - ETSEA, University of Lleida-CRA, Avenue Alcalde Rovira Roure, 191, 25198, Lleida, Spain. 2 Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 08018, Barcelona, Spain *Corresponding author (christou@pvcf.udl.es) By using combinatorial genetic transformation we recreated the carotenoid biosynthetic pathway in a South African elite inbred white corn (Zea mays) deficient for endosperm carotenoids and we identified and complemented rate-limiting steps in the pathway. We have generated transgenic plants accumulating substantial amounts of β-carotene and other nutritionally important carotenoids, ascorbic acid (vitamin C) and folate (vitamin B9) specifically in the endosperm. Here we discuss results from our current experiments focusing on: (a) stacking further nutritional components into corn; (b) the effects of genetic background on transgene expression and metabolite accumulation resulting from interactions between endogenous and induced-transgenic pathways; (c) the political dimension of the transition from the laboratory to the field and beyond,

highlighting

differences in the regulatory systems in the EU versus the rest of the world.

The multiple nutritional roles of vitamins Although many foods are said to be good sources of vitamin A, it should be noted that these generally do not contain retinal itself but derivatives that can be converted into retinol and then into either retinal or retinoic acid. Meat and dairy sources of vitamin A primarily contain the esterified form retinyl palmitate, whereas plants produce pro-vitamin A carotenoids such as β-carotene that are cleaved to produce retinol. These are abundant in a wide variety of dark green, yellow and orange fruits and vegetables such as oranges, broccoli, spinach, carrots, squash, sweet potatoes and pumpkins [1].Vitamin A deficiency (VAD) causes night blindness, i.e. the deterioration of light sensitive cells (rods) essential for vision in low light intensity and it can also damage the cornea resulting in a total form of blindness called xerophthalmia. The lack of vitamin A has a particularly severe effect on the

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

immune system leaving individuals susceptible to infections [2]. More than four million children worldwide exhibit signs of severe VAD, including 250,000–500,000 per year that become partially or totally blind [3]. During pregnancy women have a higher demand for vitamin A, and VAD in pregnancy causes nearly 600,000 deaths every year [3]. Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Lutein and zeaxanthin accumulate in the perifoeveal and foveal regions of the retinal macula, respectively [4,5]. There is very good evidence that they protect against age-related macular degeneration (ARMD) [6], a disease that affects 1.6% of 50–65-year-olds and 30% of people over 75 [7]. There is less risk of this disease in people with a carotenoid-rich diet [8,9]. Vitamin C (ascorbic acid) is a powerful electron donor (antioxidant) and a cofactor in several metabolic pathways, including those forming the mature form of collagen. It plays an important role in the synthesis and stabilization of neurotransmitters, and also reduces iron compounds, enhancing the gastrointestinal absorption of dietary non-heme iron [10]. Insufficient vitamin C in the diet causes scurvy, which involves the breakdown of connective tissue fibers and muscular weakness [10]. High levels of vitamin C are found in citrus fruits and green vegetables [2]. Vitamin B 9 (folic acid) is the source of tetrahydrofolate which is essential in DNA synthesis and many other core metabolic reactions. Deficiency in adults causes macrocytic anemia and elevated levels of homocysteine, but the impact in pregnant women is much more severe, leading to neural tube defects in the fetus. Spina bifida, in which bones of the spine do not completely enclose the spinal cord, is the most common congenital abnormality associated with folate deficiency [11].

Carotenoid synthesis in plants Carotenoids are tetraterpenoids whose synthesis in plants begins in the plastids with the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to generate the C20 intermediate geranylgeranyl diphosphate (GGPP). This reaction is catalyzed by GGPP synthase (GGPPS) [12]. The first committed step is the condensation of two GGPP molecules into 15-cis-phytoene by the enzyme phytoene synthase (PSY, or CrtB in bacteria) [13]. A series of four desaturation reactions carried out in plants by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) then generates the carotenoid chromophore. In non-green tissue this is converted to all-trans lycopene by ζ-carotene isomerase (Z-ISO) [14] and carotenoid isomerase (CRTISO), whereas in green tissue the reaction occurs spontaneously in the presence of light and chlorophyll (acting as a sensitizer) [15,16]. In bacteria, all these steps are carried out by a single enzyme, CrtI. All-trans lycopene represents a branch point in the pathway. This linear molecule can be cyclized at both ends by lycopene β-cyclase (LYCB, CrtY in bacteria) to generate the β-ionone end groups of β-carotene. Alternatively it can be cyclized at one end by lycopene ε-cyclase (LYCE) and at the other by LYCB to introduce the non-identical ε- and β-ionone end groups of α-carotene. Both these molecules can be converted into downstream products by carotene hydroxylases, such as the eponymous β-carotene hydroxylase (BCH).

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

In the β-carotene pathway this yields β-cryptoxanthin, which is further converted by the same enzyme into zeaxanthin, whereas in the α-carotene branch the conversion yields lutein, the natural pathway endpoint [17]. Furthermore, zeaxanthin enters the xanthophyll cycle through the stepwise activities of zeaxanthin epoxidase (ZEP) and violaxanthin deepoxidase (VDE). These reactions are shown schematically in Figure 1.

Biotechnology content

strategies

increase

nutritional

Recent GE strategies to increase crop yields have been highly successful [18] but the most striking advances over the last two years have involved plants engineered to produce missing nutrients or increase the level of nutrients that are already synthesized. An important trend is the move away from plants engineered to produce single nutritional compounds towards those simultaneously engineered to produce multiple nutrients, a development made possible by the increasing use of multigene engineering [19].

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Several recent reports have demonstrated how multigene metabolic engineering can increase the level of carotenoids in edible plant tissues, including the traditional target βcarotene and other carotenoids with specific functions in the human body or generally beneficial antioxidant properties. We reported the development of a combinatorial nuclear transformation method which allows the carotenoid synthesis pathway in corn to be dissected, and allows the production of diverse populations of transgenic plants containing different carotenoid profiles [20]. The system as originally reported involved the transformation of a white elite South African corn inbred (M37W) lacking endosperm carotenoids with five genes from the carotenoid pathway each under the control of a different endosperm-specific promoter. The population of transgenic plants recovered in this approach contained random combinations of transgenes, thus each unique combination had a different metabolic potential and produced a distinct carotenoid profile. Normally, the random nature of transgene integration is considered disadvantageous because hundreds of lines may need to be screened to identify one with the correct genotype and phenotype. However, random transgene integration is an advantage in this new platform because it increases the diversity of the population, resulting in plants with high levels of carotenoids such as β-carotene, lutein, zeaxanthin and lycopene alone or in combination. More recently, we demonstrated that the engineered carotenoid pathway could be introgressed from a transgenic line with a high LYCB:LYCE (lycopene β-cyclase to lycopene ε-cyclase) ratio (thus favoring the β-carotene branch) into the genetic background of a wild-type yellowendosperm corn also with a high LYCB:LYCE ratio, resulting in synergistic enhancement of the metabolic bias and creating hybrid lines producing unprecedented levels of zeaxanthin (56 µg/g dry weight) [21]. This novel strategy for combining GE and conventional breeding allows the development of “designer” hybrid lines with specific carotenoid profiles, and is equally applicable to any staple crop where nutritional improvement would be beneficial.

Multivitamin corn Whereas the enhancement of individual nutrients provides proof of principle, progress towards addressing micronutrient deficiencies in the real world will only be made once it is possible to target different nutrients at the same time. In this context, transgenic corn plants simultaneously enhanced for carotenes, folate and ascorbate provide the first example of a nutritionally enhanced crop targeting three entirely different metabolic pathways, going some way towards the goal of nutritionally complete staple crops [22] (Figure 2). This was achieved by transferring four genes into the white maize variety described above, resulting in 169 times the normal level of β-carotene (57 μg/g dry weight), 6.1 times the normal level of ascorbate (106.94 µg/g dry weight) and twice the normal amount of folate (1.94 μg/g dry weight).

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Effects of genetic background Whereas lutein is abundant in most fruits and vegetables, corn is one of very few dietary sources of zeaxanthin and even so only selected varieties produce adequate amounts of this molecule. A specific corn line we had generated through combinatorial transformation [20] expressing Zmpsy1, PacrtI and Gllycb, recapitulated the entire pathway through to β-carotene and thus accumulated β-carotene as the major carotenoid, but there were also higher levels of lycopene, lutein and zeaxanthin, the latter suggesting that endogenous BCH activity was converting some of the additional β-carotene into zeaxanthin and demonstrating there was spare capacity in the pathway. The β:ε ratio in this transgenic line was 3.51 (compared to 1.21 in wild type M37W endosperm) with a 90-fold increase in total carotenoids and a zeaxanthin level of up to 29 µg/g DW. Although many other crops have been engineered to improve carotenoid levels, previous studies have concentrated mainly on β-carotene and few have looked at the possibility of zeaxanthin production [23,24]. We introgressed the carotenoid mini-pathway from this specific transgenic line into two yellow inbreds with similar carotenogenic potential but opposite characteristics in terms of the β:ε ratio. Both EP42 and A632 accumulate carotenoids at levels up to 29 µg/g DW, with lutein and zeaxanthin as major components of the carotenoid profile. However, the low β:ε ratio of EP42 corn provides a much higher potential for lutein synthesis because ε-cyclase activity exceeds β-cyclase activity, whereas the much higher β:ε ratio of A632 corn favors the

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

synthesis of zeaxanthin because the balance of enzyme activities is reversed. Our quantitative RT-PCR experiments showed that the two inbred lines had similar levels of

Zmlycb mRNA but differed with respect to the abundance of Zmlyce mRNA, suggesting that the higher β:ε ratio of A632 corn reflected lower lycopene ε-cyclase activity. The increased β:ε ratio in the Ph-4 x A632 hybrid shows that a combination of reduced ε-cyclase activity and increased β-cyclase activity (provided by the transgene) can help skew the ratio even further, leading to the production of 56 µg/g DW zeaxanthin, more than twice the level produced in the best-performing natural varieties or the best-performing transgenic lines reported thus far. The Ph-4 x EP42 hybrid also showed an increased β:ε ratio of 2.05 compared to the wild-type parent, but in this case the higher lycopene ε-cyclase activity provided by the parental genotype prevented the balance tipping too far towards the zeaxanthin pathway. As shown in Figure 3, the transgenic line not only accumulated high levels of zeaxanthin and lutein, but also carotenoid intermediates such as phytoene, lycopene, α- and β-cryptoxanthin, and α- and β-carotene [20]. In the hybrid lines, however, these intermediates were not detected, which suggests that additional, more subtle bottlenecks present in the M37W genetic background were also alleviated by metabolic

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

complementation. M37W endosperm accumulates only traces of carotenoids due to the lack of Zmpsy1 expression, which catalyzes the first committed step in the pathway, the synthesis of phytoene. In contrast, Zmpsy1 mRNA is abundant in both yellow corn inbreds (EP42 and A632). The traces of lutein and zeaxanthin in M37W endosperm probably reflect the presence of the Zmpsy2 transcript, which is mainly responsible for carotenoid biosynthesis in green tissues but may have some residual activity in the endosperm. Interestingly, the transgenic line Ph-4 contains a significant amount of phytoene (7.36 µg/g DW), which suggests that the next step in the pathway (the conversion of phytoene into lycopene by the bacterial enzyme phytoene desaturase) is limiting. In contrast, no phytoene was detected in yellow corn, nor in the hybrids, which shows that the three enzymes carrying out the corresponding endogenous reactions in yellow corn are not limiting, and alleviate the bottleneck in the M37W background when the induced and endogenous pathways are combined in the hybrid. Similarly, the transgenic endosperm also contained significant amounts of lycopene (10.42 µg/g DW) whereas no lycopene was detected in either hybrid. These data suggest that the yellow corn backgrounds also alleviated a bottleneck in βcarotene hydroxylase activity, allowing the efficient flow of intermediates towards zeaxanthin synthesis. Our collective data indicate that the yellow corn background compensated for inefficient activity at every step of the pathway conferred by the transgenes, but the combination of reduced lycopene ε-cyclase activity and the pooled lycopene β-cyclase activity in the hybrid conferred its highly skewed β to ε ratio and its extraordinary potential to accumulate zeaxanthin. This study is the first to show that significant increases in zeaxanthin levels in a food crop can be achieved by combining conventional breeding with genetic engineering. Whereas genetic engineering provides advantages such as speed and access beyond the species gene pool, it can be difficult and/or time consuming to transform locally-adapted varieties directly and therefore make a practical impact on nutrition and health, particularly in developing countries where staples such as corn represent the predominant food source for many people. Conventional breeding for improved nutrition is slow and laborious, particularly where the intent is to modify several different metabolic pathways simultaneously [22], and is limited to the gene pools of compatible species. Our combined approach cherry-picks the advantages of both systems – the speed, power and accessibility of genetic engineering and the diversity and practicality of conventional breeding, to generate nutritionally enhanced crops with unprecedented levels of a key nutrient in the human diet.

Barriers to adoption The advances described above provide a vignette of the potential of nutritionally enhanced GE crops, and in an ideal world there would be rapid transfer of the most successful varieties from the laboratory to the field and then to deployment in areas where the benefit could be felt most. But two cases studies, Golden Rice II [25] and virus-resistant papaya [26], show that even the best ideas in the laboratory struggle to reach their targets despite a long track record of safety and performance.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

A major hurdle to the adoption of plant biotechnology is the widespread perception that only ‘big business’ benefits from it, with consumers generally failing to realize the indirect benefits to the environment. The benefits of plant biotechnology in developing countries would be more direct – addressing hunger and disease and increasing the wealth of the subsistence farmer who might be able to make a small profit from the extra yield, but take-up has nevertheless been slow particularly in Africa where South Africa was the only participant in commercial GE agriculture until 2008. One prevalent feature of contemporary biotechnology is its proprietary nature, which means that inventions (products and processes) are almost always protected with patents. These are supposed to help promote the transformation of research into marketed products but many believe they obstruct research and development by blocking access to research tools, mandating the use of material transfer agreements and attracting litigation [27]. In developing countries, many key technologies for biotechnology products appear to be unprotected; problems could arise when crops developed with such technologies are exported, but their use for subsistence agriculture is legitimate. The donation of intellectual property for humanitarian purposes in developing countries is therefore a realistic prospect, as in the case of Golden Rice [28]. The media also play a significant role in the likelihood of technology uptake, so how the public perceive GE technology depends greatly on how the information is packaged by the media. In developed countries, media access is taken for granted, but in many developing countries the news travels slowly and can be subject to political interference. Media involvement can also affect government decisions and policy (e.g. the 2002 GE food aid row that caused the Zambian government to ban GE aid from the US even though millions of its people faced starvation). It is often said that GE crops could help to address Africa’s hunger and poverty, but that farmers are being deprived of the technology and prevented from achieving agricultural success [29]. Many blame the European governments and NGOs for trying to foist their affluent values and precautionary principles on Africa’s poor [29]. Further development and adoption of biotechnology is hampered by discordant international regulations relating to research, biosafety, trade and use of GE crops and products, particularly between the EU and US [30]. Despite admirable scientific progress in the development of GE crops that have the potential to address developing world challenges, the complex regulations applying in international markets generate overlapping and sometimes contradictory requirements that are a burden to the developing country farmer [31]. This reduces the likelihood that experimental GE crops will be developed into products, a problem exacerbated by the inexperience of public sector researchers in product development and the unwillingness of companies to be involved. Furthermore, trade becomes difficult when regulatory regimes vary so widely, particularly for developing countries that may lack the resources to comply with complex regimes or develop their own. International trade becomes limited as it is more difficult for producers in developing countries to maintain their supply contracts with distributors in the developed world. The end result is that developing countries may decide it is simply easier to avoid GE products (even if they are acknowledged to be safe), with some implementing bans on GE products that not only affect market access but also make it more difficult for them to gain financial support

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from industrialized nations, particularly in order to conduct research and build human capital for biotechnology activities.

Conclusions GE strategies can be used to address micronutrient deficiency in both the developed and the developing world, as recent advances in the areas of metabolic engineering and mineral accumulation have demonstrated, particularly those studies simultaneously tackling multiple nutrients. However, this can only be achieved with the support of the public, media and politicians. Converting the current negative reinforcement cycle into a positive one will only be possible when there is less irrational hatred of GE, and this can only come about with a strenuous effort to educate the public, politicians and the media about the realistic nature of risks, and the balance between risks and benefits in all areas of life.

Acknowledgements Research at the Universitat de Lleida is supported by MICINN, Spain (BFU2007-61413; BIO2011-23324; BIO02011-22525; PIM2010PKB-00746); European Union Framework 7 Program-SmartCell Integrated Project 222716; European Union Framework 7 European Research Council IDEAS Advanced Grant (to PC) Program-BIOFORCE; COST Action FA0804: Molecular farming: plants as a production platform for high value proteins; Centre CONSOLIDER on Agrigenomics funded by MICINN, Spain.

References 1. Harrison EH. Mechanisms of digestion and absorption of dietary vitamin A. 2005. Ann Rev Nutr 25:87–103. 2. FAO/WHO. Human vitamin and mineral requirements. Report of a joint FAO/WHO expert consultation. FAO, Rome/WHO Press, Geneva 2002. 3. UNICEF. Vitamin A deficiency. 2006. http://www.childinfo.org/areas/vitamina/ 4. Landrum JT, Bone RA. Lutein, zeaxanthin, and the macular pigment. 2001. Arch Biochem Biophys 385:28–40. 5. Snodderly DM, Handelman GJ, Adler AJ. Distribution of individual macular pigment carotenoids in the central retina of macaque and squirrel monkeys. 1991. Invest. Ophthalmol Vis Sci 32:268–279. 6. Fraser PD, Bramley PM. The biosynthesis and nutritional uses of carotenoids. 2004. Prog Lipid Res 43:228–265. 7. Mozaffarieh M, Sacu S, Wedrich A. The role of the carotenoids, lutein and zeaxanthin, in protecting against age-related macular degeneration: a review based on controversial evidence. 2003. Nut. J 11:2–20.. 8. Hammond BR Jr, Johnson EJ, Russel RM, Krinsky NI, Yeum KJ, Edwards RB, Snodderly DM. Dietary modification of human macular pigment density. 1997. Invest Ophthalmol Vis Sci 38:1795–1801.

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9. Landrum JT, Bone RA, Joa H, Kilburn MD, Moore LL, Sprague KE. A one year study of the macular pigment: the effect of 140 days of a lutein supplement. 1997. Exp Eye Res 65:57–62. 10. Basu TK, Dickerson JWT. Vitamin C (ascorbic acid). 1996. In: Basu TK, Dickerson JWT (eds) Vitamins in health and disease. CAB International, Oxford, UK, pp.125–147. 11. Scott J, Kirke P, Molloy A, Daly L, Weir D. The role of folate in the prevention of neural tube defects. 1994. Proc Nutr Soc 53:631–636. 12. Rodríguez-Concepción M. Early steps in isoprenoid biosynthesis: Multilevel regulation of the supply of common precursors in plant cells. 2006. Phytochem Rev 5:1–15. 13. Misawa N, Truesdale MR, Sandmann G, Fraser PD, Bird C, Schuch W, Bramley PM. Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products. 1994. J Biochem 116:980–985. 14. Chen Y, Li F, Wurtzel ET. Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants. 2010. Plant Physiol 153:66–79. 15. Isaacson T, Ohad I, Beyer P, Hirschberg J. Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants. 2004. Plant Physiol 136:4246-4255. 16. Breitenbach J, Sandmann G. Zeta-carotene cis isomers as products and substrates in the plant poly-cis carotenoid biosynthetic pathway to lycopene. 2005. Planta 220:785–793. 17. Zhu C, Bai C, Sanahuja G, Yuan D, Farre D, Naqvi S, Shi L, Capell T, Christou P. The regulation of carotenoid pigmentation in flowers. 2010. Arch Biochem Biophys 504:132–141. 18. Farre G, Ramessar K, Twyman RM , Capell T, Christou P. The humanitarian impact of plant biotechnology: recent breakthroughs vs bottlenecks for adoption. 2009. Curr Opin Plant Biol 13:219-225. 19. Naqvi S, Farre G, Sanahuja G, Capell T, Zhu C, Christou P. When more is better: multigene engineering in plants. 2010. Trends Plant Sci 15:48-56. 20. Zhu C, Naqvi S, Breitenbach J, Sandmann G, Christou P, Capell T. Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in corn. 2008. Proc Natl Acad Sci USA 105:18232-18237. 21. Naqvi S, Zhu C, Farre G, Sandmann G, Capell T, Christou P. Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin. 2011. Plant Biotechnol J 9:384–393. 22. Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P. Transgenic multivitamin corn through biofortification of corn endosperm with three vitamins representing three distinct metabolic pathways. 2009. Proc Natl Acad Sci USA 106:7762-7767. 23. DellaPenna D, Pogson BJ. Vitamin synthesis in plants: tocopherols and carotenoids. 2006. Annu Rev Plant Biol 57:711–738. 24. Sandmann G, Römer S, Fraser PD. Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants. 2006. Metab Eng 8:291–302. 25. Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. 2005. Nature Biotechnol 23:482–487. 26. Stokstad E. Papaya takes on ringspot virus and wins. 2008. Science 320:472. 27. Lei Z, Juneja R, Wright BD. Patents versus patenting: implications of intellectual property protection for biological research. 2009. Nature Biotechnol 27:36-40. 28. Enserink M. Tough lessons from golden rice. 2008. Science 320:468-471. 29. Scoones I, Glover D. Africa’s biotechnology battle. 2009. Nature 460:797-798.

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30. Ramessar K, Capell T, Twyman RM, Quemada H, Christou P. Trace and traceability – a call for regulatory harmony. 2008. Nature Biotechnol 26:975-978. 31. Giddings LV, Chassy BM. Igniting agricultural innovation: Biotechnology policy prescriptions for a new administration. Science Progress 2009. http://www.scienceprogress.org/2009/07/igniting-agricultural-innovation/. 32. Farre G, Twyman RM, Zhu C, Capell T, Christou P. Nutritionally enhanced crops and food security: Scientific achievements versus political expediency. 2011. Curr Opin Biotechnol 22:245–251.

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1.7. Methods in Cell biology

The last advances in TEM for the in situ molecular recognition Carmen López-Iglesias*, Lidia Delgado, Gema Martínez, Yolanda Muela, Nieves Hernández Electron Cryo-Microscopy Unit. Scientific & Technological Centres, Barcelona University, Barcelona Science Park, Baldiri i Reixac 10-12, 08028 Barcelona, Spain. *clopeziglesias@ub.edu.

Introduction The spatial arrangement of macromolecular complexes within a cell is one of the main objectives of cell structural biology in our days (1). Three-dimensional studies combined with cryo-preparation methods in electron microscopy enable the study of biological specimens in a quasi in vivo “hydrated” and “three-dimensional” state. Transmission electron microscopic studies of biological samples are limited by four general factors: 1.Electrons need high vacuum to form a beam able to traverse the sample and to form an image from it; 2.The electrons heat the sample; 3.The penetration power of electrons is very low and the image is a 2D projection of the specimen. Therefore, the specimens cannot be alive, they must be immobilized or fixed. The high water content of cells (around 80%) needs to be removed or, preferably, stabilized by freezing; the samples have to be dried, embedded in a resin resistant to heating or the EM must be able to work under particular conditions (“low dose” of electrons) without damaging the sample;

the

thickness of the sample has to be between 50 and 400 nm, depending on the voltage of the electron microscope and the viewing of 3D images from the specimen requires certain strategies in working with the EM and special software, called generally: “single particle analysis/reconstruction” and “electron tomography”.

Cryoimmobilization Cryoimmobilization getting vitrification is the optimal way to immobilize or fix biological specimens in a close-to-native state, using physical procedures based on freezing (2). It is the recommendable first step in any 3D procedure. The goal is to get “vitreous” or amorphous ice (non-crystalline) from the cell water after ultra-rapid freezing, without destroying cell structures and preserving their in vivo rapidly changing interactions (3).

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Vitrification occurs in milliseconds allowing spatial and temporal resolution of cellular events. Samples can be cryo-immobilized, either by plunge freezing, impact freezing, selfpressurized freezing or high pressure freezing (HPF). For freezing suspensions or molecules or even small cells, plunging into a cooled cryogen, such as ethane or propane, is a common method (4). Another method, faster than plunging in its heat transfer, is slamming cells or tissues against a cooled metal block. For both impact- and plunge-freezing, it is possible to achieve up to 2-20 microns of good ultrastructural preservation. Beyond that, ice crystals form and destroy the tissue. However, most cells and tissues are much larger than a few microns, so we need another method to freeze them without ice crystal damage. The best method in these cases is High-Pressure Freezing (HPF), which freezes samples with liquid nitrogen under high-pressure conditions (5). Using HPF, relatively large volumes (200 microns and more) can be frozen without ice crystal damage. A new rapid freezing method uses the increase of volume of the water when it freezes naturally. It is the Self-pressurized freezing (SPRF). The increasing of water volume at the ends of a copper tube by ice crystals formation pressurizes the middle of the tube at the same time that freezing takes place in this part. SPRF is a totally new technology described by Leunissen et al (6) recently.

Single particle analysis and electron tomography Once the cells are immobilized by freezing, two strategies can provide the threedimensionality of the structures of interest:

Single particle analysis and electron

tomography. Single particles are isolated macromolecules or membranes, molecular assemblies, small organelles, small cellular structures or small organisms not needing sectioning and the procedure carried out is the reconstruction from the different orientations of the single particle on an electron microscopy grid. The best way is the analysis after vitrification and cryo-transference to an electron cryo-microscope using cryo-conditions: temperature below -170ÂşC and minimal dose of electrons (Fig. 1) (4). This strategy can be also carried out at room temperature by means of the negative staining if the structure of the particles is not depending on the water (7). Electron tomography or cryo-tomography refers to the three-dimensional reconstruction of isolated structures, cryo-sections from them, or resin sectioning after freeze substitution, acquiring images in as many directions of the electron beam as possible. This is achieved by tilting the holder supporting the grid containing the specimen and recording images at regular tilt intervals.

There are three procedures to prepare bulk specimens for three-

dimensional reconstruction. One is vitrification and direct transfer to the cryo-microscope. This approach is used when the thickness of the specimen is under the penetration power of the electrons in our electron microscope. If it is not the case, cryo-sectioning of vitreous samples allows thin sections of cells or tissues at low temperatures to be taken without loss of their vitreous state and then observed in the cryo-microscope (8). Finally, we can use freeze-substitution that refers to the substitution of the vitreous ice by an organic solvent followed by embedding in a resin which after polymerization leads to thin sections of the

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specimens (Fig. 2) (9). In this last case, the electron tomography is performed at room temperature.

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

Computational methods for 3D-reconstruction

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After image acquisition, different softwares are used for data alignment, reconstruction and viewing. The reconstruction can be considered as the inversion of the imaging process. The projections obtained from the 3D-specimen at different angles or orientations are deprojected into the reconstructed volume (Fig. 3) (10).

Recent developments and outlook Recently, the use of the dual-beam electron microscopes has been showed like a very good device for preparing samples for electron cryo-tomography (11). The focused ion beam can be used to remove part of the frozen sample to obtain a thin layer or to make a lamellae from the sample. In both cases the result is a thin vitrified sample on a grid to be load into the cryo- transmission electron microscope. On the other hand, freeze substitution, together with other techniques, like Tokuyasu and the new rehydration and VIS2FIX methods (12), can also be combined with on-section immunogold labelling for protein localization studies with reliable morphology. However, one of the main topics in the CryoEM field nowadays, is the combination of the molecular labelling, using a clonable tag, with the cryo-imaging and

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cryo-tomography to identify the components of the macromolecular machines inside the cell (13).

Acknowledgements We are very grateful to all users and collaborators of the Electron Cryo-Microscopy Unit specially to Elena Mercadé, Olga López and Cristina Risco for the valuable contribution to this manuscript.

References 1. Kühner S, van Noort V, Betts MJ, Leo-Macias A, Batisse C, Rode M, Yamada T, Maier T, Bader S, Beltran-Alvarez P, Castaño-Diez D, Chen WH, Devos D, Güell M, Norambuena T, Racke I, Rybin V, Schmidt A, Yus E, Aebersold R, Herrmann R, Böttcher B, Frangakis AS, Russell RB, Serrano L, Bork P, Gavin AC. 2009. Science. 326:1235 2.Studer D, Humbel BM, Chiquet M. 2008. Histochem. Cell Biol. 130: 889 3. Galy V, Askjaer P, Franz C, López-Iglesias C, Mattaj IW. 2006. Curr Biol. 16: 1748. 4. Rodríguez G, Soria G, Coll E, Rubio L, Barbosa-Barros L, López-Iglesias C, Planas AM, Estelrich J, de la Maza A, López O. 2010. Biophys J. 99: 48 5. Frias A, Manresa A, de Oliveira E, López-Iglesias C, Mercade E. 2010. Microb Ecol. 59: 476 6. Leunissen J.M, abd Yi H. 2009. J. Microsc. 235: 25 7. Querol-Audi J, Pérez-Luque R, Fita I, López-Iglesias C, Castón JR, Carrascosa JL, Verdaguer N. 2005. J.Struct. Biol. 151: 111 8. Pierson J, Fernández JJ, Bos E, Amini S, Gnaegi H, Vos M, Bel B, Adolfsen F, Carrascosa JL, Peters PJ. 2010. J Struct Biol. 169: 219 9. Nebot M, Deroncele V, López-Iglesias C, Bozal N, Guinea J, Mercade E. 2006 Microb Ecol. 51: 501 10. Fontana J, López-Iglesias C, Tzeng WP, Frey TK, Fernández JJ, Risco C. 2010. Virology 405: 579 11. Rigort A, Bäuerlein FJ, Leis A, Gruska M, Hoffmann C, Laugks T, Böhm U, Eibauer M, Gnaegi H, Baumeister W, Plitzko JM. 2010. J Struct Biol. 172:169 12. Karreman MA, Van Donselaar EG, Gerritsen HC, Verrips CT, Verkleij AJ. 2011. Traffic 12: 806 13. Diestra E, Fontana J, Guichard P, Marco S, Risco C. 2009. J. Struct. Biol. 165,157

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Image analysis as a service for cell-based assays and tissue cultures Rafael RodrĂguez Chief Technology Officer and co-founder of Wimasis

Abstract This essay is about image analysis as a specialized web-based service for cell-based assays and tissue cultures. It aims to show the possibilities this service offers to researchers in the fields of biology, biomedicine, drugs and biotechnology, and explores the many advantages it has over manual quantification and software products. The essay also gives a general overview of Wimasis activity, a major provider of image analysis services for the life sciences through the web. Its activity, organization and services are explained in order to give researchers a closer approach to Wimasis work and valuable tools to assist cell-based assay and tissue culture research.

Image analysis in cell-based assays and tissue cultures The relevance of image analysis for cell-based assays and tissue cultures cannot be denied. Results provided by image analysis of a cell-based assay or tissue culture determine the conclusions of the research in such a strong way that a deviation in the image analysis may lead to quite different final findings. Therefore it is very important that researchers take a reasonable time to decide who and how their image analysis will be done. When facing the choice of a way to perform image analysis on a certain assay, a few things should be taken into account. The number of images that have to be analyzed, the complexity of the analysis, the maximum time researchers can afford to wait for the results of the image analysis, the number and diversity of parameters that should be measured, the money researchers can afford to spend on the analysis, the amount of cells or tissues that might be analyzed on every image and the desired level of objectivity and comparability of the results are some of the major things that researchers should bear in mind before choosing the way to make the analysis. Depending on which of these things they value more and what the conditions of the project and research group are, they might select one or another method to conduct the image analysis. There are many options regarding the way to make the image analysis for cell-based assays and tissue cultures, but they could be all clustered into two main categories: manual and automated analysis. The manual quantification is the one performed by researchers with the single use of their mind and personal abilities to make the analysis; while the automated

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

image analysis is the one carried out by a machine equipped with a software specially designed for this kind of analysis. Manual quantification is the cheapest way of image analysis, but has two major disadvantages: it takes a lot of time, so it is totally unfeasible to choose this analysis method when there are many images and little time to analyze them; and the lack of objectivity of the results, as human brain will react differently to the image when analyzed in a second round. On the other hand, the automated image analysis provides objective results, making possible a reliable comparison between the results obtained when repeating the assay several times; this is an important advantage over manual quantification. However, automated image analysis has been traditionally offered as a software product, consisting on a program with large amounts of technical settings that are not familiar to the researcher and need to be adjusted by him to perform the right analysis output; this is not very practical for life sciences researchers, as it normally results in researchers wasting their time learning how to use the program and how to fix its settings for each kind of assay and culture, which is not what they should be devoted to. Besides, this software is usually very expensive and leads to the investment of large amounts of money, not only on the initial purchase, but also on technical maintenance of the equipment and yearly updates. To overcome the disadvantages image analysis software has, a new concept of image analysis for the life sciences has come up: the image analysis as a service, an improved form of automated image analysis that represents a remarkable innovation for the life sciences researchers.

Image analysis as a service Image analysis offered as a service is one of the multiple forms of the new paradigm of Sofware as a Service (SaaS). In this model, automated image analysis is presented as a service that you can access through the internet, using it just when needed and paying only per the individual uses. No need to own or purchase image analysis software is required to use this service, provided by image analysis experts. This way you can outsource your image analysis, gaining the expertise of image analysis developers at really low costs. Outsourcing the image analysis has a lot of advantages over traditional ways of performing the image analysis in life sciences. The first one is that it releases the researcher from the tedious analysis tasks, leaving more free time for him to do what is more important: research. Manual tracking and quantification needs a lot of time to be done and software products require a deep technical knowledge and a lot of time to understand how to make the most of the different settings for each assay. However, time is a limited resource and should be used on the most profitable way. As the image analysis tasks are not the main point of cell-based assays and tissue cultures, but just the means to extract the data, saving time automating the image analysis by outsourcing it, is a very good idea to gain time for the researcher. Other advantage of image analysis as a service is that there is no need to own or purchase software to perform the analysis, so no large amounts of money need to be spent. Unlike the software products, image analysis as a service requires no initial inversion,

HOT TOPICS IN CELL BIOLOGY - Edited by JosĂŠ Becerra and Leonor Santos-Ruiz

technical maintenance or yearly updates to be made by the user, skipping all the costs software products entail. As it is a web-based service, the updates are held in the computer servers and are run automatically and costs-free, as well as it is the maintenance. Another advantage of the image analysis as a service is that no training is needed, what means that researchers will get optimum results while saving time and money. In fact, software products require expertise technical knowledge, while the image analysis service allows researchers to get their results with no more worries than uploading the images to the server and waiting for the feedback. Besides, image analysis service gives objective results, as the analysis is solid, repeatable and reliable. This means that the analysis could be conducted once and again and it will always give the same results, which makes it a perfect method to compare the results of different assays. Furthermore, the pay-per-image system is also a way of saving money. Researchers will only pay per image analyzed, so if they are running an assay for a short period of time, they won’t need to make huge investments that will not be recovered. In this way, researchers get maximum flexibility in costs and are able to spend the money in a more profitable way. These are the main advantages any basic automated image analysis service presents. However, other benefits such as data storage, results within minutes or possibility to upload all the images at the same time, are given for free by some image analysis service providers. One of those is Wimasis.

Wimasis: the web-based image analysis service provider for the life sciences Wimasis (www.wimasis.com) is the web-based image analysis company for cell-based assays and tissue cultures. It provides image analysis as a service for life sciences researchers and counts already with a user community of more than 1.000 research groups around the world. Wimasis services focus is to bring practical image analysis solutions for life science researchers, trying to make easier their daily tasks and improve the general performance of the researching process. Its services represent a major alternative to evaluate assays made with microscopes and are delivered worldwide through its online platform, MyWim (mywim.wimasis.com). Founded in November 2009, in Munich (Germany), Wimasis has quickly expanded its analysis solutions throughout the world thanks to the reliability of its services and the strength of its hardcore values. In June 2010, a new operations center was established in Córdoba (Spain), where research and development activities are held. Since its very beginning, Wimasis working policy has been based on the principles of compromise, hard work, innovation, quality, flexibility and excellence in customer service. Researchers’ needs are the guidelines of Wimasis daily work and a qualified team of software and online platform development engineers are devoted to fulfill that mission.

HOT TOPICS IN CELL BIOLOGY - Edited by José Becerra and Leonor Santos-Ruiz

Wimasis technology To guarantee simplicity, security, reliability and efficiency, Wimasis develops, maintains and constantly improves its own processing center, supplying it with the most suitable technologies and latest technical advances. Web Service As Wimasis services are provided through the web, just an internet connection and a basic computer with minimal requirements are needed to use Wimasis platform (MyWim). But to gain access to Wimasis services, a registration might also be required as a preventing policy. Registering is fast, easy and costs-free. After registering, videos and images can be uploaded to the platform just following 3 simple steps: 1. Go onto MyWim platform and adjust the account settings: decimal separator and preferred image format. 2. Upload the images and get the results: choose the type of analysis, upload the images browsing them from the computer or dragging them into the uploader (it is possible to upload as much images as desired all at one time) and click send. In a short time, results will be ready to download from the website without any additional requirement. Security Policy As the data uploaded to Wimasis contain relevant information regarding research projects, physical protection, data encryption and secure user authentication are provided to guarantee maximum security in all data transactions and storage. Wimasis website uses Hypertext Transfer Protocol Secure (HTTPS) encryption on all web pages where personal information is required, that is why you must use an HTTPSenabled browser such as Safari, Firefox or Internet Explorer, to interact with Wimasis platform. Processing Center Several powerful servers have been designed and adapted to get a fast and efficient image analysis. A computer cluster with multiple microprocessors analyzes many images in parallel, so the time to get the final results can be highly reduced. This kind of computer clusters are able to analyze a huge amount of data at the same time, which means that researchers can upload all the images at a time and get the results in a very brief period of time.

Storage

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The images analyzed are stored in Wimasis servers. This means Wimasis servers work also as a massive and secure free storage center, which allows the researcher to gain access to its results (only his images, not anyone elseâ&#x20AC;&#x2122;s) from any part of the world.

Wimasis services Wimasis offers image analysis as a service for cell-based assays and tissue cultures. Its services are suited to every kind of image format and can be sorted in two main lines: standard services and custom solutions. Standard services Standard services are the services offered in a regular basis and are given for the most popular cell-based assays performed by researchers. Its main features are: they are adapted to every type of image format, size and illumination and they are run automatically by the computer cluster, which means that results will be available within minutes. This kind of image services have been developed in cooperation with researchers from several research units around the world, with different kind of images, which assures the service to be standardized enough to suit almost every type of images. Nevertheless, when an image does not fit the standard image analysis service, a completely free adaptation of the service can be done. The standard services are: WimScratch for Scratch assay. It evaluates the 2D cell migration on Scratch assay. Analysis data given are: cell covered area, speed of wound closure (with an overview chart), acceleration characteristics and center piece approximation. WimTube for Tube Formation assay. It evaluates the generation of new vessels in Tube Formation assay. Analysis data given are: number of branching points, tube data (length, number and statistics), loop data (number, areas and perimeter) and cell-covered area. WimSprout for Aortic and Spheroid Sprouting assays. It evaluates the generation of new sprouts in the Sprouting assay. Analysis data given are: spheroid/aortic area, sprouts area and number, cumulative sprouts length, mean sprouts length and standard deviation of sprouts length. WimTaxis for Chemotaxis and cell tracking assays. It evaluates the 2D cell migration in Chemotaxis and other migration assays. Analysis data given are: forward migration index (parallel and perpendicular), cell directionalities and cell migration activity. A free trial is available for all the standard products. Also, additional parameters can be requested when desired.

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Custom solutions As every research is unique, Wimasis offers the users the possibility to request a personally-fitted solution to meet their needs in the most suitable way. Custom solutions require a previous feasibility study, which is made completely for free. Besides, the request of a feasibility study does not compromise the researcher to agree to buy the service when he is not satisfied with the settings offered. Some of the Custom solutions Wimasis has already made were fitted to cell counting, colony forming, nucleus detection, autophagy, apoptosis, CAM assay, fibrosis grade detection, neurite and synapse detection and many others.

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In Vivo non-Invasive Bioluminescence Imaging: a Short Course Nuria Rubio, Olaia Fernández-Vila, María Alieva, Julio Rodríguez-Bagó and Jerónimo Blanco. Cardiovascular Research Center-CSIC-ICCC, CIBER-BBN, Barcelona, Spain. Optical imaging procedures are based on the use of reporters, substances that produce light photons of specific wavelengths. Detection of such specific wavelength photons in a living system identifies the presence of the specific substance.

Basic definitions According to the light generating mechanism, there are two groups of photo-reporters, fluorophores and bioluminescence generating agents. Independently of their molecular nature, reporters can be broadly classified as non-genetic and genetic. Non-genetic reporters, once introduced in cells, will become progressively diluted with each division. Conversely, genetic reporters are administered as gene coding sequences to be expressed in live cells. Except in the cases of transiently expressed reporters, the integration of a reporter coding sequence in the genome, labels not only the recipient cell, but also its descendants and no dilution but amplification of the reporter takes place by cell division.

Fluorescence Fluorescence refers to the absorption of light photons of a particular wavelength by outer shell electrons of a fluorophore, resulting in their acquisition of a higher energy level. The return of an excited electron to its ground energy level, a very fast process taking milliseconds, is accompanied by the generation of photons of energy lower than that of the incident ones. In cell biology, the most frequently used fluorescent reporters are the so called “fluorescent proteins”. Fluorescent protein reporters have found their main application in histology for the identification of cells within tissue sections and in microscopy for the identification of cells and subcellular organelles, location of specific proteins and the study of protein interactions. The application of imaging procedures in many branches of cell biology has been favored by the development of fluorescent protein reporters with excitation and emission covering most of the visible spectrum. The use of fluorescent reporters for non invasive imaging in live animals has the important advantage of not requiring the administration of a substrate. However, up to now, important difficulties have precluded its application to live animals the size of mice or larger. A great variety of biological molecules absorb light in the excitation and emission ranges, ultraviolet and infrared wavelength, of the majority of fluorescent proteins, resulting in severe

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reductions in quantum yield. This situation is complicated further by the fluorescent nature of many tissue components, that upon excitation by light generate background fluorescence, that contributes noise with considerable reductions of sensitivity. Recently, the development of fluorescent proteins with excitation in the red and emission in the red and infrared regions of the spectrum and the availability of background reducing algorithms for image treatment have generated a renaissance of in vivo applications for fluorescent reporters in small animals.

Bioluminescence Luminescence is the generation of light as a by-product of a chemical reaction. Bioluminescence is a special case of luminescent reaction catalyzed by an enzyme (luciferase). In a prototype bioluminescent reaction catalyzed by the luciferase of the American firefly Photinus pyralis (P-Luc), an organic substrate, luciferin, is oxidized in the presence of oxygen, ATP and the cofactor Ca++ with the generation of CO2, AMP, oxiluciferin and 550 nm light photons. A reaction that, in nature, takes place in the light organ of the insect (1,2). Bioluminescence is a widespread phenomenon in nature. There are bioluminescent terrestrial and aquatic organisms, ranging from bacteria, dinoflagellates, see pansies like

Renilla reniformis (R-Luc) that emits at 480 to 510 nm depending on its association with GFP, to molds eg., Jack-O-Lantern mushroom (Omphalotus olearius). There are some 2000 species of bioluminescent insects known. However, there are no known bioluminescent examples in the plant kingdom. The genes for several luciferases from Photinus piralis,

Renilla reniformis, Vargula hilgendorfii and bacteria have been cloned and their substrates identified and synthesized and are currently available for research.

Bioluminescence imaging (BLI) Use of bioluminescence reporters for in vivo imaging in mammals has important advantages over that of fluorescent reporters. Reporter photons need to travel only in the exit direction, reducing looses due to photon absorption and dispersion. More importantly, except for rare, ultralow intensity light generating redox reactions from immune system components, mammalian tissues are essentially devoid of light producing reactions, what results in a very low background, an ideal condition for light detection. However, difficult to avoid external chemoluminescent contaminants in the skin of experimental animals and some dietary components, often generate sufficient levels of background light to reduce the capacity of imaging systems beyond that of their actual physical potential. In addition to this, bioluminescence reporters have the important drawback of requiring the administration of the luciferase substrate to the experimental animal. In spite of their apparent opacity, mammalian tissues, including skin and bone are translucent. Light emitted by bioluminescent reporters at depths of 1 cm or more transverse animal tissues and can be used to generate images from cells in live animals (3,4). Light

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generated by luciferase reporters has been used in the past to detect cells in mammal embryos (5,6), transgenic fish (7) and drosophila (8). In mice, luciferases have been used to report eg.,on the activity of the AIDs virus promoter (9), to study the effect on tumor growth and metastasis of antitumor agents (10,11) and to monitor cell proliferation/differentiation in biomaterials implanted in live animals for tissue regeneration (12-14). Due to the relatively low light intensities generated by optical imaging reporters, the sensitivity of image acquisition instruments must be considerably higher than that of the human eye or photographic film. This high sensitivity requirement is compensated by the existence of light sensing phototubes and charge-coupled devices capable of detecting as few as 10 to 100 photons and responding linearly through a range of 6 orders of magnitude, a fact that facilitates considerably the quantification of biological phenomena. Imaging instruments, like video equipment, use an objective lens to project an image of bioluminescent light on the surface of a grid of photo-detectors. Whether the photo-detector is a phototube or a solid state device, the function of the detector is to amplify photon events reaching the detector into measurable electronic signals that can be recorded and displayed. In essence, detector sensitivity is characterized by its response to photons of different wavelength (quantum yield), the level of background electronic noise it generates through its different operations and its resolution. Current photo-detectors respond with quantum yields in the neighborhood of 90%, that is, they generate recordable events from 9 out of 10 incident photons, a variable depending on photon wavelength. Electronic noise is a recordable signal equivalent to that corresponding to the arrival of photons, but generated in their absence. Electronic noise, generated during different stages of image generation and detector readout, is directly proportional to temperature. In order to reduce electronic noise, photon detectors are maintained at very low temperatures. Thus, detector temperatures of the order of -80Âş C are the norm for the operation of sensitive instruments. Image resolution is also related to sensitivity. This is a consequence of larger solid-state detector pixels, as is the case with silver nitrate based photo detection, been more sensitive to light. In consequence, high resolution detectors usually come at the price of lower spatial resolution. Finally, as detector readout also generates noise, it is a common practice to reduce frequency of detector reading events by grouping of pixels (beanning), however at the expense of lower resolution (15). Other than the light detector, imaging instruments are provided with a cooling system for the detector, an absolutely dark specimen chamber, image management and storage hardware and display systems.

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In vivo imaging strategy

Monitoring options Cell distribution and number: Monitoring of the location of cells in different tissues and their number is a relatively straightforward matter that requires generation of vectors for expression of the luciferase reporter regulated by a promoter active and stable in the target

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cell type. For general purposes, unless a promoter active in the particular target cell type is available, constitutive promoters, those that are active more or less uniformly in the majority of tissues are a good choice. As tecnology stands, two luciferases, P-Luc and R-Luc, can be easily monitored in the same animal, since they use substrates that are not cross reactive. However, expression of both luciferases can be regulated by the same promoter. Of course, promoter activity should be as uniform as possible throughout the experiment and special care should be taken to ensure that silencing of genome integrated reporters, an artifact associated mainly with retroviral vectors, does not happen. Another important consideration for the interpretation of data is that luciferases regulated by constitutively active promoters report on the number of cells in a particular location, not on their proliferation state, e.g., a particular number of cells may result from a steady state of cell death and replication or from a quiescent state. Biodistribution of cells and response of tumors to drugs are typical applications. Gene expression: A very interesting application of BLI is monitoring changes in gene expression taking place in live animals and in real time. This applies specially to tissue regeneration and biomaterial studies where inductions of specific cell differentiation pathways is expected. For such type of studies, luciferase reporters should be regulated by promoters active as specifically as possible in the target tissue, although this is not always possible. Monitoring of changes in gene expression is a considerably more complex process that monitoring of cell number. As usually gene promoter-enhancer sequences are complex and large and could comprise megabase sized sequences. In consequence, a surrogate promoter for the gene of interest must be constructed, based in promoter activity studies in vitro. Such promoters are abridged sequences, containing a reduced subset of nucleotides comprising the promoter parts that

researchers have determined that have positively or

negatively regulatory function for the gene of interest. As information on promoter activity is not always complete, artificial promoters only reflect a part of the biological reality. Moreover, in the in vivo situation, changes in the activity of the reporter are a result of several factors, e.g., cell proliferation and death, as well as, transcriptional activity. Therefore, for data analysis, the cell number parameter must be taken into consideration. This can be achieved, to some degree, by introducing in the target cells two luciferase reporters, a constitutively active one to measure cell number and the tissue specific one to report on gene expression. The ratio of light generated by both procedures indicates whether changes in light production from the gene specific reporter are the result of cell number changes or reflect real changes in gene expression.

Vector construction Strategies for vector construction should be guided by considerations of factors such as target cells, intended duration of labeling, cell permissiveness for infection by viral vectors, whether integration of reporter sequences is required, levels of expression etc.. For example, retroviral vectors target replicating cells while lentiviral vectors will target quiescent, as well as replicating cells. The cell type to be labeled is another important factor

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to keep in mind; while tumor cells are usually easy to transfect, some stem cells are remarkably refractory. Alternative to viral transduction, cell transfection using classical calcium phosphate, lipofecting agents and electro or sonoporation are also possible. However, in this cases, it should be considered that procedures generating few labeled cells may require lengthy cell expansion periods that may be detrimental for future applications, eg., in the case of stem cells with limited replication potential. In all cases, it should be considered whether a pure population of labeled cells is required, or labeling of a fraction of the cells is sufficient for the intended study. In the first case, positively transduced cells can be selected by inclusion of appropriate antibiotic markers. A preferred strategy that avoids subjecting cells to antibiotic selection can be applied taking advantage of vectors that also comprise fluorescent protein reporters in addition to luciferase, and allow selection by FACS. Cells labeled with this strategy can be used for histological studies, eg. by confocal microscopy. In addition, by generating chimerical vectors of luciferase-fluorescent protein, a single promoter is used for regulation of expression with added advantages: The expressed dose of luciferase and fluorescent protein reporters is equivalent, cell cultures can be easily monitored and an economy in the size of the vector construct, a critical parameter for effective viral transfection, is also obtained.

Imaging Several factors must be taken into consideration before imaging. Image acquisition, may take from seconds to minutes and will require anesthesia of the animal to guaranty total stillness during the acquisition period in the dark receptacle of the camera. Luciferase substrates, luciferin and coelenterazine are not toxic to mice in the doses required for imaging. While luciferin is usually dissolved in water, coelenterazine the substrate of R-Luc and other luciferases is usually dissolved in alcohols and the solvent may pose a risk to the animal if injected i.v.. Also, some solvents may induce tissue necrosis if injected repeatedly in the same site. Thus, substrates should be diluted in a suitable solvent before use. Cumulative toxic effects, resulting from repeated applications, must be considered previous to initiating an experiment, at the risk of having to terminate it prematurely. While i.v. administration results in the production of the fastest and most intense light generation, i.p. inoculation, requiring less expertise, can be more convenient when sensitivity is not at a premium. However, substrates such as coelenterazine, that generates background light spontaneously, as a result of interaction with cell membranes and other components, will produce strong unspecific background images when inoculated i.p. Luciferase catalyzed reactions are very fast in vitro, but concentration dependent, nevertheless. Light production in vivo will inevitably depend on complex pharmacokinetic and pharmacodynamic considerations, determining the speed at which the substrate reaches the target cells and for how long, a large enough substrate concentration will persist to produce a detectable reaction. In experiments requiring repeated imaging or imaging of

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different luciferases, it should be taken into consideration the potential overlap of persisting bioluminescence reactions even several hours, following the administration of substrate. Finally, empirical knowledge of the light producing reaction kinetics is required to warrant reproducible and quantitative results.

Image quantification Quantification and spatial distribution of light producing reactions in live animals is the goal of imaging experiments. While image acquiring instruments are exquisitely sensitive and lineal in their response to light events, the optical properties of animal tissues are extremely complex. Tissues vary according to their content of green light absorbing hemoglobin, density, refraction index and dispersive capacity. Moreover, the light path may have to transverse different types of tissues, with non uniform matter distributions and shapes. Thus, especial preparations are required in order to quantitatively evaluate images. The best approach to these problems is again empirical and success depends on been able to standardize image acquisition parameters. The following are some issues to consider: 1- The time elapsed from substrate administration to image acquisition. Fortunately peaks of light producing activity from some luciferases/substrates combinations e.g., P-Luc and luciferin are reasonably stable during 10 to 15 minutes, in vivo. 2- Concentration of luciferase substrate. 3- Distance of specimen from objective lenses of the camera is critical. The number of photons/cm2 that reach the detector is inversely proportional to the square of the distance to the subject. 4- Depth of cell implantation should be controlled when possible, since more than 90% of the light generated at less than one centimeter depth is absorbed by the tissue. 5- Generation of standard curves of light produced versus number of light emitting cells at a particular depth in tissues. This allows the evaluation of cell number, provided that cells are in a narrow depth range. 6- Avoid anoxia since some luciferases are oxygen dependent. 7- Imaging of very low intensity chemiluminescence reactions. In theory, even reactions generating very low light intensities could be detected just by acquiring images during very long times. However, in practice, either the animals may not be kept anesthetized for very long time, or low level background chemiluminescence reactions generate background noise, limiting the time during which it is practical to acquire images. A particularly noxious problem and potential source of artifacts is that of light-activated spurious chemiluminescent reactions taking place in the surface of the skin. These complex reactions result from contamination with urine, feces and bedding materials. In addition, some materials are phosphorescent and will emit light after exposure to fluorescent tubes and other light sources. Control of this problem is particularly critical in experiments that push the limits of detection of the imaging instruments and few or poorly labeled cells must be detected, eg.,tumor metastases, stem cells, etc.

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Fortunately, most of these light generating reactions are time limited and will dissipate after a few minutes in total darkness. However, if a dark period is required before image acquisition, the kinetics of substrate use must be taken into consideration.

References 1- Campbell A. and Sala-Newby G. Bioluminescent and chemiluminescent indicators for molecular signaling and function in living cells. In: Mason WT, editor. Fluorescent and luminescent probes for biological activity. New York: Academic Press 1996.58–82. 2-Steghens JP, Min KL, Bernengo JC. Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra. Biochem. J. 1998.336: 109–13. 3. Benaron DA, Cheong WF and Stevenson DK. Tissue Optics. Science. 1997. 276:20022003. 4. Müller G, Chance B and Alfano R. editors. Medical optical tomography: Functional imaging and monitoring. Bellingham, WA: SPIE Press Optical Engineering Press. 1993 5. Matsumoto K, Anzai M, Nakagata N, Takahashi A, Takahashi Y and Miyata K. Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm. Mol. Reprod.Dev.1994. 39:136–140. 6. Thompson EM, Adenot P, Tsuji FI and Renard JP. Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase. Proc. Natl. Acad. Sci. USA. 1995. 92:1317–1321. 7. Tamiya E, Sugiyama T, Masaki K, Hirose A, Okoshi T and Karube I. Spatial imaging of luciferase gene expression in transgenic fish. Nucleic Acids Res. 1990. 18:1072–1076. 8. Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood KV, Kay SA and Hall JC. Novel features of drosophila period transcription revealed by real-time luciferase reporting. Neuron. 1996. 16:687–692. 9. Contag C. H., Spilman S. D., Contag P. R., Oshiro M., Eames B., Dennery P., Stevenson D. K. and Benaron D. A. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem.Photobiol. , 1997; 66:523–531. 10. Vilalta M, Dégano IR, Bagó J, Aguilar E, Gambhir SS, Rubio N, Blanco J. Human adipose tissue-derived mesenchymal stromal cells as vehicles for tumor bystander effect: a model based on bioluminescence imaging. Gene Ther. 2009. 16: 547-57. 11. Alieva M, Bagó JR, Aguilar E, Soler-Botija C, Vila OF, Molet J, Gambhir SS, Rubio N, Blanco J. Glioblastoma therapy with cytotoxic mesenchymal stromal cells optimized by bioluminescence imaging of tumor and therapeutic cell response. PLoS One. 2012;7(4):e35148. 12. Román I, Vilalta M, Rodriguez J, Matthies AM, Srouji S, Livne E, Hubbell JA, Rubio N, Blanco J. Analysis of progenitor cell-scaffold combinations by in vivo non-invasive photonic imaging. Biomaterials. 2007. 28: 2718-28. 13. Dégano IR, Vilalta M, Bagó JR, Matthies AM, Hubbell JA, Dimitriou H, Bianco P, Rubio N, Blanco J. Bioluminescence imaging of calvarial bone repair using bone marrow and adipose tissue-derived mesenchymal stem cells. Biomaterials. 2008. 29: 427-37. 14. Vilalta M, Jorgensen C, Dégano IR, Chernajovsky Y, Gould D, Noël D, Andrades JA, Becerra J, Rubio N, Blanco J. Dual luciferase labelling for non-invasive bioluminescence imaging of mesenchymal stromal cell chondrogenic differentiation in demineralized bone matrix scaffolds. Biomaterials. 2009. 30:4986-95. 15- http://www.hamamatsu.com

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2. APPLIED CELL BIOLOGY Cell biology and beyond: Applications of cell biology Leonor Santos-Ruiz Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine, (CIBER-BBN). BIONAND-UMA. Department of Cell Biology, Genetics and Physiology, Faculty of Sciences, 29071 Málaga, Spain. Cell biology is a scientific discipline that studies cells. As every single living being is composed of, at least, one cell, cell biology somehow entwines all biological sciences. Starting as a descriptive science, cell biology has greatly evolved in the last two centuries, benefiting from other disciplines such as molecular biology or genetics, and from technological developments. Today, cell biology is broad and diverse as ever, and it is difficult to establish its boundaries with other scientific disciplines. Every problem that requires the use of cells for its experimental settings is a cell biology problem. Every experiment that requires the knowledge of cell behaviour and physiology for the interpretation of its results is a cell biology experiment. Thus, cell biology has become “a blend of advanced cytology, molecular biology, genetics, biochemistry, computation and engineering” 1. Although traditionally regarded as a basic science, the fact is that fundamental discoveries in cell biology research have made their way to the market, sometimes in unpredictable ways, producing an indelible effect on human life. It is hard to find a home in the developed countries where cell biology has not silently entered in the form of food, cosmetics or medicines. Understanding how cells work in health and how they don’t work in disease has benefitted not only human, but also animal and plant health, fostering the development of novel diagnostic methods, new vaccines, and new drugs and therapies to fight disease. Allied with genetics, plant cell biology has given us food and medicines. Plant biotechnology deserves special attention for what it has already provided us, and for what it may still provide to the development of the Third World. Among the many drugs obtained by plant biotechnology, alkaloids are a classical example that deserves special mention. These are a group of plant compounds exploited from ancient times as pharmaceuticals, stimulants, narcotics and poisons. Numerous plantderived alkaloids are common in the clinical practice, including analgesics like morphine and codeine, anticancer agents like vinblastine and taxol, the gout suppressant colchicine, the

Quoted from Tom Misteli at J. Cell Biol. Vol. 184 No. 1 11–12

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muscle relaxant tubocurarine, the antiarrythmic ajmaline, the antibiotic sanguinarine and the sedative scopolamine. Other important plant-alkaloids include caffeine, nicotine, cocaine and heroin, which is cocaineâ&#x20AC;&#x2122;s O,O-acetylated derivative. In the biotechnology era, alkaloid production has been increased by isolating genes and using them to genetically engineer the accumulation of alkaloids and other secondary metabolites in plants, thus increasing the production and facilitating purification. Food industry has benefited as well from the alliance between biotech and cell biology. Plant biotechnology has produced improved, more productive or more resistant crops to feed people. Research is still ongoing to develop crops that can endure cold or draught, poor soils and plagues. An interesting form of transgenesis consists on introducing genes encoding nutritional supplements to compensate poor diets. A good example is the introduction of vitamin A gene from carrot into widely consumed crops like rice. Shortage of vitamin A causes blindness and paves the way to death due to measles and diarrhoea in half of all countries, specially in Africa and South East Asia. Transgenic vitamin A-rich rice is helping to prevent these maladies. Far over the suspicions of the well-fed first-world citizens, it is undoubted that these crops raise a hope today for underdeveloped countries. An apparently less dramatic application of cell biology, but significantly important due to its economic impact, is cosmetics. Most face creams contain collagen, elastin, hyaluronic acid and other components of the extracellular matrix, as a result of a deeper understanding attained in skin biology. Comprehension of melanocyte physiology is at the base of a successful market of self-tanning lotions, skin clarifying masks, sun protection creams and other related products. Cell biology is present in virtually all aspects of cosmetology, from product development to publicity. Cosmetic companies hire cell biologists for developing, producing and testing their products. They have incorporated techniques developed for scientific research into their routine methodologies. For example, human cell cultures are replacing the massive use of laboratory animals for cosmetic research and testing. Last, but not least, cosmetic industry uses science for their publicity: cosmetics are presented as the result of scientific innovation as a guarantee of quality and safety. Basic cell biology concepts like cell proliferation or extracellular matrix have reached peopleâ&#x20AC;&#x2122;s everyday conversations not through school learning or interaction with clinicians, but through TV ads of cosmetic creams. As a scientist and a teacher, I must find this food for thought. Finally, it is worth mentioning that cell biology is not only involved in the development of the above-mentioned health, food or cosmetic products. It is involved in industrial manufacturing as well. The production of many drugs implies the use of cells as living reactors. The use of living cells as protein factories in biopharmaceutical manufacturing is at the heart of a $50 billion growth industry.

Interdisciplinarity and technology The past few decades have seen a remarkable advance in the study and the use of living cells. Fundamental discoveries in basic cell biology are impacting medicine and industry at an ever-quickening pace. Part of the credit for this advance must be granted to two causes: technological breakthrough and interdisciplinarity.

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The development of novel technologies has enabled researchers to perform experiments that had been possible only in their imagination, getting new and deeper insights into biological problems. Innovative applications of these technologies are also providing new potentialities for translation to the clinic and industry. In the last decade, advances in physics, microscopy and mass spectrometry have revolutionized the ability to detect and quantify protein abundance, modifications and interactions, as well as the concentrations of intracellular metabolites. High-throughput sequencing has provided the complete sequence of many genomes. RNA interference (RNAi) has allowed efficient knocking down of gene function. Cell-based screening has become a standard approach in drug discovery. All these techniques have provided new insights into epigenetics, have enabled new discoveries, such as the existence of miRNAs in the genome, and have elucidated a molecular understanding of altered gene regulation in disease, driving the identification of new targets for many different disease states. More interestingly, the development of new advanced sophisticated research tools has prompted interdisciplinarity, which has proven itself as the most powerful tool scientists can use. Last decades have seen an increasing collaboration between different research communities inside and outside cell biology. The interaction between geneticians, clinicians and cell biologists has led to the identification of frequent mutations in particular types of disease. Interactions between structural biologists, medicinal chemists and cell biologists have allowed to clarify the complex interactions of proteins in cell signalling, facilitating the development of small molecules to manipulate these processes in a therapeutic context. This, together with advances in small-molecule design and screening, has led to fruitful collaborations with pharmaceutical chemists, and they are beginning to design new drugs to specifically inhibit the molecule or molecules responsible for altered cell behaviours in disease situations.

Stem cells, cell therapy and tissue engineering Few topics in cell biology have drawn attention as much as stem cells. Up till now, the st

21 century is being the century of stem cells. In the last decades, intense research on embryonic and adult stem cells has provided important information on the mechanisms driving tissue and organ formation during development and on the processes that maintain or repair tissues after injury. Stem cell research has also provided clues about certain pathological conditions. It is now under consideration that loss or altered stem cell functions may underlie numerous degenerative disorders and diseases, including hematopoietic and immune system disorders, cardiovascular diseases, diabetes, chronic hepatic injuries, gastrointestinal disorders, brain, eye, and muscular degenerative diseases, and cancers. A major breakthrough in stem cell biology was the finding that the induced expression of a few key genes could revert a differentiated phenotype into an embryonic stem cell-like state. The so-derived induced pluripotent stem cells (iPS cells) have opened new paths to both basic and applied cell biology. Reprogramming differentiated cells into stem-like cells is a good tool to study the genetic and epigenetic mechanisms of cellular self-renewal,

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pluripotency and differentitation. It has also allowed researchers to derive patient-tailored iPS cells to study the biology of a variety of diseases. Patient-derived iPS cells might one day be used to cure patients, used either for cell therapy, gene therapy or tissue engineering. At present, they are not considered safe for clinical research, due to the genetic manipulation needed for iPS cell generation. Not-integrating gene vectors are being investigated, but it is still unclear if iPS cells could become cancerous within the patient’s body. Since the beginning of stem cell biology, most efforts have been focused on exploiting their potential therapeutic uses. Their capacity for self-renewal and multilineage differentiation offers the possibility of obtaining huge amounts of tissue-specific progenitors that can be used for cell-replacement or tissue engineering. Presently, there are more than four thousands clinical trials with stem cells, including embryonic, fetal, aminiotic fluid, umbilical cord and adult stem cells. The therapies under investigation aim at alleviating a wide variety of pathological disorders, among them hematopoietic and immune disorders, dysplastic anemia, autoimmune diseases, cancer, heart failures, muscular disorders, skeletal diseases, nervous system disorders, lung disorders, retinal diseases, liver, skin and hair disorders, diabetes, etc. However, before stem cell-based therapies become routine in the clinic, several issues have to be taken into consideration. First, it is still necessary to determine how stem cell therapies work, i.e., which biological mechanisms are responsible for any observed positive effect not previously attained with other therapies. Second, questions regarding the cell population to be used and optimal dose and routes of delivery will have to be addressed. In fact, most current stem cell-based clinical trials are Phase I and Phase II trials with the purpose of determining the safety and the efficacy of a particular procedure. Hopefully, these questions will be solved out in the next years so that the promise of stem cell-based therapies will turn into reality.

Nanotechnology Cells function in a nanoscale world. Intra and intercellular communication occur through single molecules or supramolecular complexes. The extracellular matrix is an interconnected fibrous network with nanoscale architecture. It was predictable that scientists would sooner or later work at a nanoscale level, and recent technological advances have produced the instrumentation needed to start unravelling the structure and dynamics of cells at a nanoscale detail. Nanotechnologies are technologies that use engineered materials or devices typically ranging from 1 to ~100 nanometres (1 nanometer is one billionth of a metre). At least some aspect of these nanomaterial or nanodevices can be manipulated by physical and/or chemical means at nanometre resolutions, resulting in unique functional properties. Nanobiotechnology has been defined as the application of nanotechnology for the study of biological and biomedical systems. Achievements in the field of nanobiotechnology include advances in cell transfection, cell isolation and sorting, tissue engineering, cell tracking and imaging, and molecular detection. “Nano” tools available for these purposes

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include nanoparticles and self-assembling peptides for gene delivery, micro/nanofluidic devices for stem cell electroporation, magnetic nanoparticles for cell sorting, nanofibers and micro/nano structured scaffolds for tissue engineering, micro/nanoencapsulation for cell therapy, carbon nanotubes optical probes, superparamagnetic iron oxide nanoparticles for cell labelling and MRI tracking and detection, quantum dots for cell tracking, and a long etcetera. Nanodevices and nanomaterials can interact with biological systems at molecular levels with a high degree of specificity. The advantage of this molecular specificity is that nanodevices and nanomaterials can interact with target cells or tissues in highly controlled ways, inducing the desired physiological responses without affecting other cells/tissues, therefore minimizing undesirable effects. These properties make nanobiotech products very attractive to the health arena, where there is a constant need of improvements in terms of better diagnostics, drug delivery approaches, and imaging technologies. Current clinical applications of nanobiotechnology include nanoparticles and nanogels that specifically transport drugs and small molecules across the blood-brain barrier, functionalized nanoparticles used for spatial and temporal tracking of molecules or cells within the body, and functionalized nanoparticles that specifically target tumour cells for their posterior destruction via hitting or intracellular delivery of cytotoxic compounds. The progresses of basic research coupled to the advances in nanobiotechnology are making available new diagnostics and therapeutic tools that are rapidly transiting from bench to bedside. It is expected that in the years to come nanobiotechnology will assist the integration of diagnostics/imaging with therapeutics and will facilitate the development of personalized medicine.

Education As cell biology research widens our knowledge and comprehension of the living world, and drives the development of new foods, drugs, and health services and products, its impact on society needs to be properly recognized. In the era of information and globalization, an adequate presentation of the scientific work to the general public has become indispensable. It is vital that we make the effort of educating the public and policy makers about the impact that basic sciences have in improving human condition. As the British Society of Cell Biology puts it: “It is […] important that everyone feels informed about how the increase in knowledge about cell biology could affect him or her and society in general. Society will have to make informed decisions […]. A basic understanding of cell biology including genetics will be as important as having some knowledge about computers and the Internet”. Fortunately, the amount and variety of available resources for communicating with the public has grown with the advent of the internet. Web pages, blogs, communities, ejournals… all of them can help scientists come closer to the general public. But a resource that is particularly powerful is Wikipedia. As an open access source of information, Wikipedia is truly global, reaching people in virtually all countries and in practically all sectors of the general public, including policy makers, voters, and students. By becoming wikipedians we

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can both draw attention to our field of research and contribute to the dissemination of accurate scientific information.

Suggested readings 1. Akhtar A, Fuchs E, Mitchison T, Shaw RJ, St Johnston D, Strasser A, Taylor S, Walczak C, and Zerial M. A decade of molecular cell biology: achievements and challenges. Nat Rev Mol Cell Biol. 2012; 12(10): 669–674 2. Chakraborty M, Jain S, Rani V. Nanotechnology: emerging tool for diagnostics and therapeutics. Appl Biochem Biotechnol. 2011;165(5-6):1178-87 3. Engel A and Miles M Nanotechnology at the interface of cell biology, materials science and medicine. Nanotechnology 2008; 19 380201 4. Escalera J. Stem cell-based drug therapies: A discussion on emerging technologies and future developments. Yale J Biol Med. 2009; 82(3): 111–112 5. Facchini PJ. Alkaloid biosynthesis in plants: Biochemistry, Cell Biology, Molecular Regulation, and Metabolic Engineering Applications. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52:29–66 6. Mimeault M, Hauke R, and Batra SK. Stem cells: A revolution in therapeutics- Recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin Pharmacol Ther. 2007;82(3):252-64 7. Misteli T. The changing world of modern cell biology. J Cell Biol. 2009; 184(1): 11–12 8. Misteli T. JCB meeting: Cell Biology of Disease. J Cell Biol. 2009 October 19; 187(2): 155–156 9. Pérez López S, Oter Hernández J. (2012). Advances in stem cell therapy. In by Carlos López-Larrea, Antonio López-Vázquez and Beatriz Suárez-Álvarez (Eds.) Stem Cell Transplantation (pp. 290-313) Landes Bioscience and Springer Science+Business Media. 10. Silva GA. Neuroscience nanotechnology: progress, opportunities and challenges. Nat Revs. Neuroscience. 2006 (7):65-74 11. The American Society for Cell Biology (ASCB) 48th annual meeting report. J Cell Biol. 2009; 184(2): 12. The British Society for Cell Biology Website. (http://www.bscb.org) 13. The Spanish Society for Cell Biology Website (http://www.sebc.es) 14. World Health Organization Website on Nutrition Topics (http://www.who.int/nutrition/topics/vad/en/) 15. Xie Y. The application of nanotechnology in stem cell research. 2008. Nanotech Columns (www.nanotech-now.com/columns/column=23) 16. Xie Y. Nanobiotechnology: From Stem Cell, Tissue Engineering To Cancer Research. 2009. Nanotech Columns (www.nanotech-now.com/columns/column=23)

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2.1. Cell biology of cancer

Cancer cell biology: key points in future treatments of cancer Guillermo López-Lluch Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, CIBERER, Instituto Carlos III, Carretera de Utrera Km. 1, 41013 Sevilla, Spain.

Introduction The explosion of highthroughput techniques during the last decade is permitting us to explore complex interactions in biological systems at very different levels. Today we can study the complexity of cancer cells not only from their specific characteristics but also from the point of view of changes in the gene expression levels with transcriptomic and proteomic approaches and also the metabolic changes affecting these cells with metabolomics and the different changes at the molecular level by using different omics approaches such as glycomics, phosphoproteomics, interactomics, etc… With these and other techniques we already know that cancer is a very heterogeneous disease not only taken into consideration the origin or the type of cell affected but also considering the diversity of tumor cells into a specific cancer and their changes in characteristics during cancer development. In fact, we can currently establish a hierarchy inside tumor where some of the cells maintain tumorigenic capacity whereas others don’t. Cancer cells that maintain tumorigenic capacity receive the name of cancer stem cells (CSC) and are thought to be responsible for resistance to chemo- and radiotherapy (1). This complexity of cancer at molecular, cellular and tissue levels obligates us to design specific treatments for each patient accordingly to the characteristics of their respective cancer process. In fact, the enormous capacity of tumor cells to survive and activate synergistic pathways that cooperate to regulate cell survival as an adaptive resistance indicates that therapies based on one of a few targets can be ineffective in the treatment of some types of cancer. In fact, in the case of acute myeloid leukemia (AML), recent works demonstrate that the combination of different specific inhibitors of survival pathways instead of monotarget therapies must be used depending on the individual characteristics of AML in each patient (2). Taken into consideration the complexity of this system, we briefly summarize in this section some new findings on cancer cell metabolism in relationship with the main characteristics of tumor cells involved in their survival capacity, cell growth and metastatic dissemination.

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Cancer cell metabolism Transformation of cells is accompanied by changes in metabolism increasing glucose consumption by glycolysis accompanied by the decrease in the mitochondrial respiratory metabolism (3). This change, known as the Warburgâ&#x20AC;&#x2122;s effect, represents a mechanism important in the increase of growth in cancer cells. However, the relationship between carcinogenesis and metabolism is currently under controversy. The lower metabolic state in tumoral cells has been also considered as results of the hypoxic environment surroundings tumors but not an intrinsic characteristic of tumour cells (4). Further, different metabolic characteristics of cells have been shown in tumors depending on the localization of cells inside the tumor. However, it has been also indicated that tumor promotion requires the repression of biogenesis and functional activity of mitochondria and a higher glycolytic phenotype (5). Then, it seems that mitochondria-related metabolic changes can be importantly involved in tumor evolution and, for this reason, mitochondria have been recently considered as main targets for anti-cancer drugs since mitochondria work in cancer cells in a different way than in normal cells (6). Treatments targeting on mitochondrial activity are based on the fact that mitochondrial activity is not completely blocked in tumoral cells but the ratio of fermentation to respiration is higher in these cells in comparison with normal cells. In fact, the metabolic state of the cells, defined by the bioenergetic mitochondrial index relative to the cellular glycolytic potential, is being considered a a signature of carcinogenesis and probably a prognostic value for many carcinomas (7). Probably, cancer cells maintain a minimum threshold of respiration and they are quite vulnerable to further reduction induced by different compounds while non-transformed cells are able to survive since they maintain a high oxidative metabolism. For this reason, the use of natural or new bioactive compounds able to modify tumor cell metabolism affecting mitochondrial-dependent oxidative metabolism is a promising strategy for the design of new cancer therapies. In this field, polyphenols and, more specifically, resveratrol and its derivatives, which are able to affect mitochondrial biogenesis and induce oxidative metabolism, could be used in future therapies against cancer (8).

Ribosomal biogenesis in cancer progression Cells respond to changes in the environment by modifying their whole physiology. In this process, ribosome activity is significantly involved. Protein synthesis represents one of the more energetic processes in the cells. The activity of ribosomes must be balanced with the ratio of transcription at the nucleus. Abnormal ratios between mRNA and ribosomes must alter protein synthesis disrupting then the flux of information between DNA and protein synthesis. Malfunction of this flux of information by ribosomal defects can cause aberrant effects in growth. In fact, excessive ribosome production and translational activity has been found in tumor cells and hematologic disorders (See Teng Teng and George Thomas in this book) (9).

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This enhanced translational activity found in tumoral cells may give them a competitive edge by increasing the capacity to produce proteins to support the higher growth ratio and to increase survival capacity. It seems also that an enhanced translational capacity is linked to a higher capacity to proliferation since one of the most known proto-oncogenes, c-Myc, is a master regulator of ribosome biogenesis (10). The high ratio of protein synthesis needed in tumoral cells make us to suppose that affecting ribosome synthesis in tumoral cells will stop cell cycle progression by activating a checkpoint. In fact, induction of cell cycle inhibitors such as p53 and its targets as p21, BAX and MDM2 are induced in riboproteinS6 conditioned depletion in liver cells (12). Translation control seems to be regulated by cell metabolism through nutrient sensors such as the mammalian target of rapamycin (mTOR) in cancer cells (13). In prostate cancer cells, the mTOR-depending signaling has revealed that this sensor controls the expression of many cell proliferation, metabolism and invasion related genes. It seems that the control of mTOR by an ATP site inhibitor such as INK128, can reprogram gene expression reducing the metastatic capacity or prostate cancer cells (14). Then, it seems clear that there is a link between metabolic control, translational capacity at the ribosomal machinery and metastatic capacity in cancer cells.

Metastasis One of the most important characteristics of cancer cells is their capacity to leave primary tumors and disseminate in the organism through metastasis. The importance of metastasis in cancer progression is clear since most of the cancer patients die due to untreatable disseminated metastasis. Metastasis is a very complex process that has been extensively studied at molecular, cellular and tissue levels in the different cancer types (see Amparo Cano chapter in this book). However, to date, scientific literature is full of different studies focused on the heterogeneous factors involved in metastasis but not a clear integrative picture has been found yet probably due to the complexity and diversity of tumor cells. As the specific metabolic profile of tumor cells seems to be an important factor to be taken into consideration in tumor treatment, we consider that changes in metabolism can be also related to metastasis in tumors. In fact, very recently, tumor growth and metastasis promotion by senescent fibroblasts has been suggested. It seems that the increase of autoand mitophagy in tumor surrounding fibroblasts stimulate the release of high effective mitochondrial fuel such as ketone bodies and L-lactate to the tumor environment. This release causes mitochondrial metabolism changes in tumor cells inducing higher cell growth and increasing their metastasis capacity (15). Further, a compartmental model for tumor metabolism has been recently proposed. In this model, glycolytic stromal cells produce mitochondrial fuels such as L-lactate and ketone bodies that are then transferred to oxidative cancer cells (16). Furthermore, it seems that stromal cells such as cancer-associated fibroblasts, adipocytes and

inflammatory cells are more glycolytic and fuel tumor

progression and metastatic dissemination by feeding mitochondrial metabolism in cancer

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cells (17). This can be the cause by which targeting mitochondrial bioenergetics seems to be a potent stimulus to induce caspase-independent cell death in ovarian cancer stem cells (18). These complex metabolic relationships between cancer cell proliferation and metastatic capacity must be studied in deep and clarified. It seem clear that the glycolitic characteristics described by the Warburg effect cannot be ascribed to the whole population of cells in a tumor since there is a hierarchy in cell types and metabolism that is related to their specific growth and metastatic capacity. Therefore, it has been recently suggested that the Warburg effect must be revisited by using proteomic and other omic approaches to deep in our knowledge of cancer cell metabolism and of cancer cell biology in general (19). This knowledge will help us to focus treatments on the specific characteristics of CSCs, responsible of the progression of tumor and metastasis.

Conclusion Cancer is a common disease but that shows a high heterogeneity due to the different source of cancer cells and the different evolution and levels of adaptation of these cells. In fact, in a very recent paper, the study of driver mutations in 100 breast cancer tumors have highlighted the substantial genetic diversity underlying this common disease (20). It seems clear that cancer cells develop a high capacity of adaptation to new environment and that they activate mechanisms to modulate their own but also surrounding metabolism to increase their capacity to survive, proliferate and disseminate. Even more, in a primary tumor we can find types of cancer cells that seem to cooperate between themselves to assure the survival of the whole population. In this complex system, the study of the metabolism of cancer cells will highlight the common targets to develop new therapies specially focused on the induction of cell death of those cells involved in relapse and metastasis such as CSCs. The development of new and more individualized diagnostic tools to clarify the specific characteristics of tumor cells in each patient will help us to develop therapies adapted to the individual situation of each patient. Probably, the discovery of new and promising targets for therapy will help us to design more accurate and effective treatments for each type and/or evolution state of cancer.

References 1. La Porta CA. Thoughts about cancer stem cells in solid tumors. 2012. Workd J Stem Cells. 4(3):17-20. 2. Siendones E, Barbarroja N, Torres LA, BuendĂa P, Velasco F, Dorado G Torres A, LĂłpezPedrera C. Inhibition of Flt3-activating mutations does not prevent constitutive activation of ERK/Akt/STAT pathways in some AML cells: a possible cause for the limited effectiveness of monotherapy with small-molecule inhibitors. 2007. Hematol Oncol. 25(1):30-37. 3. Annibaldi A, Widmann C. Glucose metabolism in cancer cells. 2010. Curr Op Clin Nutr Metab Care. 13(4):466-470.

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4. Zu XL, Guppy M. Cancer metabolism: facts, fantasy, and fiction. 2004. Biochem Biophys Res Commun. 313:459-65. 5. Sanchez-Arago M, Chamorro M, Cuezva JM. Selection of cancer cells with repressed mitochondria triggers colon cancer progression. 2010. Carcinogenesis 31:567-576. 6. Ralph SJ, Rodriguez-Enriquez S, Neuzil J, Moreno-Sanchez R. Bioenergetic pathways in tumor mitochondria as targets for cancer therapy and the importance of the ROS-induced apoptotic trigger. 2010. Mol Aspects Med. 31:29-59. 7. Cuezva JM, Krajewska M, de Heredia ML, et al. The bioenergetic signature of cancer: a marker of tumor progression. 2002. Cancer Res. 62:6674-6681. 8. L贸pez-Lluch G, Santa Cruz-Calvo S, Navas P. Resveratrol in cancer: Cellular and mitocondrial consequences of proton transport inhibition. 2012. Curr Pharm Des. 18: 13381344. 9. Shenoy N, Kessel R, Bhagat T, Bhattacharya S, Yu Y, McMahon C, Verma A. Alterations in the ribosomal machinery in cancer and hematologic disorders. 2012. J Hematol Oncol. 5(1):32. 10. Kenneth NS, Ramsbottom BA, Gomez-Roman N, Marshall L, Cole PA, White RJ. TRRAP and GCN5 are used by c-Myc to activate RNA polymerase III transcription. 2007. Proc Natl Acad Sci U S A. 104:14917-14922. 12. Volarevic S., Stewart MJ, Ledermann B, Zilberman F, Terracciano L, Montini E, rompe M, Kozma SC, Thomas G. Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6. 2000. Science. 288:2045-2047. 13. Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM. A unifying model for mTORC1-mediated regulation of mRNA translation. 2012. Nature. 485(7396):109113. 14. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, Shi EY, Stumpf CR, Christensen C, Bonham MJ, Wang S, Ren P, Martin M, Jessen K, Feldman ME, Weissman JS, Shokat KM, Rommel C, Ruggero D. The translational landscape of mTOR signalling steers cancer initiation and metastasis. 2012. Nature. 485(7396):55-61. 15. Capparelli C, Guido C, Whitaker-Menezes D, Bonuccelli G, Balliet R, Pestell TG, Goldberg AF, Pestell RG, Howell A, Sneddon S, Birbe R, Tsirigos A, Martinez-Outschoorn U, Sotgia F, Lisanti MP. Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production. 2012. Cell Cycle. 11(12):2285-302. 16. Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, Casimiro MC, Wang C, Fortina P, Addya S, Pestell RG, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. 2009. Cell Cycle. 8(23):3984-4001. 17. Sotgia F, Whitaker-Menezes D, Martinez-Outschoorn UE, Flomenberg N, Birbe RC, Witkiewicz AK, Howell A, Philp NJ, Pestell RG, Lisanti MP. Mitochondrial metabolism in cancer metastasis: visualizing tumor cell mitochondria and the "reverse Warburg effect" in positive lymph node tissue. 2012. Cell Cycle. 11(7):1445-1454. 18. Alvero AB, Montagna MK, Holmberg JC, Craveiro V, Brown D, Mor G. Targeting the mitochondria activates two independent cell death pathways in ovarian cancer stem cells. 2011. Mol Cancer Ther. 10(8):1385-1393. 19. Scatena R, Bottoni P, Pontoglio A, Giardina B. Revisiting the Warburg effect in cancer cells with proteomics. The emergence of new approaches to diagnosis, prognosis and therapy. 2010. Proteomics Clin Appl. 4(2):143-158. 20. Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, Wedge DC, Nik-Zainal S, Martin S, Varela I, Bignell GR, Yates LR, Papaemmanuil E, Beare D, Butler A, Cheverton A, Gamble J, Hinton J, Jia M, Jayakumar A, Jones D, Latimer C, Lau KW, McLaren S, McBride

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DJ, Menzies A, Mudie L, Raine K, Rad R, Chapman MS, Teague J, Easton D, Langerød A; Oslo Breast Cancer Consortium (OSBREAC), Lee MT, Shen CY, Tee BT, Huimin BW, Broeks A, Vargas AC, Turashvili G, Martens J, Fatima A, Miron P, Chin SF, Thomas G, Boyault S, Mariani O, Lakhani SR, van de Vijver M, van 't Veer L, Foekens J, Desmedt C, Sotiriou C, Tutt A, Caldas C, Reis-Filho JS, Aparicio SA, Salomon AV, Børresen-Dale AL, Richardson AL, Campbell PJ, Futreal PA, Stratton MR. The landscape of cancer genes and mutational processes in breast cancer. 2012. Nature. 486(7403):400-404.

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Ribosome biogenesis, cell cycle progression and tumorigenesis: Key points in ribosomopathies and future treatment of cancer Teng Teng1,2 and George Thomas2,3 1. Department of Cancer and Cell Biology, 2. Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, University of Cincinnati, OH 45220, USA 3. Catalan Institute of Oncology, Bellvitge Biomedical Research Institute, IDIBELL, Gran Via de l'Hospitalet, 199, 08908 Hospitalet de Llobregat, Barcelona, Spain

Overview of ribosome biogenesis and associated pathologies The ribosome is an essential and complex intracellular organelle that translates mRNA into protein. It is estimated that a proliferating eukaryotic cell expends up to 80% of its energy on nascent ribosome biogenesis [1], which requires the synthesis, modification, assembly and transport of large and small ribosomal subunits, composed of

ribosome

RNAs (rRNAs) and ribosome proteins (RPs). This well-coordinated process requires the utilization of all three RNA polymerases to synthesize rRNAs and RPs [2]. RNA polymerase I transcribes the polycistronic 47S rRNA in the eukaryotic nucleolus, a sub-compartment of the nucleus which acts as the site of ribosome biogenesis [3]. The 47S precursor rRNA is chemically modified and processed by numerous small nucleolus RNAs (snoRNAs) and protein co-factors such as endo- and exo-nucleases, into the smaller 18S, 28S and 5.8S rRNAs which serve as both a ribosomal scaffold and the catalytic core [3,4]. Different from the other rRNAs, 5S rRNA is produced by RNA polymerase III in the nucleoplasm, which is also the site of the synthesis of the approximate 80 RP mRNAs, which are transcribed by RNA polymerase II. These RPs are then transported into the cytoplasm where they are translated into mature proteins, before re-entering the nucleolus with the mature 5S rRNA and being assembled into nascent ribosomes [2,5]. Recent studies strongly suggest that RPs are more than just structural components of the ribosome, but also participate in prerRNA processing and pre-ribosome assembly within the nucleolus, nuclear transport of pre40S and pre-60S subunits, and in the final maturation steps within the cytoplasm [6-8]. A change in the ratio of ribosomes to mRNAs can alter the pattern of protein synthesis within the cell [9,10], eventually leading to a rupture between the genetic program and protein expression, which may disrupt cellular homeostasis and cause aberrant effects in growth. Both the over-production of ribosomes and defects in ribosome biogenesis have been linked to pathological conditions. Excessive ribosome production and enhanced translational capacity have been associated with the increased proliferation rate of cancer cells [11,12]. Since the 1970s, pathologists have used the increased size and disorganized

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morphology of the nucleoli as an indicator of cancer malignancy [13]. On the other hand, the importance of intact ribosome biogenesis is underscored by two hematopoietic disorders which are linked to mutations or allelic deletion of ribosomal protein genes: Diamond Blackfan Anemia (DBA) [14] and 5q- syndrome[15]. DBA is an inherited congenital bonemarrow-failure syndrome that is characterized by macrocytic anemia and insufficiency of erythroid precursors in an otherwise normal cellular bone marrow. Besides the defects in red blood cell maturation, about 40% of patients present with growth retardation and congenital malformations, including craniofacial, thumb, limb and heart defects [14]. Since the first report in 1999 that ribosomal protein S19 (RPS19) was mutated in DBA, heterozygous mutations in other ribosomal protein genes, including RPS24, RPL35a, RPS17, RPL5, RPL11, RPS7, RPS10 and RPS26, have been identified in multiple unrelated families, accounting for ~50% of all DBA patients[16-22]. More recently, deletions at several ribosomal protein gene loci have been identified through a genome-wide SNP array in 17% of the patients without known ribosome protein gene mutations from previous sequencing analyses [23]. 5q- syndrome is a distinct subtype of myelodysplastic syndrome (MDS), which is characterized by isolated interstitial deletion surrounding a common deleted region (CDR) on the long arm of chromosome 5. 5q- syndrome patients display refractory macrocytic anemia, hypolobulated micromegakaryocytes, with often-elevated platelets count[24]. Of ~40 genes deleted in the CDR, haploinsufficiency of RPS14 is responsible for the macrocytic anemia in 5q- syndrome, further strengthening the link between impaired ribosome biogenesis and erythroid hypoplasia [15].

Cell proliferation, but not cell growth is blocked by RPS6 deletion Given the pivotal role of maintaining homeostasis of ribosome production, it was originally hypothesized that the cell may have evolved a surveillance system to sense the state of ribosome biogenesis. To examine this hypothesis, Volarevic et al. developed an inducible conditional knockout mouse model of RPS6, an essential component of the 40S ribosomal subunit [25]. In this model, the mouse RPS6 gene loci were flanked by loxP sites. Homozygous floxed S6 (fS6) mice were crossed with transgenic mice expressing the CRE recombinase under the control of an Mx promoter, which can be induced upon administration of IFN-Îą [26]. Southern blot analysis showed a complete deletion of RPS6 in the mouse liver upon intraperitoneal injection of IFN-Îą compared to partial excision in other tissues, providing a unique model to specifically examine the effect of RPS6 depletion in hepatocytes [25]. fS6/CRE- and fS6/CRE+ animals were first challenged with a fasting and refeeding regime to examine the effect of RPS6 deletion on hepatocyte growth. During fasting, a reduction of ~50% of liver mass occurs due to the degradation of cellular proteins, ribosomes, and organelles through autophagy, which maintains essential cellular processes during nutrient deprivation [27,28]. Upon refeeding, ribosome biogenesis becomes activated while degradation is inhibited, such that the total ribosomal content is restored within a day,

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leading to re-growth of the liver to its original size without changing cell number [29]. Consistent with previous literature, the average liver weight of IFN-Îą treated fS6/CRE- and fS6/CRE+ mice after a two day fast, decreased by greater than 40%. Unexpectedly, recovery of liver mass and protein content in both genotypes occurred at the same rate despite the absence of 40S subunit production and decreased mean polysome size in fS6/CRE+ mice upon on refeeding compared to fS6/CRE- mice.

P pulse-labeling of rRNA from livers of

both genotypes under the same regime also revealed a lack of newly synthesized 18S rRNA in fS6/CRE+ animals and an accumulation of the 34S rRNA precursor after refeeding [25]. Thus, although deletion of RPS6 effectively impaired 40S ribosome biogenesis upon refeeding, the remaining ribosomes were sufficient to support hepatocyte growth. A different paradigm to challenge hepatocyte ribosome biogenesis is partial hepatectomy (PH), where removal of ~70% of the liver stimulates the remaining hepatocytes to re-enter cell cycle and proliferate through 1 to 1.5 rounds to regenerate the lost liver mass [30]. Livers of fS6/CRE- mice completely recovered lost mass 10 days post PH, whereas livers of fS6/CRE+ mice failed to regenerate in the same time frame. Consistent with a requirement for cell proliferation during liver regeneration, hepatocytes from fS6/CRE+ mice post PH were devoid of mitotic figures and resembled the quiescent hepatocytes in livers before PH. Neither 3H-thymidine nor BrdU incorporation was detected in fS6/CRE+ livers 60 hours post-PH, whereas two peaks of 3H-thymidine incorporation were readily observed in fS6/CRE- mice post-PH, indicating the remaining hepatocytes in fS6/CRE+ mice failed to enter S phase in the absence of 40S subunit biogenesis [25]. These elegant studies by Volarevic et al. demonstrated that the proper execution of ribosome biogenesis is essential for cell proliferation, but is dispensable for cell growth.

RPS6 depletion induces dependent p53 induction

RPL11-translation

Oscillation of the levels of various cyclins and their associated cyclin-dependent-kinase (CDK) activities provides the biochemical switch to regulate the order and timing of cell cycle progression [31]. Molecular analysis of cell cycle regulators in fS6/CRE- and fS6/CRE+ mouse livers post-PH revealed normal accumulation of cyclin D1 in both genotypes but a lack of cyclin E synthesis in fS6/CRE+ mice [25]. Expression of cyclin E and activation of the cyclin E/CDK2 complex is essential for cell cycle progression through the G1/S checkpoint and inhibition of this complex has been demonstrated in response to radiation induced p53dependent cell cycle arrest [32,33]. Thus, it was plausible that impairment of ribosome biogenesis elicits a cell cycle checkpoint, in agreement with the hypothesis that cells possess a surveillance mechanism to sense the state of ribosome production. To test this possibility, Fumagalli et al. examined the gene expression profile of livers from fS6/CRE+ and fS6/CRE- mice post PH using microarray analysis. Consistent with a defect in G1/S transition, a number of S-phase promoting factors such as MCM3, MCM5 and CDC6 failed to accumulate in fS6/CRE+ mice. In parallel, p21, BAX and MDM2, cell cycle inhibitors and established p53 targets, were up regulated in RPS6 deleted livers. In

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agreement with this finding, ELISA analysis revealed a significant increase in p53 levels in livers of fS6/CRE+ mice compared to fS6/CRE- mice post PH. More strikingly, the expression of p53 targets was induced even before PH, suggesting that stress induced by impaired ribosome biogenesis was sufficient to induce p53 [34]. Consistent with the in vivo data, depletion of RPS6 in cultured human A549 cells with siRNA also led to a robust induction of p53 and p21. The p53 response was not specific to loss of RPS6, as depletion of RPS23, another essential component of the 40S subunit, or of RPL7a, a 60S subunit ribosomal protein, also led to increased expression of p53 and p21, indicating this phenomenon was a general response to inhibition of ribosome biogenesis. Incorporation of BrdU, which was reduced by 60% upon RPS6 depletion, was reversed by co-depletion of RPS6 and p53 [34]. Previous studies have proposed that inhibition of rRNA synthesis disrupts the structure of the nucleolus, releasing RPL11, an essential RP of the 60S subunit, into the nucleoplasm where it interacts with MDM2, inhibiting its E3-ligase activity towards p53 [35,36]. However, stabilization of p53 in cells depleted of RPS6 does not depend on nucleolar disruption. Immunofluorescent staining of two nucleolus markers, NHP2 and nucleolin, and the 60S subunit protein RPL7a confirmed that the nucleolar structure was intact in cells depleted of RPS6 [34], suggesting that the p53 induction upon impaired 40S biogenesis does not require nucleolar disruption. Interestingly, co-depletion of RPL11 together with RPS6 was able to abolish the induction of p53 and rescue the S-phase entry defect observed in cells depleted of RPS6 alone. Moreover, co-depletion of RPL11 with either RPS23 or RPL7a suppressed the accumulation of p53 and p21 induced by RPS23 or RPL7a depletion alone [34]. In support of the essential role of RPL11 in the p53 response, significantly more RPL11 co-immunoprecipitated with MDM2 upon depletion of RPS6 or RPL7a compared to control non-silencing siRNA treated cells. Thus, cells are able to sense impairment of ribosome biogenesis and invoke a p53 response in an RPL11dependent manner. If RPL11 is required for the p53 response, and impaired ribosome biogenesis occurs in the absence of nucleolar disruption, by what mechanism is free RPL11 available to bind to MDM2? Cells depleted of an RP of the large subunit, such as RPL7a, have impaired 60S ribosomal biogenesis; RPL11 which is not incorporated into the pre-60S subunit is likely to accumulate as ribosome-free RPL11. However, 60S subunit production is unperturbed upon depletion of a RP of the small subunit, such as RPS6, so that simple accumulation of ribosome-free RPL11 is unlikely. This suggests that a mechanism to increase levels of RPL11 must exist when 40S ribosome biogenesis is impaired. Polysome analysis of RPL11 transcripts revealed that more RPL11 mRNA transcripts were recruited to actively translating polysomes in RPS6 depleted cells, compared to control cells. Moreover, western blot analysis also showed a significant increase in total RPL11 protein level upon RPS6 depletion. The translational up-regulation of RPL11 is a general response to defective 40S biogenesis, as RPL11 transcripts shifted to heavier polysome in cells depleted of a second RP of the small subunit, RPS23, but not in cells depleted of the large subunit RP, RPL7a [34]. Interestingly, other RP transcripts including RPS8, RPS16 and RPL26 were also translationally upregulated upon RPS6 depletion [34]. Translation of RP mRNAs is

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negatively regulated by a sequence motif at the beginning of their 5' untranslated regions (5’UTR) called the 5'-terminal oligopyrimidine (5'-TOP) motif, which consists of a stretch of uninterrupted pyrimidine bases [37]. Exchange of pyrimidine bases with purines renders translation of 5’-TOP messages constitutively active [38,39]. Using reporter constructs under the control of either the first 29 nucleotides (nt) of the 5’-UTR of RPS16 (wt reporter) or a mutant where five of the pyrimindines were replaced with purines (cm5 reporter), Fumagalli

et al. demonstrated that depletion of RPS6 induced a shift of wt reporters onto the polysomes whereas most cm5 reporter transcripts were already loaded onto the polysomes in non-silencing siRNA transfected cells. Therefore, inhibition of 40S ribosome biogenesis led to translational up-regulation of 5’-TOP messages, allowing production of excess RPL11 to induce a p53 response through inhibition of MDM2[34].

p53 in DBA and 5q- syndrome? Studies by Volarevic et al. and Fumagalli et al. unraveled a p53-dependent surveillance mechanism to sense insults to ribosome biogenesis, which may provide molecular insights for ribosomopathies such as DBA and 5q- syndrome[25,34]. The clear question is whether induction of p53 in DBA and 5q- syndrome contributes to the pathogenesis of erythroid hypoplasia? The first supporting evidence of a role for p53 came in zebrafish models, where depletion of RPS19, the most frequently mutated ribosome protein in DBA patients, led to developmental defects and a block in erythropoiesis associated with increased expression of p53. Moreover, co-depletion of p53 and RPS19 alleviated the phenotype[40]. It was anticipated that heterozygous deletion of RPS19 in mice would phenotypically resemble DBA. Unexpectedly, mice heterozygous for the RPS19 allele were hematologically normal, potentially due to transcriptional compensation; homozygous RPS19 knockout mice were embryonically lethal [41,42]. In contrast, a mouse model with a missense mutation in one RPS19 allele, presented with a mildly reduced (~8%) red blood cell (RBC) count, slightly increased (~2%) mean corpuscular value (MCV), and dark skin, phenotypes which were rescued in a p53 negative background[43]. Other efforts to create an RPS19 deficient mouse model for DBA include a transgenic mouse that conditionally expresses mutant human RPS19, and a transgenic RPS19 doxycycline-inducible RNA interference (RNAi) model. Both models are characterized by significant anemia in peripheral blood and impaired proliferation of hematopoietic progenitors in bone marrow [44,45]. In the latter model, p53 target genes were upregulated in hematopoietic progenitors, and complete inactivation of p53 in the transplanted bone marrow was able to reverse these defects, although the extent of rescue depended on the level of RPS19 depletion[45]. A mouse model of 5q- syndrome with conditional deletion of RPS14 and other genes within the CDR, was also characterized by severe macrocytic anemia, impaired development of bone marrow progenitor cells, and elevated p53 levels in hematopoietic progenitors. Importantly, the hematologic defects were completely rescued in a p53 null background [46]. Similar results were observed in primary human hematopoietic progenitor cells depleted of either RPS19 or RPS14 by shRNA; p53 was induced specifically in erythroid progenitor cells, contributing to cell cycle arrest and increased apoptosis. Treatment of these cells with the p53 inhibitor Pifithrin-α, prevented

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induction of p21 and rescued erythroipoiesis in a dose-dependent manner. Finally, analysis of bone marrow biopsies from DBA and 5q- syndrome patients showed increased nuclear p53 staining in erythroid progenitor cells[47]. Although there is increasing evidence of a direct link between p53 and the pathogenesis of DBA and 5q- syndrome, questions remain as to whether other factors contribute to the erythroid dysplasia. Recently, mutations in RPL11 were identified in DBA patients. Earlier studies suggest that the induction of p53 by defective ribosome biogenesis occurs in an RPL11-dependent manner, raising questions as to whether the mechanism of bone marrow failure might be different in RPL11-mutant patients. Moreover, RPL5, the second most mutated gene in DBA patients [19,21,48], is another ribosome protein which interacts with and inhibits MDM2, potentially by functioning in a pre-existing complex with RPL11. Is p53 stabilized in the absence of positive regulators like RPL11 and RPL5 in these patients? And what is the relative contribution of RPL11 and RPL5 to the inhibition of MDM2? Do they function separately or together as a complex? Undoubtedly, these and other questions will be the focus of future studies. Interestingly, a recent study in a p53-deficient mouse erythroblast line demonstrated that proliferation and differentiation of erythroblasts were impaired upon RpS19 or RpL11 depletion in the absence of p53. Furthermore, they identified candidate mRNAs messages which were under-translated upon ribosome stress, which may contribute to impaired erythroipoiesis in DBA patients[49]. Thus, in addition to p53â&#x20AC;&#x2122;s role, the general reduction in overall translational capacity caused by mutation in RPs may also play a pivotal role in the ribosomopathies.

Too many ribosomes or too few ribosomes: all roads lead to cancer? Enhanced translational capacity may give cells a competitive edge by increasing levels of proteins needed for proliferation or survival. For example, it has been shown that forced expression of Brf1, an RNA polymerase III specific transcriptional factor, specifically increased cellular tRNA and 5S rRNA levels, leading to enhanced protein synthesis and increased proliferation rate. Moreover, Brf1 over-expressing MEFs showed a transformed morphology which was sufficient to sustain anchorage independent growth in vitro and cause tumor formation in vivo . A similar phenotype was also observed when tRNAiMet, the tRNA responsible for polypeptide chain initiation, was mildly over-expressed [50]. Another example linking enhanced translational capacity to proliferation can be found in cells expressing the proto-oncogene c-Myc. c-Myc is a master regulator of ribosome biogenesis: it regulates transcription of 45S rRNA by RNA polymerase I, and activates transcription of 5S rRNA by RNA polymerase III [51,52]. Moreover, all ribosome protein genes are direct targets of c-Myc [53-56]. In Drosophila melanogaster, image disc cells with increased dMyc expression had a competitive proliferative advantage over surrounding cells with less dMyc expression. This competitive edge was lost when high-expressing dMyc cells were placed in an RPL19 heterozygous background, supporting the idea that the advantage

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was dependent on translational capacity [57]. In higher eukaryotes, mice with increased levels of c-Myc in B-lymphocytes (Eµ-Myc model), developed aggressive lymphomas within 200 days. The Eµ-Myc B-lymphocytes had increased protein synthesis and increased cell size compared to lymphocytes from wild-type mice. Crossing the Eµ-Myc mice to an RPL24 heterozygous background normalized the translational rate and cell size, and delayed the onset of lymphoma [58]. Interestingly, Eµ-Myc mice which were crossed to mice homozygous for a mutated MDM2 which cannot bind RPL11 and RPL5, developed a much more aggressive lymphoma, and none of the lymphoma-bearing mice survived more than 11 weeks [59]. Taken together, increased ribosome biogenesis plays an important role in EµMyc driven tumor initiation. Potentially, the imbalance between rRNA and RP production, allows an accumulation of ribosome-free RPL11 and RPL5, which elicits an early p53dependent tumorigenesis barrier. Any breach of the p53 ‘barrier’ would potentially result in enhanced tumorigenicity, similar to what has been described as the dual role of DNA overreplication in RAS-driven tumor formation[60]. Surprisingly, decreased ribosome biogenesis has also been linked to tumorigenesis. Both DBA patients and 5q- syndrome patients have an increased propensity to develop cancer, particularly acute myeloid leukemia (AML) [61]. Consistent with this, P-element insertions in the Drosophila dRPS6 gene caused over-proliferation in selective tissues and formation of melanotic tumors [62,63]. In zebrafish, heterozygous mutation of 17 RPs caused malignant peripheral nerve sheath tumors (MPNSTs), a tumor type rarely observed in laboratory zebrafish strains [64,65]. Although it is not obvious how cells with impaired ribosome biogenesis and a p53-dependent checkpoint would become malignant, further analysis revealed that cells derived from the RP-mutated MPNSTs did not synthesize p53 protein [65]. Therefore, one possible explanation could be that reduced translational capacity and stabilized p53 impose a selection pressure on cells with impaired ribosome biogenesis, until mutated clones are eventually selected with the potential to bypass these pressures (Fig. 1). In conclusion, recent reports have expanded our understandings of ribosome biogenesis as a tightly monitored and regulated process. Both impaired and uncontrolled ribosome biogenesis are associated with diseases ranging from erythroid hypoplasia to cancer progression. Further studies to unravel the molecular pathways underlying these pathologies will greatly benefit our understanding of the link between ribosome biogenesis, cell cycle regulation and cancer progression, and will aid in the development of new therapeutic strategies.

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51. Poortinga, G., K.M. Hannan, H. Snelling, C.R. Walkley, A. Jenkins, K. Sharkey, M. Wall, Y. Brandenburger, M. Palatsides, R.B. Pearson, G.A. McArthur, and R.D. Hannan. 2004. MAD1 and c-MYC regulate UBF and rDNA transcription during granulocyte differentiation. EMBO J. 23:3325-35. 52. Kenneth, N.S., B.A. Ramsbottom, N. Gomez-Roman, L. Marshall, P.A. Cole, and R.J. White. 2007. TRRAP and GCN5 are used by c-Myc to activate RNA polymerase III transcription. Proc Natl Acad Sci U S A. 104:14917-22. 53. Coller, H.A., C. Grandori, P. Tamayo, T. Colbert, E.S. Lander, R.N. Eisenman, and T.R. Golub. 2000. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci U S A. 97:3260-5. 54. Guo, Q.M., R.L. Malek, S. Kim, C. Chiao, M. He, M. Ruffy, K. Sanka, N.H. Lee, C.V. Dang, and E.T. Liu. 2000. Identification of c-myc responsive genes using rat cDNA microarray. Cancer Res. 60:5922-8. 55. Boon, K., H.N. Caron, R. van Asperen, L. Valentijn, M.C. Hermus, P. van Sluis, I. Roobeek, I. Weis, P.A. Voute, M. Schwab, and R. Versteeg. 2001. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO J. 20:1383-93. 56. Menssen, A., and H. Hermeking. 2002. Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes. Proc Natl Acad Sci U S A. 99:6274-9. 57. Moreno, E., and K. Basler. 2004. dMyc transforms cells into super-competitors. Cell. 117:117-29. 58. Barna, M., A. Pusic, O. Zollo, M. Costa, N. Kondrashov, E. Rego, P.H. Rao, and D. Ruggero. 2008. Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency. Nature. 456:971-5. 59. Macias, E., A. Jin, C. Deisenroth, K. Bhat, H. Mao, M.S. Lindstrom, and Y. Zhang. 2010. An ARF-independent c-MYC-activated tumor suppression pathway mediated by ribosomal protein-Mdm2 Interaction. Cancer Cell. 18:231-43. 60. Di Micco, R., M. Fumagalli, A. Cicalese, S. Piccinin, P. Gasparini, C. Luise, C. Schurra, M. Garre, P.G. Nuciforo, A. Bensimon, R. Maestro, P.G. Pelicci, and F. d'Adda di Fagagna. 2006. Oncogene-induced senescence is a DNA damage response triggered by DNA hyperreplication. Nature. 444:638-42. 61. Narla, A., and B.L. Ebert. 2010. Ribosomopathies: human disorders of ribosome dysfunction. Blood. 115:3196-205. 62. Watson, K.L., K.D. Konrad, D.F. Woods, and P.J. Bryant. 1992. Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system. Proc Natl Acad Sci U S A . 89:11302-6. 63. Stewart, M.J., and R. Denell. 1993. Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth. Mol Cell Biol. 13:2524-35. 64. Amsterdam, A., K.C. Sadler, K. Lai, S. Farrington, R.T. Bronson, J.A. Lees, and N. Hopkins. 2004. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol. 2:E139. 65. MacInnes, A.W., A. Amsterdam, C.A. Whittaker, N. Hopkins, and J.A. Lees. 2008. Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene mutations. Proc Natl Acad Sci U S A. 105:10408-13.

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Epithelial plasticity and metastasis: New regulatory mechanisms Amparo Cano and Gema Moreno-Bueno Departamento de Bioquímica, UAM. Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM. Instituto de Investigación Sanitaria IdiPAZ. c/Arzobispo Morcillo, 2. 28029 Madrid. Spain

Introduction Metastasis is the major live-threatening consequence of cancer because most of the cancer patient deaths are due to untreatable disseminated metastasis rather than to the primary tumor. Generation of metastasis is an extraordinary complex biological process at molecular, cellular and tissue levels in which not only genetic and epigenetic alterations/adaptations of tumor cells are involved but also complex tumor-stroma interactions are required for the success of the metastatic process (1, 2). For solid tumors and particularly in carcinomas (epithelial-derived tumors, representing at least 85% of all human tumors) the metastatic cascade involves several sequential steps including: invasion of adjacent tissues by tumor cells, intravasation, dissemination through the blood stream, arrest and extravasation at the parenchyma of the secondary organ and finally colonization to generate macrometastasis (1, 3, 4). The complexity of the metastatic process has hindered the knowledge of the mechanisms involved, but significant progress has been obtained in the last decade on the cellular and molecular events underlying some of the steps of the metastatic cascade, in particular on invasion. Local invasion of tumor cells requires dissociation of cell-cell interactions (mainly through the functional loss of Ecadherin), remodeling of the extracellular matrix and acquisition of migration. These coordinated changes frequently result in profound alterations of the cellular phenotype reminiscent of the process of Epithelial-Mesenchymal Transition (EMT), firstly described in embryos and found to be essential for embryonic development (reviewed in ref. 5). The EMT process is, actually, considered as a key event for metastasis, being involved in invasion, intravasation and extravasation (1, 5, 6) and probably representing a particular subtype of EMT, called type 3 by some authors, that may exhibit some distinct properties from developmental EMT (7). One important characteristic of tumoral EMT, shared with embryonic EMT, is its transient nature being a dynamic event that seems to occur only in focal invasive areas of carcinomas. Indeed, the reverse Mesenchymal-Epithelial Transition (MET) process has been proposed to be required for the generation of macrometastasis, and perhaps for other steps of the metastatic cascade (5, 8-10). These observations lead support to the concept of epithelial plasticity as a more general definition of the cellular changes that carcinoma cells may experience during their long journey from the primary tumor to their metastatic sites.

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Regulation of EMT and epithelial plasticity: a new role for lysyl oxidase-like 2 (LOXL2) During the last decade a great body of knowledge has been generated on the regulation of EMT since the original description of Snail1 as an E-cadherin repressor and EMT inducer, followed by the identification of additional E-cadherin repressors also acting as EMT inducers (reviewed in refs. 6, 8) presently described as EMT-TFs. A plethora of signaling pathways have also been identified as regulating EMT at transcriptional and posttranscriptional level, including many different growth factor signaling pathways, miRNAs (i.e, miR-200 family) or phosphorylation events (6, 8-13). Many of the described posttranslational regulatory mechanisms impinge on the protein stability of EMT-TFs, such as Snail1, Snail2 or Twist, while regulation by miRNAs have been so far mainly described for the ZEB and Snail1 factors (8, 10, 13-15). Among the post-translational regulation of Snail1 we described a few years ago lysyl oxidase-like 2 (LOXL2) as an interacting Snail1 partner that increases Snail1 protein stability and repressor activity and induces EMT (16). LOXL2 is a member of the lysyl oxidase gene family constituted by the prototypical LOX and four related members, lysyl oxidase-like 1 to 4 (LOXL1-4) (17). All members of the family contain a highly conserved C-terminal catalytic domain but diverge at the N-terminal region. They catalyze the oxidative deamination of peptidil-lysine groups in substrates that generate highly reactive aldehyde groups that initiate inter and intramolecular cross-linkages (17). While LOX and LOXL1 are required for extracellular matrix assembly and cardiovascular system homeostasis, intracellular functions of LOX and several LOXL members in tumorigenesis have been also recently reported (reviewed in refs. 18, 19).

Indeed, our

previous studies identified intracellular LOXL2 as a prognostic marker in larynx squamous cell carcinomas and associated to tumor progression in a model of mouse skin carcinogenesis (20). Interestingly, functional studies in squamous carcinoma cells showed that LOXL2 is a negative regulator of epidermal squamous differentiation while it promotes tumor growth through Snail1-dependent and Snail1-independent mechanisms (20). That previous study also showed increased LOXL2 overexpression associated to poor survival in N0 breast carcinomas in public datasets (20). These observations, together with other studies showing the involvement of LOXL2 in invasion of breast carcinoma cells (19, 21), supported a role of LOXL2 in breast tumors. Other studies have indeed shown an important role for extracellular LOXL2 in several tumor types (22). We have focused our recent studies on the role of LOXL2 in breast cancer.

LOXL2 is carcinomas

overexpressed

basal-like

breast

Breast carcinomas are a highly heterogeneous group of tumors containing different subclasses. In the last decade, molecular profiling has allowed the classification of different breast tumor types into at least five different subtypes: the luminal A and luminal B

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(characterized by the expression of luminal markers); the ErbB2/neu (overexpressing the Her2/neu oncogene); the normal-like (closely similar to the normal mammary gland); and the basal-like group (with expression of myoepithelial markers) (reviewed in ref. 23). Basal-like tumors are highly aggressive tumors with visceral metastasis and high recurrence; they are frequently high grade and triple negative tumors (ER-/PR-/Her2-) (24, 25) for which no specific targets have been yet defined. Basal-like breast tumors and a new subclass, defined as claudin-low tumors, have been recently characterized by us and others as prone to express a defined set of EMT markers being likely candidates to suffer EMT processes (26, 27). We designed an initial study to identify the molecular signature associated to highly aggressive breast tumors in a sample of 58 grade 3 infiltrative ductal carcinomas (IDC). Unsupervised hierarchical clustering sub-classified the samples into two clusters: cluster A that included the luminal and Her2neu tumors (n=43) and was defined as a non-basal breast carcinoma cluster, while cluster B contained tumors lacking expression of hormone receptors and the Her2neu oncogene (n=15), thus representing the basal-like cluster (28). A supervised analysis identified a set of 311 genes able to classify the non-basal and basallike tumors (Fig. 1A). The basal-like tumors showed very low levels of ESR, PR and

Her2neu transcripts as compared to the mean values obtained for the non-basal tumors, indicating that the basal-like cluster indeed contains the triple negative tumors (28). Interestingly, LOXL2 mRNA was found up-regulated in the basal-like cluster (Fig. 1A) and this was confirmed by qRT-PCR in a sample of 46 tumors (26 with non-basal and 18 with basal-like phenotype) in which LOXL2 expression was found up-regulated in basal tumors

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by a mean of 5-fold compared to non-basal tumors (Fig. 1B). To confirm these observations, the expression of LOXL2 was analyzed in a panel of breast carcinoma cell lines that include luminal (MCF7), ErbB2+ (BT474, SkBR3) and basal-like carcinoma (MDA-MB231, BT549) cells (Fig. 1C-D). Expression of LOXL2 was only detected in basal-like carcinoma cells as well as in the non-tumorigenic basal-like HBL100 cell line at the mRNA and protein levels (Fig. 1C, and data not shown). Interestingly, intracellular LOXL2 protein was detected in a punctate and perinuclear pattern in basal-like cells (Fig. 1D), in agreement with previous observations in other cell and tumor systems (16, 20). These data indicate that LOXL2 is overexpressed in basal-like tumors and derived cell lines.

LOXL2 expression is a negative regulator of the epithelial phenotype of basal-like carcinoma cells. To obtain insights into the role of LOXL2 in basal-like carcinoma cells, loss of function studies were performed by stable shRNA. LOXL2 silencing in MDA-MB-231 cells leads to a

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dramatic phenotypic change by which shLOXL2 cells acquire an epithelioid phenotype as compared to mesenchymal control shEGFP cells (Fig. 2A), suggesting that a reverse MET process has occurred. In agreement with the phenotypic reversion, LOXL2 silencing was associated to decreased motility and invasiveness of MDA-MB-231 cells. Similar results were obtained after LOXL2 silencing in additional basal-like cell lines (BT549 and HBL100) (28). Importantly, LOXL2 silencing also led to a significant decrease in the tumor growth potential (Fig. 2B) and to suppression of lung metastatic dissemination (Fig. 2C) of MDAMB-231 cells. Surprisingly, the MET-like phenotype induced by LOXL2 silencing occurs independently of E-cadherin and Snail1 regulation (Fig. 2D), suggesting that other cell adhesion mechanisms and EMT factors might be at play. Indeed, N-cadherin participates in the maintenance of cell-cell contacts of shLOXL2 cells (data not shown). On the other hand, mechanistic studies indicated that LOXL2 downmodulates the expression and organization of tight junctions (ZO1, claudin1) and cell polarity components (Lgl2) of basal-like carcinoma cells as well as participates in activation of the FAK pathway (28). Interestingly, LOXL2 downregulates claudin1 and Lgl2 at transcriptional level through mechanisms independent of Snail1 and of LOXL2 catalytic activity (28). These data uncover a new and unexpected molecular action of LOXL2 in the regulation of migration and epithelial cell polarity.

LOXL2 is overexpressed in metastatic basal-like carcinomas To investigate the biological relevance of LOXL2 in basal-like carcinoma cells, the expression of LOXL2 was analyzed in a cohort of grade 3 IDC (n=195) by immunohistochemistry. Intracellular LOXL2 was detected in two distinct staining patterns: a diffuse stain all over the cytoplasm (79.5% tumors) (Fig. 3Aa) and an increased heterogeneous stain in the cytoplasm and perinuclear region (20.5% tumors) (Fig. 3Ab). The intracellular LOXL2 stain is in agreement with our previous observations in SCC (20). Comparison of the staining patterns with clinico-pathological features of the breast tumor sample indicated that the cytoplasmic/perinuclear LOXL2 stain is significantly associated to basal-like tumors (Fig. 3B) and to the incidence of distant metastasis of basal-like tumors (Fig. 3B, C). Together, these results indicate that intracellular LOXL2 is a new marker of metastatic basal-like breast carcinomas.

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Discussion/conclusions The present data strongly support the active participation of intracellular LOXL2 as a negative regulator of the epithelial phenotype both in squamous cell carcinomas and in basal-like breast carcinomas. The results obtained in the basal-like breast tumors are also in agreement with a recent report showing overexpression of LOXL2 associated to breast metastasis (29) and with previous studies relating LOXL2 with invasiveness of breast carcinoma cells (19, 21). Interestingly, the LOXL2 action in squamous cell carcinoma cells is mediated both by Snail1-dependent and Snail1-independent mechanisms while it seems to be completely independent of Snail1 in basal-like breast carcinoma cells. This raises the issue of whether LOXL2 can mediate its action by interaction with other EMT-TFs, an aspect that is being presently investigated in our group. On the other hand, LOXL2 is able to repress the expression of tight junction and cell polarity genes at transcriptional level independent of Snail1 and of LOXL2 catalytic activity (28) through still undefined mechanisms. LOXL2 modulation of cell differentiation independent of its catalytic activity has been also recently reported in other model systems (30), although other studies have shown the requirement of the catalytic activity of extracellular LOXL2 for FAK and Src activation

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and metastasis in gastric carcinomas (31). The mechanisms underlying transcriptional repression by LOXL2 independent of its catalytic activity is an open question that we are presently investigating. One interesting possibility is the implication of intracellular LOXL2 in the secretion pathway and/or nuclear protein traffic that may indirectly influence transcriptional regulation, as suggested from the intracellular staining pattern observed in carcinoma cells and tumors (see Figs. 1 and 3). Regardless the specific molecular mechanisms by which LOXL2 exert its function, the present evidence supports a meaningful role for intracellular LOXL2 as a negative regulator of epithelial phenotype and epithelial differentiation in different tumor model systems (squamous, basal-like), thus opening new and unexpected avenues for the regulation of epithelial plasticity. Moreover, the strong association of LOXL2 perinuclear expression with metastatic basal-like carcinomas and the dramatic suppression of lung metastasis induced by LOXL2 knockdown in basal carcinoma cells defines a new role for intracellular LOXL2 to facilitate tumor invasion and metastasis. It can not be excluded at present that intracellular LOXL2 can work in a coordinated fashion with the established extracellular actions of LOXL2 (22, 29, 31). Although additional information from other tumor types and cell systems is clearly required, the present information supports LOXL2 as a potential new therapeutic target for distant metastasis.

Acknowledgements The authors like to thank all members of A. Cano and G. Moreno labs for their excellent work and continuing support. This work has been supported by grants from the Ministry of Innovation and Science (MICINN) (SAF2007-63051; SAF2010-21143; SAF2010-20175; Consolider-Ingenio CDS2007-0017) and Fundación Mutua Madrileña 2007, 2009 to AC and GMB.

References 1. Gupta GP, Massagué J. Cancer metastasis: building a framework. 2006. Cell. 127:67995. 2. Joyce JA, Pollard JW. Microenvironment regulation of metastasis. 2009. Nat Rev Cancer 9:239-49. 3. Nguyen DX, Bos PD, Massagué J. Metastasis: from dissemination to organ-specific colonization. 2009. Nat Rev Cancer 9:274-84. 4. Valaystan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. 2011. Cell 147:275-92. 5. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. 2002. Nat Rev Cancer2:442-54. 6. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. 2009. Cell 139:871-90. 7. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. 2009. J Clin Investig 119:1420-28. 8. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? 2007. Nat Rev Cancer 7: 415-28.

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9. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. 2009. Nat Rev Cancer 9:265-73. 10. Nieto, MA. The ins and outs of the epithelial to mesenchymal transition in health and disease. 2011. Ann Rev Cell Dev 10: 347-76. 11. De Craene B, van Roy F, Berx G. Unraveling signalling cascades for the Snail family of transcription factors. 2005. Cell Signal 17:535-47. 12. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. 2006. Nat Rev Mol Cell Biol 7:131-42. 13. Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop—a motor of cellular plasticity in development and cancer? 2010. EMBO Rep11:670-7. 14. Nieto MA, Cano A. The Epithelial-Mesenchymal Transition under control: global programs to regulate epithelial plasticity. 2012. Cancer Biol, May 18 [Epub ahead of print]. 15. Brabletz T. miR-34 and SNAIL: Another double-negative feedback loop controlling cellular plasticity/EMT governed by p53. 2011. Cell Cycle 11:215-6. 16. Peinado H, Iglesias-de la Cruz MC, Olmeda D, et al. A molecular role for lysyl oxidaselike 2 enzyme in snail regulation and tumor progression. 2005. EMBO J 24: 3446-58. 17. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. 2001. Prog Nucleic Acid Res Mol Biol 70: 1-32. 18. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. 2006. Cell Mol Life Sci 63:2304-16. 19. Payne SL, Hendrix MJ, Kirschman DA. Paradoxical roles for lysyl oxidases in cancer-a prospect. 2007. J Cell Biochem 101:1338-54. 20. Peinado H, Moreno-Bueno G, Hardisson D, et al. Lysyl oxidase-like 2 as a new poor prognosis marker of squamous cell carcinomas. 2008. Cancer Res 68: 4541-50. 21. Hollosi P, Yakushiji JK, Fong KS, Csiszar K, Fong SF. Lysyl oxidase-like 2 promotes migration in noninvasive breast cancer cells but not in normal breast epithelial cells. 2009. Int J Cancer 125(2):318-27. 22. Barry-Hamilton V, Spangler R, Marshall D, et al. Allosteric inhibition of lysyl oxidase-like2 impedes the development of a pathologic microenvironment. 2010. Nat Med 16:1009-17. 23. Hergueta-Redondo M, Palacios J, Cano A, Moreno-Bueno G. “New” molecular taxonomy in breast cancer. 2008. Clin Tranls Oncol 10:777-85. 24. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. 2004. Clin Cancer Res 10:5367-74. 25. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. 2010. N Engl J Med 363:1938-48. 26. Sarrio D, Rodriguez-Pinilla SM, Hardisson D, et al. Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. 2008. Cancer Res 68:989-97. 27. Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, et al. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. 2009. Cancer Res 69:4116-24. 28. Moreno-Bueno G, Salvador F, Martin A, et al. Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas. 2011. EMBO Mol Med 3:528-44. 29. Barker HE, Chang J, Cox TR, et al. LOXL2-mediated matrix remodeling in metastasis and mammary gland involution. 2011. Cancer Res 71:1561-72. 30. Lugassy J, Zaffryar-Eilot S, Soueid S, et al. The Enzymatic Activity of Lysyl Oxidase-like2 (LOXL2) Is Not Required for LOXL2-induced Inhibition of Keratinocyte Differentiation. 2012. J Biol Chem 287:3541-9. 31. Peng L, Ran YL, Hu H, et al. Secreted LOXL2 is a novel therapeutic target that promotes gastric cancer metastasis via the Src/FAK pathway. 2009. Carcinogenesis 30:1660-9.

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2.2. Cell Therapy and Tissue Engineering

Regenerative Medicine in the Context of Cell Biology: Technical and Practical Approaches José A. Andrades1 and María J. Gómez-Lechón2,3 1. Department of Cell Biology, Genetic and Physiology, Faculty of Sciences, Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), University of Málaga, 29071-Málaga, Spain. 2. Unidad de Hepatología Experimental. IIS Hospital La Fe, Avda. Campanar 21, 46009-Valencia, Spain. 3. CIBEREHD, Fondo de Investigaciones Sanitarias, Barcelona, Instituto de Salud Carlos III, Spain.

Cells for Regenerative Medicine Regenerative medicine helps natural healing processes to work faster and better. These technologies and techniques create an environment in which missing or damaged tissue that would not ordinarily regrow in fact regenerates fully. Within just a few years, the possibility that the human body contains cells that can repair and regenerate damaged and diseased tissue has gone from an unlikely proposition to a virtual certainty. Some of the most impressive demonstrations of regenerative medicine since the turn of the century have used varying forms of stem cells-embryonic, adult, and most recently induced pluripotent stem cells-to trigger healing in the patient. Regenerative medicine promises to extend healthy life spans and improve the quality of life by supporting and activating the body’s natural healing. By definition, a stem cell (either adult or embryonic) is characterized by its ability to selfrenew and its ability to differentiate along multiple lineage pathways. Ideally, a stem cell for regenerative medicinal applications should meet the following criteria: Can be found in abundant quantities. Can be harvested by a minimally invasive procedure. Can be differentiated along multiple cell lineage pathways in a regulatable and reproducible manner. Can be safely and effectively transplanted to either an autologous or allogeneic host. Finally, can be manufactured in accordance with current Good Manufacturing Practice guidelines. Are human adult and embryonic stem cells equivalent in their potential for generating replacement cells and tissues? Current science indicates that, although both of these cell types hold enormous promise, adult and embryonic stem cells differ in important ways. What is not known is the extent to which these adult stem cell. An adult stem cell is an undifferentiated (unspecialized) cell that occurs in a differentiated (specialized) tissue,

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renews itself, and becomes specialized to yield all of the specialized cell types of the tissue from which it originated. Adult stem cells are unspecialized cells that are found in different parts of the body and, depending on the source tissue, have different properties. These cells are capable of selfrenewal and give rise to daughter cells that are specialized to form the cell types found in the original body part. Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and other non-embryonic sources, and have demonstrated a surprising ability for transformation into other tissue and cell types and for repair of damaged tissues. However, the term â&#x20AC;&#x153;adult stem cellâ&#x20AC;? is somewhat of a misnomer, because the cells are present even in infants and similar cells exist in umbilical cord and placenta. More accurate terms have been proposed, such as tissue stem cells, somatic stem cells, or post-natal stem cells. Adult stem cells may have more plasticity than originally thought. Stem cell plasticity is the ability of a stem cell from one tissue to generate the specialized cell type(s) of another tissue. For example, adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells (MSCs). These cells are a good example of cells which have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, MSCs have the potential to give rise, either in vitro or in vivo, to bone cells, cartilage cells, fat cells and other types of connective tissue (which is expected), but they may also differentiate into cardiac muscle cells and skeletal muscle cells (this was not initially thought possible) which suggest these cells as an attractive cell source for tissue engineering and regenerative medicine approaches. Human embryonic stem cells (ESCs) are derived from embryos, specifically the inner cell mass of a blastocyst, a hollow ball of cells that forms approximately five days after conception. ESCs can be induced to differentiate into a wide range of tissues that soon could be used for therapeutic applications in regenerative medicine. Despite their developmental potential, sources used to generate human ESC lines raise serious practical and ethical concerns, for clinical application, which recently prompted efforts to reprogram somatic cells back to a pluripotent state. These efforts resulted in the generation of conditions allowing specialized adult cells to be genetically "reprogrammed" to assume a stem cell-like state. These adult cells, called induced pluripotent stem cells (iPSCs), were reprogrammed to an embryonic stem cell-like state by introducing genes important for maintaining the essential functional properties of ESCs. Since this initial discovery, researchers have rapidly improved the techniques to generate iPSCs, creating a powerful new way to "de-differentiate" cells whose developmental fates had been previously assumed to be determined. However, the genetic manipulations required to generate iPS cells may complicate their growth and developmental characteristics, which poses serious problems in predicting how they will behave when used for tissue-regenerative purposes.

Biomaterials for Tissue Engineering Tissue engineering is an interdisciplinary field that combines the knowledge and technology of cells, engineering materials, and suitable biochemical factor to create artificial

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organs and tissues, or to regenerate damage tissues. It involves cell seeding on a scaffold followed by culturing in vitro prior to implantation in vivo. While stem cells obviate the shortcomings of using a patientâ&#x20AC;&#x2122;s differentiated cells, the rate-limiting step in successful cell therapy is not only the number of transplanted cells but their survival rate post-transplantation. In short, the transplanted stem cells need help to stay alive long enough for their therapeutic effect to be seen. The majority of stem and progenitor cells in the transplanted bolus die shortly after transplantation. In some cases, more than 95% of transplanted stem cells die within two weeks of transplantation. Since tissues contain cells encapsulated in a carbohydrate and protein-rich extracellular matrix (ECM), one approach to significantly improve stem cell survival is to include a biomaterial carrier that acts as an ECM mimic upon in vivo delivery. These carriers have been prepared from synthetic or naturally sourced polymers and provide an adhesion surface which not only localizes cells but also provides a template for new tissue formation. Natural biomaterials have been extensively used for tissue engineering since they have advantages over synthetic materials such as similarity with natural ECM. For example, alginate, chitosan, collagen and its derivatives, fibrin, heparin, and a widely used hyaluronic acid (HA) have been investigated for the fabrication of three-dimensional scaffolds. However, difficulty in adjusting the properties and their source-related immunogenicity remains a problem. In contrast, synthetic biomaterials composed of artificially synthesized polymers, although most reveal poor biocompatibility, can be designed with precise control of their physiochemical properties to give better performance when biomedically applied. Aliphatic polyesters and polyanhydrides are the most commonly used synthetic polymers for tissue engineering and drug delivery. By combining hydrophilic and hydrophobic segments within the structure of the polymers, a variety of synthetic biomaterials with the desired mechanical properties and degradation behaviours can be generated. The ideal scaffolds provide a framework and initial support for the cells to attach, proliferate and differentiate, and form an ECM. It should be noted that scaffold surface topography and chemistry (wettability, softness and stiffness, roughness), microstructure (porosity, pore size, pore shape, interconnectivity, specific surface area) and mechanical properties have been shown to significantly influence cell behaviours such as adhesion, growth and differentiation, and to affect the bioactivity of scaffolds used for in vivo regeneration applications of various tissues, such as cartilage, skin and peripheral nerves. For tissue engineering purposes, understanding cell behaviours and responses on extracellular scaffolds within physiological relevant 3D construct can aid the design the design of optimal bioactive tissue engineering scaffolds. Controlling cell behaviour and remodeling by modulating the local engineered extracellular environment process is also a critical step in the development of the next generation of bioactive tissue engineering scaffolds. From a researcherâ&#x20AC;&#x2122;s perspective, the matrix choice will be driven by maintaining consistency with i) the therapeutic stem cell mode of action, ii) the properties of the ECM from both the transplanted cell and the target tissue. There are two primary modes of action for a therapeutic stem cell after transplantation: direct, or transplanted cell engraftment into the host tissue, and indirect, or secretion of trophic bioactive factors which induce host tissue

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repair. The former requires the matrix to be degradable in concert the remodelling and cell proliferation of the transplanted cells. Alternatively, the latter requires a release of soluble factors as well as a longer-term protective environment from the host immune system. Since alginate and polyethylene diacrylate (PEGDA) degrade very slow, they are well suited for the latter indication. Matrix choice is also dependent on the properties of the therapeutic cell ECM since it can provide cues for proper cellular function. In particular, the matrix biopolymer backbone is an essential property to consider since it can contain a great deal of biological information crucial to the cell type. For example, HA has a dual role in cellular biology; it plays a key structural role in the ECM through its interaction with members of the lectican family of proteoglycans while affecting cellular signalling important for development. In cartilage, HA interacts with the lectican aggrecan which stabilizes the cartilage ECM. In addition, HA also interacts with the chondrocyte CD44 receptor to induce genes involved in matrix degradation. In cardiac development, HA interacts with another lectican, versican, in the cardiac ECM while playing a crucial signaling role in endothelial cell migration and transformation.

Advanced Toward Clinical Therapies In the last two decades, regenerative medicine has shown the potential for "bench-tobedside" translational research in specific clinical settings. Therefore, cell-based therapies have been a particularly active area of investigation in recent years. Progress made in cell and stem cell biology, material sciences and tissue engineering enabled researchers to develop cutting-edge technology which has lead to the creation of nonmodular tissue constructs such as skin, bladders, vessels and upper airways. The goal of cell therapy, overlapping with that of regenerative medicine, is to repair, replace or restore damaged tissues or organs. Strategies presently under development include transplants of stem cells, the manipulation of the patient's own stem cells, and the use of scaffold materials that emit biochemical signals to spur stem cells into action. Regenerative therapies have been demonstrated (in trials or the laboratory) to heal and repair broken bones, blindness, deafness, heart damage, Parkinson's disease, replacement of skin for burn victims, restoration of movement after spinal cord injury and regeneration of pancreatic tissue to produce insulin for people with diabetes and a range of other conditions. Stem cell-based therapies are a major area of investigation in cancer research. The idea that a patient's tissues could provide him/her copious, immune-matched supply of pluripotent cells has captured the imagination of researchers and clinicians worldwide. Furthermore, ethical issues associated with the production of ESCs do not apply to iPSCs, which offer a non-controversial strategy to generate patient-specific and disease-specific cell lines for clinical applications. Thus, iPS cell technology has been received with great interest by research and medical communities. Gene therapy has the potential to become an important treatment regimen. In principle, it allows the transfer of genetic information into patient tissues and organs. Consequently, diseased genes can be eliminated or their normal

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functions rescued. Stem and iPS cells are already being explored as a vehicle for delivering genes to specific tissues in the body. Harnessing stem cells for use as drug delivery systems is another goal of researchers. Cell therapy may take the form of a stem cell transplant such as a hematopoietic cell transplant that is used to restore the blood and immune system of patients with leukemia, lymphoma or other blood disorders. Bone marrow transplants have been used for the past 40 years to regenerate the blood and immune systems of patients with leukemia, lymphoma, severe aplastic anemia or inherited metabolic diseases. Unfortunately, the major limitation with allogenic bone marrow transplants is the availability of matched donors. Stem cells from umbilical cord blood have emerged as an alternative to bone marrow transplants, providing an easily obtainable and readily available source of treatment. Umbilical cord blood transplants may result in a lower incidence of transplant complications, specifically graftversus-host disease, common in patients receiving a transplant from an unrelated donor. In addition to regenerating the blood and immune systems, scientists anticipate that stem cells will be used to replace damaged or diseased tissues and organs. Clinical trials are ongoing to repair scarred or dying heart muscle after a heart attack or during congestive heart failure. On-going research in diabetes is focused on understanding how stem cells might be trained to become the type of pancreatic islet cells that secrete needed insulin. Repair of debilitating spinal cord injuries is also a goal of researchers through the regeneration of neurons, myelin and nerve cells. In summary, stem cells and iPSC cells represent important sources of cell-based therapies, their translation to clinical trials still requires the scale-up production, or â&#x20AC;&#x153;bankingâ&#x20AC;?, of large numbers of the desired cell type. Several important advantages of these cells should be highlighted, they provide an excellent model to establish the proof-of concept that genetic defects can be corrected in vitro by vectors expressing missing or defective genes, thus paving the way for "personalised medicine". Hepatocyte transplantation has become a viable alternative treatment to liver transplantation and has been performed for a variety of indications, including acute liver failure, end-stage liver disease, and inborn errors of metabolism. Cells derived from other tissues, such as bone marrow, monocytes, endothelium, and pancreas, are also being explored as potential therapies for liver-based metabolic disorders. Stem cells from extra- or intrahepatic sources have been recently characterized and their usefulness for the generation of hepatocyte-like lineages has been demonstrated. Therefore, they are being increasingly considered for future applications in liver cell therapy. Finally, models of functioning hearts and livers have been engineered using "natural tissue" scaffolds and efforts are underway to produce kidneys, pancreas and small intestine. Creation of custom-made bioengineered organs, where the cellular component is exquisitely autologous and have an internal vascular network, will theoretically overcome the two major hurdles in transplantation, namely the shortage of organs and the toxicity deriving from lifelong immunosuppression.

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Ethical Considerations in Clinical Therapies Stem cell-based treatments have been the clinical standard of care for some conditions, such as the mentioned hematopoietic stem cell transplants for leukemia, but research on these therapies has greatly expanded in recent years. Investigational New Drug (IND) applications have been field for several therapies, and clinical trials are underway. As might be expected, problems have already developed. In late 2009, the FDA asked the Geron Corporation to halt its trial on a groundbreaking stem cell therapy for spinal cord injury when a review revealed the formation of cysts in some (animal) trial subjects. Research on the development of stem cell therapies poses ethical challenges for several reasons: - Stem cells are novel therapeutic agents whose development and manufacture require innovative procedures for ensuring purity and homogeneity. - Cell types may differ in their ability to proliferate and safely implant in the body. Their use poses risks of infusional toxicity, severe immune responses, and tumorigenesis. - Animal models may not accurately reflect toxicity in humans. In response to these challenges, in 2008, a multidisciplinary, international task force of the International Society of Stem Cell Research proposed ethical guidelines for clinical translational research (ISSCR, 2008). The task force issued 39 recommendations, most of which fall into five broad categories: (1) cell processing and manufacture, where these be conducted scrupulous, expert, and independent review and oversight. As a rule, minimally manipulated products (cell maintained in culture under non-proliferating conditions for short periods of time) require less oversight than those subjected to extensive manipulations, such as genetic alterations. (2) Preclinical studies, that are meant to provide evidence of product safety and proof-of-principle of therapeutic effect. This normally requires sufficient studies in animal models, including larger animals where structured tissue needs to be tested in a load-bearing model. (3) Clinical trials of stem-cell research, which must conform to internationally accepted principles governing the protection of human subjects, including regulatory oversight, peer review by an expert panel independent of the investigators and sponsors, fair subject selection, informed consent, and patient monitoring. (4) Stem cellbased medical innovation. The task force condemned the widely prevalent practice of marketing unproven stem cell interventions. Such unproven stem-cell interventions require a written plan explaining, among other things, the interventionÂ´s scientific rationale, clinical justification, a description of procedures, and plans for follow-up, data collection, identification of adverse effects, and assessment of efficacy. (5) Considerations of social justice, apply to all research involving human subjects but receive special importance in view of public involvement in this emergent research are. Among the recommendations of the task force are public engagement in the policy making of governmental agencies.

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Industrial Approaches in Tissue Engineering The technical challenges of tissue engineering are, of course, intellectually and scientifically interesting and can add substantial and previously unattainable knowledge to our understanding of biological systems. Tissue engineering models of biological systems can even provide insight into pathologic processes. However, perhaps the major attraction of academic researchers and industrial organizations to this field is the potential of the technology to be readily converted to clinical applications. For this to happen, the technology almost always will be transferred from an academic environment to an industrial organization that will lead the comprehensive translational studies and convert scientific observations into a manufactured product. As a technology, tissue engineering has been shown to be feasible in vitro and in vivo, but the true demonstration of the potential value of the technology is in its clinical applications. Although the field is still in its infancy, there are already tissue-engineered products on the market, addressing previously unmet clinical needs in wound care and in orthopedics and demonstrating that the attractiveness and motivation of the field is justified. Perhaps one of the next major challenges is demonstration that the technology can lead to commercially feasible products, with manageable investment, product development costs, and time to market and, finally, a revenue generation that justifies the expense. The close connection between new technology, clinically effective treatment, and commercially feasible product is obvious and is no better demonstrated than in tissue engineering. All three of these areas, each complex in itself, must be aligned and achieved before tissue engineering can be regarded as successful. The initial thrust of tissue engineering was driven by the envisioned commercial opportunities, and this has seen development of some new products, particularly in wound care and in orthopedics. In wound care, TransCyte, a dermal fibroblast-derived extracellular matrix on a sheet biomaterial, was introduced in 1997 for the treatment of third- and seconddegree burns. Apligraf and Dermagraft, introduced in 1999 and 2001, respectively, are products containing allogeneic live cells and human ECM and are used for the treatment of chronic wounds such as venous and diabetic foot ulcers. In orthopedics, the Carticel product (autologous cultured chondrocytes) was introduced in 1997 for the treatment of focal articular defects. In 2004, Infuse was approved for spinal fusion and consisted of the growth factor BMP2 within a collagen sponge (to regulate growth factor release), placed within a metal cage. A commonality of these producs is that they have been expensive to develop and produce, and once the product has been introduced in the market, they have required substantial commercial commitment by their manufacturing companies to allow the products to become established within the clinical marketplace. All but Infuse products have struggled to attain profitability, in spite of their demonstrated clinical value to certain patient populations. There are many new tissue engineering product development programs now ongoing, some incremental compared to products on the market and some that will be novel and technologically advanced and will provide new treatment. It is expected that these new products will reach the market in the next 5 to 10 years, particularly in the fields of wound care, orthopedics, and cardiovascular disease, but it may be that tissue engineering

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approaches will also have impact in neurologic disease, cancer, diabetes, and other pathologies previously unrepresented by tissue engineering.

Dr. Andrades’ group is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2009-13903-C02-01 and FIS PI10/02529), Red de Terapia Celular (RD06/0010/0014), and the Andalusian Government (P07-CVI2781, PAIDI BIO-217 and PI-0729-2010). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Dr. Gómez-Lechón’s group acknowledges the financial support of the European Union (CT-2005-037499 and CT-2011-278152), the Spanish Ministry of Economy and Competitiveness (IF08/3638, PI-10/0923 and PI-070550), Program Cenit, CDTI (Melius and Dendria Projects); and CIBEREHD funded by the Instituto de Salud Carlos III.

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The Stem Cell And Its Neighborhood: The Niche Of Stem Cells In Animal And Plant Organisms José Becerra BIONAND-UMA. Department of Cell Biology, Genetics and Physiology, Faculty of Sciences, UMA, Malaga, Spain. Networking Research Center for Bioengineering, Biomaterials and Nanomedicine, (CIBER-BBN), 29071 Malaga, Spain Entering the term "stem cell", limited to the title, in PubMed in the year 2012, the computer search engine yields approximately 31,600 results. The first registered article is from 1946 and relates to hematology, "Stem cell lymphoma of the newborn". Selecting articles published before 1998, 52 years, there are 6,367 results, representing 122 articles per year. This means that since the time Thomson and colleagues (1) published their paper on human embryonic stem cells in 1998, 14 years ago, an average of 2000 items each year have been published, containing in its title the term "stem cell", and raising. Only during the first three month of 2012, 1344 sources have been published. Therefore, the topic stem cell (SC) is perhaps one of the most influential scientific concepts in recent scientific history, at least in terms of the number of researchers involved and the number of pages written in scientific journals. If we include articles written about SC in mainstream newspapers and social magazines, we can consider it to be one of the most widespread scientific events and, therefore, one of the issues on which most money has been invested at a global scale, in a short period of time. However, this data and expectations have not matched either the results achieved or their scientific quality. Perhaps it can be said that the chapter written about the stem cell niche is one of the highest scientific excellence. By means of the stem cell niche we can comprehend the biological

significance

the

presence

during

postnatal

development,

maintenance/exit of the quiescent state, regulation of cell proliferation and leaving the niche area to initiate a differentiation pathway, preceded by a progenitor phase in which proliferation is very high. The term “stem cell niche” (SC niche) was first proposed by Schofield in 1978 (2). He proposed that other cells should accompany the bone marrow stem cells in order to explain the formation of mature hematopoietic cell clones when they are injected in the spleens of irradiated mice. The first SC niche described in molecular terms was that of the Drosophila

melanogaster germarium (3). An SC niche is defined as a complex, multi-factorial local microenvironment required for the maintenance of SC biology. The SC niche consists of SCs, non-SCs, extracellular matrix and molecular signals originating in both types of cells. Inside the niche, the SC can divide symmetrically or asymmetrically giving rise to both new SCs and proliferating progenitor cells (4). Committed stem cell progeny become indispensable components of the niche in a wide range of stem cell systems. These daughter SC provide different feedback signals to their stem cell parents (5). Understanding

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the molecular mechanisms underlying the crosstalk between different components of the SC niche we can move forward; not only we will learn about the biology of stem cells but also we will gain more knowledge about the therapeutic possibilities they may have. Some of the best-characterized SC niche models are Drosophila germarium or testis, vertebrate hair follicle, epiderm, intestinal crypts, bone marrow, skeletal muscle and brain subventricular zones (6-11). All these studies provide information on SC niche biology and SC dependence on this tissue microenvironment in several tissues and organisms throughout the animal kingdom. Although stem cells and their niche have been described during development, regeneration, tissue homeostasis and injury, the discovery that only a small fraction of tumor cells is capable of initiating a new tumor, cancer SC was defined and recent data suggests that a similar microenvironment, called the cancer SC niche, controls their self-renewal and differentiation (12). Obviously, SC niche of various tissues and organs does not work in the same manner. They behave in accordance with the peculiarities of each situation. Sometimes normal activity is the continuous replenishment of new cells from their corresponding stem. In such cases the proliferative activity of stem and progenitor cells are very active in order to ensure the required daily turnover. That is the case of the epidermis, intestinal epithilium or hematopoiesis. In other cases, such as that of the hair follicle, activity is cyclical and therefore the regulation of the system is subjected to this behavior. A third type of stem cells undergoes extremely low or no division during normal homeostasis but can respond efficiently to stimuli or injury. The brain and skeletal muscles belong to this type. Perhaps the latter may occur in any tissue injury, such as connective, bone, tendon, etc. where even within a relatively dormant situation, any aggression triggers the activity of stem cells in their niche. Among all known types of adult stem cells, one deserves further explanation: mesenchymal stem cells (MSC). MSCs have been defined "as a diverse subset of multipotent precursors present in the stromal fraction of many adult tissues" (13). This cell type has aroused special interest for its potential in vitro culture and the ability to differentiate into various mesenchymal cell phenotypes, making them interesting for application in regenerative medicine. However, their identity in vivo and their functional role in the homeostasis of adult tissues is still enigmatic and subject to controversy. In addition to the possibility of MSC differentiation, its ability to secrete a large amount of bioactive molecules such as growth factors, cytokines and chemokines, should be included, which is probably the most important biological role of tissue injuries. Due to its ubiquity, it is difficult to determine the MSC niche, and even if there is a unique niche for the MSC. It is likely that there are at least two situations in which the MSC niche can be defined differently. One would be in the bone marrow where MSCs play an essential role in the regulation of both hematopoietic stem cell (HSC) progression and in their own differentiation into osteoblasts. Thus, some authors propose that in the bone marrow there is a sole niche for both types of stem cells (14). Furthermore, a cell type in the neighborhood of blood vessels of many organs has been identified, including the own bone marrow, capable of growing as plasticadherent fraction that can give rise to colonies of fibroblastic morphology known as colony-

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forming unit fibroblasts (CFU-Fs) and has been shown to express pericyte-specific markers (CD146, NG2 and platelet-derived growth factor receptorÎ˛ (PDGFRÎ˛) (15-17). In the last few years in vitro results from different culture conditions and sparse in vivo data, together with many ongoing clinical trials, show mixed results in the literature, making it difficult to clearly understand the reality of the MSC in the body function and its significance in reparative/regenerative cell therapies for clinical applications (13, 18).

Plant stem cell niches Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. But terms and concepts associated with stem cell biology have spread from animal to plant research only in the last few years. If in animal development the concept "SC niche" has been in existence for over 20 years, it is not found in plant development until recently. In the early years of this century even the term "niche" does not appears in scientific literature which discusses plant morphogenesis (19), Nevertheless, a few years later this concept is of central importance, even appearing in the title of articles (20). From then to now, the differences and similarities between the SC niche of animals and plants have been continuously brought to light (21). Logically, plant stem cells are associated with meristems, as the group of cells ensuring the continued growth of plants, classically distributed in certain parts of the plant. The bestknown meristems are associated with the tips of stems and roots, respectively called shoot apical meristem (SAM) and root apical meristem (RAM). Although these meristems are not the only in the plant body, they are best studied and are currently providing abundant information to understand plant development and growth. From the classical studies of plant anatomy meristems are considered as the areas of the plant where active cell division occur, resulting in cells that initiate differentiation to produce the various tissues and organs. This concept has been pointed throughout the past years so that now in the meristem there are distinguished cells with a low rate of division that form the organizing center (OC) in the shoot or the quiescent center (QC) in the root. Groups of cells around OC or QC, known as stem cells, and cells derived from these that are actively dividing, that may be called progenitor cells or transit amplifying cells forming the meristem peripheral zone, capable of differentiating into various cell tissues (22). Thus, plant SC niche is located in the meristem where the stem cells provide a continuous molecular crosstalk with OC or QC cells and the differentiation zones. In the last few years this intense intercellular signalling in the niche of the plant SC has been dissected with great precision (23). Key regulatory genes that respond to intercellular signaling, e.g. WUS and CLAVATA in shoot and the WUS homologue WUS-RELATED HOMEOBOX5 (WOX5) in root, ultimately control rates of cell growth, division and the decision to enter a differentiation pathway. Auxin and cytokine signals are integrated with each other and with regulatory genes in the shoot and root meristems. Other novel intercellular signals as small RNAs have also emerged in the balance of cell proliferation/differentiation and initiate tissue patterning. Since Clowes discovered the quiescent center in the early 1960s (24), who propose almost intuitively its importance in regulating root meristem, accepted until today, there have

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been frequent discoveries that have taken this structure by various ups and downs. It has been proposed that QC is a group of differentiated cells producing hormones that induce cell proliferation in their environs (25). More recently it was thought that the quiescent center were the genuine stem cells and their derivatives give rise to the concentrically organized tissues (19). Now experiments show that the QC of roots and OC in shoots are homologous and they are crucial in plant growth regulation of the apical meristems (23).

References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145– 1147. 2. Schofield R (1978). The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells; 4:7–25. 3. Lin H, Spradling AC (1993). Germline stem cell division and egg chamber development in transplanted Drosophila germaria. Dev. Biol.; 159:140–152. 4. Hsu YC, Fuchs E (2012). A family business: stem cell progeny join the niche to regulate homeostasis. Nat Rev Mol Cell Biol. ; 13(2): 103–114 5. Mondal BC, Mukherjee T, Mandal L, Evans CJ, Sinenko SA, Martinez-Agosto JA, Banerjee U (2011). Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance. Cell; 147:1589–1600. 6. Fuchs E, Tumbar T, and Guasch G (2004). Socializing with the neighbors: stem cells and their niche. Cell, 116, 769-78. 7. Mitsiadis TA, Barrandon O, Rochat A, Barrandon Y, De Bari C (2007). Stem cell niches in mammals. Experimental Cell Research, 313, 3377-85. 8. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009). Single Lgr5 stem cells build crypt–villus structures invitro without a mesenchymal Niche. Nature, 459, 262-266. 9. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell; 122:289–301 10. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell.; 97:703–716 11. de Cuevas M, Matunis EL (2011). The stem cell niche: lessons from the Drosophila testis. Development 138, 2861-2869 12. Borovski T, De Sousa E Melo F, Vermeulen L, Medema JP (2011). Cancer stem cell niche: the place to be. Cancer Res;71(3):634-9. 13. Nombela-Arrieta C, Ritz R and Silberstein LE (2011). The elusive nature and function of mesenchymal stem cells. Nat Rev Mol Cell Biol. Feb;12(2):126-31.

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14. Méndez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma'ayan A, Enikolopov GN, Frenette PS (2010). Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 15. da Silva Meirelles L, Chagastelles PC, Nardi, NB (2006). Mesenchymal stem cells reside in virtually all postnatal organs and tissues. J. Cell Sci. 119, 2204–2213 16. da Silva Meirelles L, Caplan AI, Nardi NB (2008). Insearch of the in vivo identity of mesenchymal stem cells. Stem Cells, 26, 2287–2299. 17. da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI (2009). Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev; 20(56):419-27. 18. Becerra J, Santos-Ruiz L, Andrades JA, Marí-Beffa M (2011).The stem cell niche should be a key issue for cell therapy in regenerative medicine. Stem Cell Rev. and Rep; 7 (2): 248255. 19. Irish VF and Jenik PD (2001). Cell lineage, cell signaling and the control of plant morphogenesis. Current Opinion in Genetics & Development, 11:424–430 20. Stahl Y and Simon R (2005). Plant stem cell niches. Int. J. Dev. Biol. 49: 479-489 21. Sablowski R (2004). Plant and animal stem cells: conceptually similar, molecularly distinct? Trends Cell Biol;14(11):605-11. 22. Singh MB and Bhalla PL (2006). Plant stem cells carve their own niche. Trends in Plant Science 11 (5):241-46 23. Sablowski R (2011). Plant stem cell niches: from signalling to execution. Curr Opin Plant Biol;14(1):4-9. 24. Clowes FA (1964). The quiescent center in meristems and its behavior after irradiation. Brookhaven Symp Biol. Mar;16:46-58. 25. Torrey JG (1972). On the initiation of organization in the root apex. In The Dynamics of Meristematic Cell Populations. M. W. Miller and C. C. Kuehart, editors. Plenum Press, New York. I - 13.

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Efficiency and Biosafety in Cell Therapy Agustin G. Zapata Department of Cell Biology. Faculty of Biology. Complutense University. 28040 Madrid, Spain From the first report by James Thomson (1) on the production of human embryonic stem cells (hESCs), many data have been published on the molecular mechanisms that determine the properties of these cells, pluripotency and autorenewal capacity, permitting promising therapeutic applications as well as the appearance of new experimental approaches, including cell reprogramming. Nevertheless, the principal problems for the therapeutic use of hESCs and/or their derivates: specificity, safety differentiation of hESCs to the necessary cell type and their immunogenicity still remain. This can explain why only one phase I clinical trial using hESC-derived committed cell progenitors is currently in progress around the world and important biomedical companies have cancelled their scientific programmes on Cell Therapy (2). In this brief review we will summarize current information on the benefits and problems, in terms of efficiency and safety, of using different stem cells, i.e., embryonic, reprogrammed and adult, for Cell Therapy.

hESCs are not safe and provoke immunological responses in recipients The identification of numerous parameters, largely transcription factors, concerned with the differentiation of hESCs to committed progenitor cells or even differentiated cells of distinct cell lineages has opened the gates to their use in Cell Therapy. However, there is growing evidence that the in vivo injection of hESCs or their derived committed progenitors has frequently induced tumours, a problem that remains to be definitively resolved. A second problem for the therapeutic use of hESCs or their derivates is their immunogenicity, which could provoke a host immune reaction against the injected cells. In an attempt to resolve these problems on ESC immunogenicity, researchers assayed the transfer of host nuclei in enucleated oocytes to generate pre-implanted embryos capable of providing ESCs with the nuclear genome of the patient to be further treated. As known, this approach, which was successful in numerous mammals, failed in humans and has been abandoned.

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Reprogrammed stem cells are excellent models to study the pathogeny of both monogenic and multifactorial diseases, but cannot be used in Cell Therapy The identification of Nanog, Oct3/4 and Sox2, the so-called core pluripotency network, as transcription factors critical for conferring pluripotency and auto-renewal capacity to ESCs gave rise to

a new approach to obtain ESCs from the reprogramming of somatic

differentiated cells. This method avoided the ethical problems associated with the use of human embryos and the immunogenicity of allogeneic ESCs. Previously, some studies had obtained cells exhibiting properties of ESCs resulting from the fusion of ESCs and adult somatic cells. Although the resulting cells were obviously tetraploid and, therefore, not viable for therapy, these experiments clearly showed that ESCs and embryonic germ cells contained factors capable of inducing reprogramming and pluripotency in somatic cells, although accountable molecules remained unknown (3). Surprisingly, Yamanaka and colleagues found that a combination of only four transcription factors generate cells similar to ESCs, the so-called induced pluripotent stem cells (iPSCs), first from fibroblasts of mouse, and later of humans (4, 5). Remarkably, these factors were reported in ESCs to govern their pluripotency and auto-renewal, i.e Oct3/4 and Sox2, with Nanog as a master controller, and other transcription factors (c-myc, Klf4), not key to the reprogramming process, but capable of improving it. These first studies used retro or lentiviruses to transfect fibroblasts (or other cell types) with the transcription factor genes, but over the last few years the protocols have been importantly modified to avoid insertional mutagenesis and to improve their efficiency. For example, reprogramming proteins (6) and micro RNAs (7) have been incorporated to the standard protocols of cell reprogramming. It is important to mention some key steps of the reprogramming processes to understand the true nature and therapeutic potential of iPSCs. Although we do not intimately know the underlying mechanisms of cell reprogramming, we can describe several stages of the process: The reprogramming entails intermediate steps that include multiple cycles of ADN replication, cell divisions and epigenetic changes that together result in the silencing and activation of specific endogenous genes. In fact, transfected transcription factor genes are only necessary during the reprogramming process; they are later silenced and the endogenous genes, masters of ESC properties, are activated to control the stemness of iPSCs. Unfortunately, IPSCs do not currently represent a hopeful therapeutic alternative to ESCs, although they have other interesting applications (see below). Any reprogrammed cell is in risk of suffering mutations and epigenetic changes that make the evaluation of every generated clone mandatory, something that could invalidate their therapeutic applications just for economic reasons. In fact, iPSCs support important stress and cell reprogramming inhibits tumour suppressor genes and activates oncogenes. We know that elimination or alterations of the tumour suppressor gene p53 favours the efficiency of cell reprogramming, but also promotes genetic instability and tumour development (8). In confirming these

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results, a genetic screening of 22 hiPSCs generated in 7 different laboratories around the world found about 5 point mutations/exome, most occurring in genes related to cancer. In addition, although around 50% of mutations found were already present in the original fibroblasts in a low frequency, the other half appeared during or after the reprogramming process. On the other hand, as mentioned above, one of the presumptive advantages of iPSCs compared to ESCs is that the former are not immunogenic for patients from which they derive. However, a recent result that needs further confirmation, questions this assumption because autologous iPSCs derived from fetal fibroblasts generate teratomas that are rejected after being grafted in genetically matched mice with the donor cells (9). According to this study, iPSCs were more immunogenic than ESCs. Apparently, several genes overexpressed in the iPSC-derived teratomas were responsible for the activation of specific T cells that eventually resulted in the teratoma rejection. It is important to note that, despite these problems, which question the possible therapeutic application of iPSC or their derivates, iPSCs derived from patients suffering from monogenic or multifactor diseases constitute excellent tools for drug screening and/or for providing human models for the study of human diseases that do not currently have adequate experimental animal models. Furthermore, the production of iPSCs for Cell Therapy implicates, in any case, their safety differentiation to the required mature cell type. Therefore, over the last few years other approaches have tried to directly differentiate mature cells to other cell lineages by transfection with transcription factors known to be involved in the specific development of the required cell types. Previously, a few studies had reported that this kind of direct reprogramming only functioned when the cells involved belong to closely-related cell lineages. Thus, transcription factors of the C/EBP family allow the conversion of lymphocytes in macrophages and the absence of transcription factor Pax 5 reverted B lymphocytes to primitive lymphoid progenitors. More recently, however, the direct reprogramming of murine fibroblasts to neurons was reported (10). Nevertheless, the underlying mechanisms governing these processes are still largely unknown and reported results are frequently suprising. In the mentioned study, while the combination of several transfected genes (Ascl 1, Brn 2 and Myt 1L) induced mature neurons, the transfection of fibroblasts with Ascl 1 alone provided immature neurons. Furthermore, most neurons obtained in this study were excitatory whereas, during the development of the central nervous system, Ascl 1 expression is involved in the generation of inhibitory neurons. Bathia and colleagues (11) were the first ones to directly convert human fibroblasts in multilineage hematopoietic progenitors. In this study, a new and interesting approach was used based on the partial reprogramming of human fibroblasts, followed by the treatment of these reprogrammed intermediates with a cocktail of hematopoietic cytokines. More studies are, however, necessary to confirm the reliability of this protocol and the genetic, epigenetic and functional nature of the cells produced. Fetal stem cells, relatively similar to the adult mesenchymal stem cells present in connective tissues (see below), could constitute an alternative to ESCs and iPSCs but have been little studied.

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Adult stem cells exhibit limited therapeutic capacities Over the past ten years, numerous pre-clinical and clinical studies emphasized the capacity of different adult progenitor cells to transdifferentiate to unrelated cell lineages. In these studies, by contrast to direct reprogramming, the cell conversion was accounted for by the influence of an altered microenvironment, rather than any genetic manipulation of original progenitors. Unfortunately, numerous clinical trials performed by using hemopoietic stem cells as a source of cell progenitors failed, and the concept of transdifferentiation is currently little accepted. More recently, mesenchymal stem cells (MSCs) have become the most frequently used cell type in studies and clinical trials on cell therapy although again the results are limited. MSCs are an enigmatic cell type initially described as the adherent fraction of cultures of total bone marrow cell suspensions that form clonogenic colony forming unit-fibroblasts (12). Later, they have been reported to be a component of all connective tissues. MSCs exhibit high, but limited, self-proliferation capacity and osteogenic, adipogenic and, some clones, chondrogenic capacities. There are no specific markers for identifying MSCs, but most express CD29 and CD105 and other cell markers (i.e., CD133, SSEA-1, SSEA-4, CD271). They also express TLRs and receptors for numerous cytokines and chemokines (13). Remarkably, MSCs have immunoregulatory properties that have been successfully exploited for therapeutic proposals in the treatment of GvH disease and autoimmunity. Although, frequently reported as immunosuppressive cells, this is not constitutive and seems to be dependent on the microenvironment, especially on IFNÎł levels. MSCs can negatively affect every step of the immune response, from antigen presentation to the activation of T and B lymphocytes, affecting the TH1-TH2 balance and the activity of effector TH17 cells and Treg cells, in an opposite manner (14-18). The in vivo origin of MSCs remains to be confirmed. Presumably, they are pericytes that appear to be associated with the microvessels of connective tissue, and in the bone marrow, the main source for obtaining these cells, we know them as adventitial reticular cells or, more recently, nestin positive cells (19). However, other cell types that form part of the hematopoietic niche, especially CAR cells (CXCL12 rich cells), could also be MSCs. On the other hand, it is possible that the heterogeneity observed in cultures of MSCs reflects these presumptive different origins, but also the self-plasticity of this cell type (20-23). MSCs have substituted HSCs in clinical cell therapy trials to produce not only osteoblasts, adipocytes and chondrocytes, but also skeletal and cardiac muscle cells, neurons and hepatocytes. Nevertheless, the success is very limited and the underlying mechanisms involved in this recovery of cell types of different lineages are largely unknown. More promising applications, as indicated above, could correspond to the clinical application of MSCs based on their immunoregulatory and anti-inflammatory properties, which permits their use even in allogeneic conditions. Clinical trials that employ the immunoregulatory properties of MSCs to control GvH disease have provided controversial results with highly variable response rates (24, 25). These discrepancies could be due to the different GvHD

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characteristics of treated patients and/or to the different protocols used. The time of MSC application seems to be particularly important (26). The therapeutic effects of MSCs in autoimmune diseases first reported in numerous experimental models (27-29), and later in clinical studies (30-32) are also a matter of discussion.

Conclusions We can summarize these results by concluding that neither ESCs nor iPScs can currently be used for therapeutic applications, although the latter are good tools as experimental models to study the pathogeny of severe human diseases that lack animal experimental models. In the case of cell therapy performed with adult cell progenitors, MSCs seem to currently be the elective cell type, although results are very limited, restricted to mesoderm-derived tissues or related to the immunoregulatory properties of this cell type. A better phenotypic and functional characterization of MSCs as well as well controlled protocols are necessary to obtain more successful results.

Acknowledgments This work was supported in part by grants: S-B10-0204/2006 (Regional Government of Madrid), BFU 2010-16250 (Spanish Ministry of Science and Innovation), RD06/0010/0003 (Spanish

Ministry

Health

and

Consumption),

GR35/10A-910552

(Complutense

University), Spanish Association for Cancer Research.

References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282:1145-1147. 2. Baker M. Stem-cell pioneer bows out. Nature 2011; 479:459. 3. Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008; 132:567-582. 4. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676. 5. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors . Cell 2007; 131:861-872. 6. Sommer CA, Mostoslavsky G. Experimental approaches for the generation of induced pluripotent stem cells. Stem Cell Res Ther 2010; 1:26. 7. Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 2011; 8:633-638. 8. Kawamura T, Suzuki J, Wang YV, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming . Nature 2009; 460:1140-1144. 9. Zhao T, Zhang ZN, Rong Z, et al. Immunogenicity of induced pluripotent stem cells. Nature 2011; 474:212-215. 10. Vierbuchen T, Ostermeier A, Pang ZP, et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2011; 463:1035-1041.

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11. Szabo E, Rampalli S, Risueno RM, et al. Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 2010; 468:521-526. 12. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4:267-274. 13. Deschaseaux F, Pontikoglou C, Sensebe L. Bone regeneration: the stem/progenitor cells point of view. J Cell Mol Med 2010; 14:103-115. 14. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105:1815-1822. 15. Stagg J. Immune regulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 2007; 69:1-9. 16. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008; 8:726-736. 17. Ghannam S, Pene J, Torcy-Moquet G, et al. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol 2010; 185:302-312. 18. Tatara R, Ozaki K, Kikuchi Y, et al. Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation. Cytotherapy 2011; 13:686-694. 19. Mendez-Ferrer S, Michurina TV, Ferraro F, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466:829-834. 20. Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone marrow . Blood 2007; 109:1743-1751. 21. Kiel MJ, Morrison SJ. Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol 2008; 8:290-301. 22. Trumpp A, Essers M, Wilson A. Awakening dormant haematopoietic stem cells. Nat Rev Immunol 2010; 10:201-209. 23. Ehninger A, Trumpp A. The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in. J Exp Med 2011; 208:421-428. 24. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroidresistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008; 371:15791586. 25. von Bonin M, Stolzel F, Goedecke A, et al. Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium. Bone Marrow Transplant 2009; 43:245-251. 26. Tisato V, Naresh K, Girdlestone J, et al. Mesenchymal stem cells of cord blood origin are effective at preventing but not treating graft-versus-host disease. Leukemia 2007; 21:19921999. 27. Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005; 106:17551761. 28. Madec AM, Mallone R, Afonso G, et al. Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells. Diabetologia 2009; 52:1391-1399. 29. Sun L, Akiyama K, Zhang H, et al. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells 2009; 27:1421-1432. 30. Carrion F, Nova E, Ruiz C, et al. Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients. Lupus 2010; 19:317-322. 31. Liang J, Zhang H, Hua B, et al. Allogenic mesenchymal stem cells transplantation in refractory systemic lupus erythematosus: a pilot clinical study. Ann Rheum Dis 2010; 69:1423-1429.

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32. Yamout B, Hourani R, Salti H, et al. Bone marrow mesenchymal stem cell transplantation in patients with multiple sclerosis: a pilot study. J Neuroimmunol 2010; 227:185-189.

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New strategies for cardiac regeneration Felipe Prósper Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain *Corresponding author, Felipe Prósper is to be contacted at Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Avda Pío XII n36, E31008 Pamplona, Spain. Tel.: +34 948 255400x5807; fax: +34 948 296500. E-mail address: fprosper@unav.es

Abstract Cardiovascular diseases are the main cause of morbidity and mortality worldwide. A huge effort has been made to improve current standard approaches for treating patients with ischemic heart disease. However, despite the greater efficacy of new drugs and clinical techniques, which have decreased the number of acute patients and prolonged the life of chronic ones, the classic treatments are still not able to regenerate the diseased heart. For this reason, alternative therapies based on the use of gene, protein and stem cells have been developed in combination with bioengineering techniques, with the aim not only of protecting but also repairing the damaged heart. All these new therapies, especially stem cell therapy and the possibility of combining these cells with biomaterials in order to reinforce their potential or even create new tissues, are reviewed in this chapter.

Introduction According to the World Heart Organization, more people die annually from cardiovascular diseases than from any other cause, since they represent 29% of all deaths. By 2030, almost 23.6 million people will probably die from cardiovascular diseases (CVD), this being the first cause of death, representing 42% of deaths. The major modifiable risk factors associated with ischemic heart disease (IHD) are tobacco and alcohol use, hypertension, high cholesterol, obesity, diabetes and physical inactivity. Other nonmodifiable factors related to CVD include aging, family history of cardiovascular disease, gender and ethnic origin. Therapies driven to improve myocardial function in IHD include pharmacological treatment, percutaneous intervention and surgery. Most of these are aimed at minimizing the symptoms and preventing progression of the disease, but are able neither to regenerate the tissue nor to restore the heart function in a maintained form. In fact, the last and only resort

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for severe cases is heart transplantation with the concomitant limitations of the donor waiting lists and the need for a immunosuppressive regimen to prevent rejection, which obviously has its own significant deleterious side effects. The failure of these therapies to rescue the damaged heart and the inconvenience of heart transplants have led to the emergence of alternative treatments, including gene (1), protein (2) and stem cell (SC) (3) therapies. Combination of these therapies with tissue engineering (TE) could boost their benefits, through strategies that could increase cell function, survival and cell homing. Thus, cells, biomaterials and/or biologically active molecules could be applied with the main objective of restoring, maintaining and/or enhancing tissue and organ function (4) gathering engineering, medical and biological applications.

Protein Therapies For Cardiovascular Disease There have been significant efforts to introduce novel therapeutic strategies in IHD based on the use of growth factors, which are able to enhance the intrinsic capacity of the heart to repair itself or regenerate after damage. Angiogenic cytokine therapy has been widely regarded as an attractive, straightforward treatment for ischemic heart disease. The main goal of this therapeutic approach in myocardial ischemia is coronary collateral development by means of the administration of angiogenic cytokines. Research in preclinical models has screened the potential use of molecules such as FGF2, VEGF, PDGF, Neuregulin 1 or SHH. One of the first angiogenic growth factors related to tumor vascularization to be discovered was FGF, which was later linked to angiogenesis and cardiac repair through its action on different cell types including endothelial cells, smooth muscle cell and myoblasts that express FGF receptors (5). The most widely used protein to induce angiogenesis both in preclinical models and in clinical assays is VEGF, a factor that induces vascular hyperpermeability and acts as an endothelial cell-specific mitogen. PDGF participates in angiogenesis and vessel stabilization and its angiogenic synergism in combination with FGF has already been proven in a myocardial infarction model in swine. Other proteins such as Neuregulin-1,

triggers

multiple

responses

including

proliferation

and

survival

cardiomyocytes, promotion of regeneration and decrease of hypertrophy among others (6). Despite its complexity, some investigations in mice have elucidated the critical role of SHH signaling in the maintenance of adult coronary vasculature by promoting angiogenesis and cell survival (7). Its therapeutical potential has also been proven in myocardial ischemia models both in mice and rats (7). Although several clinial trials have explored the use of these proteins, the major hurdle found in these angiogenic clinical trials is the short-lived effect of the administered molecules due to the high instability of proteins when injected as a bolus (8). The evanescence of these compounds in heart tissue has led to unsatisfactory results and studies have failed to demonstrate significant amelioration in treated patients. To overcome these limitations, several technologies have been explored to allow the encapsulation of factors by developing drug delivery systems that permit a controlled and localized release of the growth factors for

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longer time. These systems can also protect proteins from degradation, preserving their bioactivity during release (reviewed in (9)). Particles prepared with the Poly(lactic-co-glycolic acid) (PLGA) copolymer have been widely used due to the excellent biocompatibility and biodegradability of the material. Benefits of using PLGA particles for angiogenesis have been shown in hindlimb ischemia models, resulting in increased blood vessel formation. Also, the effect of delivery of PLGA microparticles loaded with VEGF-A165 has been studied in a rat model of cardiac ischemiareperfusion, demonstrating an increase in heart tissue angiogenesis and arteriogenesis, besides positive remodeling of the heart (10). Moreover, PLGA has been also used to encapsulate heat shock protein 27 (HSP27), which exerts protective effects in cardiac cells under hypoxic conditions. Finally, PLGA microparticles have also been combined with other delivery systems in order to optimize the patterns of growth factor controlled release. Alginate gel/PLGA microsphere combination system containing VEGF enhanced the angiogenic response after hind limb ischemia in rats and mice. This combination system also allowed a dual delivery strategy and improved the effects of single factors.

Tissue Engineering Despite the general benefit that stem cell therapy offers, some important limitations like the low degree of cell engraftment and survival in the heart have been evidenced in cell therapy. As an average, more than 70% of the transplanted cells are lost during the first 48 h, progressively disappearing during the following days. Cell injection implies that a great percentage of cells directly leak through capillaries and also die through anoikis due to the lack of matrix anchorage-dependent survival signals. Furthermore, MI imposes a hypoxic, pro-inflammatory and/or fibrotic environment that harms the transplanted cells. Despite this aspect, a functional improvement has generally been demonstrated after cell treatment in the settings of AMI, chronic ischemic heart failure and dilated cardiomyopathy (reviewed in (11)). Therefore, it has been hypothesized that an increase in the survival rate of the cells would improve their positive effects by reinforcing their trophic effect or even their in vivo differentiation. With this in mind, different scaffold-based approaches (which have received the general term of tissue engineering) have been tested in order to favor cell retention. Tissue engineering (TE), has been defined as the process of creating living, functional tissues to repair or replace the tissue or organ function lost due to age, disease, damage, or congenital defects. Novel biomaterials are being designed to direct cell organization, growth and differentiation in the process of forming functional tissue by providing physical, mechanical and chemical cues. In the setting of cardiac regeneration, the ideal material should be biocompatible, biodegradable (at a rate coupled to cell proliferation and nativetissue deposition), allow cell proliferation, stimulate its differentiation and maturation, and present similar mechanical and physical properties to the healthy heart. Notably, this would include the capacity to sustain rhythmic contraction, variations in frequency and impulse propagation, which are key features of the cardiac tissue.

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Initial studies focused on the use of injectable materials that could improve cell retention and provide structural anchorage (12) while allowing the use of less invasive ways of delivery like catheter-based injections. Early experiments were performed by combining the cells with biomaterials derived from the extracellular matrix, like collagen, fibrin or gelatin. Also, matrigel or other factors that provided a favorable environment rich in cytokines and growth factors were tested. In general, an increased survival rate of the transplanted cells was shown and consequently, a greater improvement of the cardiac function of the treated hearts (13). Thanks to this relatively simple approach, the trophic effect exerted by the cells was boosted by increasing their survival and engraftment in the tissue. Moreover, importantly, it has been observed that some of the injected materials can exert a positive effect themselves, as has been shown, for example, for alginate. This material is liquid, but suffers a phase transition to hydrogel when injected into the desired tissue, as the local calcium concentration increases. Thus, the groups of Dr. Cohen and Dr. Leor have shown that when recent (7 days) or old (60 days) rat infarcts were treated with this alginate solution, wall thickness was significantly increased, while both systolic and diastolic dilatation and dysfunction were prevented. Interestingly, the effect was even superior to that of neonatal CM transplantation (14). Furthermore, alginate also provides means for material modification, as demonstrated by the same group. The former approach was altered by linking IGF1 and HGF to the hydrogel, supplying these cytokines with proteolysis protection. When injected in a rat model of acute MI, modified alginate sequentially released the molecules, which preserved ventricle thickness, attenuated infarct expansion and fibrosis deposition, and also increased angiogenesis and induced CM-cycle re-entry (15). In a different approach, the group led by Randall Lee conjugated the adhesion-promoting motif arginine-glycine-asparagine (RGD) sequence to alginate and showed its therapeutic capacity to treat a model of chronic MI in rat (16).

Cell patches The in vitro construction of 3D grafts and their epicardial implantation has been studied by several groups worldwide. In general, this approach provides cells with a structural support, which helps to increase their retention within the desired area, but also hinders remodeling processes that eventually end up in chamber dilatation. The creation of cellular patches has been developed by using different materials characterized by their biocompatibility and/or biodegradability. Two types of materials in particular have been tested: porous biomaterials or hydrogel/extracellular matrix (ECM)-based matrices. Regarding the first ones, for example, Leor and coworkers tested the putative benefit of treating infarcted rats with a porous 3D alginate scaffold seeded with rat CM previously matured in vitro. Nine weeks after transplantation, graft-implanted animals showed a significant improvement of heart function and decrease in LV dilatation, which was accompanied by extensive vascularization of the scaffold by host-derived vessels, notwithstanding the fact that transplanted CM were mostly replaced by collagen, and no

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evidence of structural integration was found (17). Similarly, Piao et al. seeded BM-MNC on a poly-glycolide-co-coprolactone scaffold to treat MI in rats. The treatment prevented LV dysfunction and adverse remodeling, and stimulated BM-MNC to migrate towards the diseased tissue (18). Fitzpatrick et al employed a mesh of poly-glicoid acid seeded with human fibroblast. Implanted in a rat model of MI, the patch preserved cardiac function through an increased wall thickness and a smaller infarct, mostly related to the paracrine action of the cells (19). Finally, in an interesting approach, the group of Dr. Levenberg reported the in vitro generation of a cardiac tissue by a tri-culture of hESC-derived CM, hESC-derived endothelial cells and embryonic fibroblasts on a sponge of PLGA. The engineered tissues showed high interaction between cell-types, with endothelial cells promoting CM proliferation, and fibroblasts stabilizing endothelial cell-derived capillaries. The constructs also proved their in vitro functionality, but have not been subjected to in vivo testing yet (20).

Cell sheets In year 2002, Shimizu and coworkers first published the application of the cell sheet technology for the treatment of MI (21). In their work, they employed a temperatureresponsive polymer poly(Nisopropylacrylamide). When cells were cultured on this material at 37 ºC, they were able to attach and grow to confluence. Then, when temperature was lowered below 32ºC, the polymer rapidly hydrated and swollen, allowing cells to detach, forming the so-called cell sheet. Furthermore, with a simple procedure, it was also possible to stack sheets, thus increasing the thickness of the graft or its composition. Their first paper (21) showed the production of a four layer graft composed of neonatal rat CM which contracted in vitro. When it was subcutaneously transplanted in nude rats, it continued to beat and promote cell maturation. This group also proved that when transplanted into an injured myocardium, grafts integrated and transmitted impulse propagation without evidence of arrhythmia. On the other hand, Sekine and coworkers showed that when CM were cultured in the same cell sheet as endothelial cells, there was a significant increase in the survival of the CMs and paracrine activity of the sheets. Moreover, when employed to treat a rat model of MI, the effect of the mixed-culture sheets was significantly better than the obtained with CM-only sheets (22). Similarly, when co-cultures of fibroblast and endothelial cells were used, the effect was greater than that of cell sheets composed of endothelial cells alone. Finally, the feasibility of this therapy was established in two pre-clinical relevant models. Bel et al. employed a Rhesus monkey model of MI in which animals were treated with cell sheets composed of adipose-derived stromal cells and ESC-derived cardiac progenitors (SSEA1+). Cells showed a robust engraftment in diseased organs even 2 months postgrafting, inducing an increase of angiogenesis and what is more relevant, no evidence of teratoma formation (23). In a second paper, Miyagawa et al demonstrated that skeletal myoblast sheets induce a significant benefit upon cardiac function, fibrosis and angiogenesis (24) in a pig model of MI.

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Conclusions And Final Remarks In the last two decades, science has assayed new approaches for treating cardiovascular diseases. Among others, stem cell, gene and protein therapies have been shown to present an enormous potential, although, of course, many aspects still need to be solved or better understood. In the case of stem cell therapy, hopes were initially directed towards the differentiation capacity of the cells, which, ideally, could replace the injured heart with new cardiac and vascular tissue. However, the results have not been as positive as expected and data obtained from many in vivo and even clinical studies have shown that the main mechanisms of action of the cells are not through differentiation but through cytokine and factor secretion. There are several reasons for this lack of translation from in vitro to in vivo differentiation, including, together with the variable degree of real differentiation potential among the stem cell populations, the lack of an adequate microenvironment to host the cells and guide their differentiation. Thus, it has been shown that one of the main limitations that stem cell therapy has presented is the low level of engraftment and survival of the transplanted cells, which greatly diminish their efficacy. New strategies, like the combination of stem cells with the bioengineering or micro/nano-technologies, are intended to solve this problem and furthermore, allow to more complex tissues to be created, which can be transplanted into the tissue. Importantly, the employment of materials has proven useful to limit infarct expansion, maintain ventricle geometry and compensate loss of functional capacity. Thus, although many aspects like the electro-mechanical properties of the cardiac cell/tissues will need to be strictly controlled and obtaining a real source of cardiac progenitor cells without tumor or immunological risks is still not straightforward, this new approach for treating cardiovascular disease appears to be a very promising alternative that will boost the established positive benefits of stem cell transplantation.

Acknowledgments Funding sources: Instituto de Salud Carlos III (ISCIII PI050168, PI10/01621, CP09/00333 and ISCIII-RETIC RD06/0014), Ministerio de Ciencia e Innovación (PLE20090116 and PSE SINBAD, PSS 0100000-2008-1), Gobierno de Navarra (Departamento de Educación), Comunidad de Trabajo de los Pirineos (CTP), European Union Framework Project VII (INELPY), Agencia Española de Cooperación Internacional para el Desarrollo (AECID), Caja de Ahorros de Navarra (Programa Tu Eliges: Tu Decides) and the “UTE project CIMA”.

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3. Passier R, van Laake LW, Mummery CL. Stem-cell-based therapy and lessons from the heart. Nature 2008; 453:322-329. 4. Langer R, Vacanti JP. Tissue engineering. Science 1993; 260:920-926. 5. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies. Circulation 2004; 109:2692-2697. 6. Bersell K, Arab S, Haring B, Kuhn B. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 2009; 138:257-270. 7. Pola R, Ling LE, Silver M, Corbley MJ, Kearney M, Blake Pepinsky R, Shapiro R, Taylor FR, Baker DP, Asahara T, Isner JM. The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat Med 2001; 7:706-711. 8. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation 2002; 105:788-793. 9. Formiga FR, Tamayo E, Simon-Yarza T, Pelacho B, Prosper F, Blanco-Prieto MJ. Angiogenic therapy for cardiac repair based on protein delivery systems. Heart Fail Rev 2011. 10. Formiga FR, Pelacho B, Garbayo E, Abizanda G, Gavira JJ, Simon-Yarza T, Mazo M, Tamayo E, Jauquicoa C, Ortiz-de-Solorzano C, Prosper F, Blanco-Prieto MJ. Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia-reperfusion model. J Control Release 2010; 147:30-37. 11. Menasche P. Cell-based therapy for heart disease: a clinically oriented perspective. Mol Ther 2009; 17:758-766. 12. Wall ST, Walker JC, Healy KE, Ratcliffe MB, Guccione JM. Theoretical impact of the injection of material into the myocardium: a finite element model simulation. Circulation 2006; 114:2627-2635. 13. Ryu JH, Kim IK, Cho SW, Cho MC, Hwang KK, Piao H, Piao S, Lim SH, Hong YS, Choi CY, Yoo KJ, Kim BS. Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium. Biomaterials 2005; 26:319326. 14. Leor J, Tuvia S, Guetta V, Manczur F, Castel D, Willenz U, Petnehazy O, Landa N, Feinberg MS, Konen E, Goitein O, Tsur-Gang O, Shaul M, Klapper L, Cohen S. Intracoronary injection of in situ forming alginate hydrogel reverses left ventricular remodeling after myocardial infarction in Swine. J Am Coll Cardiol 2009; 54:1014-1023. 15. Ruvinov E, Leor J, Cohen S. The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction. Biomaterials 2011; 32:565-578. 16. Yu J, Gu Y, Du KT, Mihardja S, Sievers RE, Lee RJ. The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model. Biomaterials 2009; 30:751-756. 17. Leor J, Aboulafia-Etzion S, Dar A, Shapiro L, Barbash IM, Battler A, Granot Y, Cohen S. Bioengineered cardiac grafts: A new approach to repair the infarcted myocardium? Circulation 2000; 102:III56-61. 18. Piao H, Kwon JS, Piao S, Sohn JH, Lee YS, Bae JW, Hwang KK, Kim DW, Jeon O, Kim BS, Park YB, Cho MC. Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model. Biomaterials 2007; 28:641-649.

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Stem cells in Skin Tisular Engineering Sara Guerrero-Aspizua1,2,3, Marcela del Río1,3

Marta

Carretero1,3

and

1. Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT, Madrid, 2. Department of Bioengineering, Universidad Carlos III (UC3M), Madrid, 3. Centro de Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER), Madrid, Spain.

Introduction The presence of adult stem cells have been described in a lot of tissues and organs, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. Stem cells usually remain in a quiescent state for long periods of time but in the presence of the needed stimulus they are activated by a normal need for more cells to maintain tissues, or by pathological conditions such as disease or tissue injury. Typically, there is a very small number of stem cells in each tissue, making isolation of large quantities of stem cells difficult. Scientists in many laboratories are trying to find better ways to grow substantial numbers of adult stem cells in cell culture and to manipulate them to generate specific cell types so they can be used to treat damaged tissues. The epidermis is the outermost component of the skin formed mostly by a particular kind of epithelial cells known as keratinocytes. It is morphologically divided into different layers or strata. From the bottom (innermost), these layers are basal, spinous, granular, and cornified cell layer (Fig. 1). Keratinocytes produced in the basal layer, where cell proliferation is confined, move upward to the outer surface in a process named as epidermal differentiation. The stem cell´s niche of the skin is located in the basal layer of the epidermis (Fig. 1) and at the base of hair follicles, and knowledge and maintenance of this compartment is crucial for the study of the skin, in both physiological and pathological conditions. Given the importance of these cells, the study of the skin has been widely developed over the past decades, for the design of future therapeutical cutaneous strategies and for replacement therapies, where its wide use is a direct consequence of its easy access [1], [2], [3]. Current cell culture techniques have optimized in vitro expansion of cells obtained from skin biopsies to be assembled in three-dimensional matrices and engineered skin equivalents amenable to clinical use.

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Epidermal Stem Cells The constant renewal of the epidermis and hair follicle, together with its ability to respond efficiently to external aggressions that lead to their loss always constituted a clear indication of the existence of epidermal stem cells. The search for molecular markers that funtionally characterize the epidermal stem cell population is a key point has been investigated extensively during the last decades. However, these studies did not lead the way to the identification of bona fide functional markers for human epidermal stem cells (ESCs). Markers including, α6bri/CD71dim, and Lrig1+ were suggested to be useful to enrich for highly clonogenic cells [4]. However, the in vitro proliferative capacity of keratinocytes remains the most consistent way to identify stem cells of human interfollicular epidermis, since more than 20 years ago [5]. Thus, clonogenic assays appear to be the best predictors of “stemness”, at least in terms of extensive proliferative capacity of the putative interfollicular epidermal stem cells [5], [6]. Although previous attempts to assess the putative stem cell behaviour of single genetically modified human clones in vivo were unsuccessful [6], recent advances in organotypic cultures and surgical techniques have now made it possible to achive this goal [7].

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Although skin biopsies are the most common source of keratinocytes and epidermal stem cells, recent studies have deepened in the generation of these cells from human embryonic stem cells (hESc) and induced pluripotent stem cells (iPSc) [8]. Thus, much of the current enthusiasm comes from the possible therapeutic use of somatic cells (including keratinocytes) derived from them. Howard Green and co-workers followed the time- and migration-dependent development of the keratinocyte lineage from human embryonic stem cells in culture [9]. They also showed that optimization of culture conditions improves the proliferation, but not sufficiently to permit their clonal isolation. More recently, in collaboration with our group, french researchers achieved the differentiation of bona-fide epidermal stem cells from hESc as demonstrated after stringent in vivo tests. Progress in this field is really fast, and we must be alert in the coming years to move in a realistic way all these advances to day-to-day clinic.

Skin Bioengineering: Applications

Pre-Clinical

And

Clinical

In vivo studies in the skin of human beings are obviously limited by ethical and practical constraints. Current knowledge comes mainly from the use of murine models, including knockout and transgenic strategies. However, due to the differences existing between human and murine skin architecture and physiology, it is necessary to search for models closer to the human context. In that search, scientists have also performed, studies in large animals such as pigs, whose skin architecture and dynamics resemble that of humans, although such an alternative is expensive and requires special animal facilities [10]. On the other hand human skin organotypic cultures [11], [12], [13] are restricted by their short culture life span and the absence of responses such as angiogenesis. Researchers have also

used

xenogenic

immunocompromised

transplantation

mice

[14],

but

donor/patient

cutaneous

biopsies

addition

difficulties

sourcing

the biggest disadvantage here, is the great heterogeneity existing between skin samples. A possibility to overcome these drawbacks involves the stable regeneration of normal or diseased human skin in appropriate hosts by means of tissue engineering [15]. This approach represents a significant challenge that involves adequate human epidermal stem cell manipulation in vitro.

The Skin Humanized Mouse Model Our team has developed an improved whole autologous bioengineered skin based on the use of a fibrin three-dimensional dermal scaffold in which fibroblasts are embedded (World Patent WO/2002/072800). The method involves the deconstruction-reconstruction of the skin of healthy donors or patients suffering from different diseases (Fig. 2). That is, in

vitro isolation and amplification of skin cells (fibroblasts and keratinocytes, including the population of epidermal stem cells) from biopsies and their assembly as a bioengineered

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skin that is subsequently transplanted to immunodeficient mice [16], [17]. It has been demonstrated by immunohistochemical studies over long follow-up periods that a healthy and mature skin with human architecture is able to persist after a large number of epidermal turn-overs [17] demonstrating that the model fulfils the requirement of stem cells preservation in vivo . This was even further tested by the achievement of regenerated skin after a secondary transplant protocol [7]. Another advantage of the model is that it allows the generation of a large number of engrafted mice containing a homogeneous and significant area of human skin, from a small skin biopsy

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The so-called skin-humanized mouse model based on the permanent engraftment of a bioengineered human skin onto the back of immunodeficient mice aimed originally to the study the physiopathology of the skin evolved also toward disease modeling. [16], [17], [18] (Fig.2). In this regard, the skin-humanized mouse model has been really useful for modeling several human monogenic skin diseases, such as the skin fragility disease epidermolysis bullosa (EB) [20], [21], the cancer-prone disease xeroderma pigmentosum (XP) [22], and the Netherton syndrome (NS), an epidermal differentiation disorder caused by mutations in the

SPINK5 gene [23] and Pachyonychia Congenita (PC) [24], a debilitating skin disorder. This model is also usefull for driving studies in normal human skin, both in a physiological or pathological context, such as wound healing [25], [26], [27]. Finally, our team were also able to generate a skin humanized mouse model for complex pathologies such as psoriasis, where the immune component plays a central role [28]. All these humanized mouse models have been a unique platform on which to evaluate innovative therapeutic strategies in dermatology such as cell therapy using ESCs [29], [8], [30], [31] and gene therapy [32], [16], [26], [25], [33], [34].

Preclinical Humanized Mouse Models Modeling physiological process: Wound healing. Human cutaneous wound healing is a complex process not completely understood [35], [36]. Development of chronic ulcers coupled to different diseases is nowadays a major problems of the health care system and carry a high social cost [37]. The development of effective treatment of chronic ulcers is one of the greatest medical challenges [38]. Reliable human wound-healing models are obviously necessary to address both mechanistic and therapeutic matters. To this end, our team has developed an in vivo wound-healing model by creating excision wounds on the skin humanized mouse model that recapitulates all major features of cutaneous wound healing (Fig. 3). Monitoring the expression of various epidermal and mesenchymal markers showed that re-epithelialization, dermal matrix remodeling and basal membrane reorganization accurately mimic the process in humans [25]. The bioengineered skin humanized mouse model of wound healing emerges as a unique platform for evaluating pharmacological, cell and gene therapy strategies for wound healing [39], [26].

Modeling rare monogenic skin diseases Development of rare disease models, especially in a human context, would contribute to the basic and translational research toward individualized. In recent years, our group has focused its interest in the modeling of different skin diseases that are detailed below.

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Modeling response to UV light in normal regenerated human skin and Xeroderma Pigmentosum (XP): Studies of UVB effects on the skin of volunteers are precluded by ethical and technical reasons and are unimaginable in cancer-prone patients. Molecular changes associated to UVB irradiation have been extensively characterized by in vitro studies in keratinocytes [40], [41]. We challenged our system to assess whether it responds adequately to UV irradiation. Moreover, by using Caucasian or African-descent donor keratinocytes we were able to confirm the well known modulation of the UVB responses by the amount of skin pigmentation [42], [43]. The model also proved suitable to test topic photoprotective agents as well as to study DNA damage repair kinetics after UVB irradiation in terms of epidermal hyperplasia and keratin K6 induction [44], [45]. Our model recapitulated the human phenotype and appears suitable to study chronic effects including mutagenesis and carcinogenesis (Fig. 3). We have also established a photosensitive humanized skin model by grafting bioengineered skin containing cells from Xeroderma Pigmentosum (XP) patients. Xeroderma Pigmentosum (XP) is an autosomal and recessive disorder characterized by a severe deficiency in DNA-repair, caused by the mutation of nucleotide excision repair (NER) enzymes. The first in vivo evidence of XP keratinocyte deficiency in nucleotide excision repair (NER) was obtained after acute UVB irradiation [22].

Recessive Dystrophic Epidermolysis Bullosa (RDEB): Epidermolysis Bullosa (EB) includes a clinically and genetically heterogeneous group of rare skin diseases characterized by skin fragility [46]. One of them, the recessive subtype of DEB (RDEB; OMIM: 120120), a very severe form of EB, is due to mutations in the gene coding for type VII collagen (COL7A1). General blistering of regenerated human skin was recapitulated in the humanized mouse model containing collagen VII-null RDEB keratinocytes similarly to skin biopsies from RDEB patients. Complete and permanent reversion of this phenotype was obtained by genetic modification of RDEB epidermal stem cells using retroviral vectors carrying the human collagen VII gene (COL7A1) used in the generation of the skin equivalents. Phenotypic correction was also attained by a cell-based therapy based on the use of a chimerical bioengineered skin (Fig. 3).

Pachyonychia Congenita (PC) Pachyonychia congenita (PC) is a rare autosomal dominant keratin disorder characterized by thickened and dystrophic nails as well as painful palmoplantar keratoderma [47], [48]. PC is caused by dominant-acting mutations in any one of the genes encoding the differentiation-specific and stress-inducible keratins, K6a, K6b, K16, or K17 [49], [50]. The dominant-negative mutations on these genes lead all of the epithelial fragility symptoms associated with PC. Our group has contributed with the establishment of two skin-

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humanized models of PC (Fig. 3). One of them involves the use of bioengineered skin from an uninvolved area of PC patients carrying the same K6a mutation, displayed epidermal phenotypic changes consistent with a hyperproliferative response. On the other hand, the use of keratinocytes from affected skin from another patient carrying a different mutation in the same codon of the keratin K6a gene resulted in the development of a constitutively expressed, bona fide PC phenotype. Currently we are evaluating the amenability of these humanized PC models to genetic intervention similar to that recently reported in PC patients [51].

Netherton Syndrome (NS) Netherton syndrome (NS) is a hereditary skin disorder caused by mutations in the

SPINK5 gene encoding the protease inhibitor LEKTI [52],

[53] that causes defective

keratinization, recurrent infections, and dehydration. In the more severe cases, NS is mortal in about 10% of the patients in the first year of life [54],; [55], [56]. Grafting of human NS bioengineered skin onto immunodeficient mice made it possible to recapitulate the characteristic histological features of NS [23]. By using a lentiviral vector to direct SPINK5 expression in keratinocytes resulted in reversal of skin phenotype (Fig. 3) [23]. In this study we found that limited numbers of LEKTI-expressing cells mediate valuable beneficial effects likely through paracrine effects.

Modeling a complex inflammatory skin disease: Psoriasis Inflammatory and autoimmune cutaneous disorders have a high prevalence among the population. This is the reason because it represents a major health and social concern worldwide. Skin infiltrating T lymphocytes play a central function in triggering and maintaining common chronic inflammatory skin diseases such as psoriasis and atopic dermatitis, where a deregulation in the Th1/Th2/Th17 balance exists. In psoriasis this equilibrium is skewed towards Th1, whereas a Th2 phenotype is predominant in atopic dermatitis. Th17 cells are more abundant in both disorders [57]. Animal models for inflammatory cutaneous pathologies will contribute to the complete understanding of the essential mechanisms underlying the epidermal-immune cell interactions and the development of new therapeutic strategies [58], [59], [60], [61], [62]. Xenotransplantation models of psoriasis closely mimic human disorders and have been used extensively [63], [64], [14]. However, this approach presents ethical and practical limitations, in addition to the wide heterogeneity existent between samples. Within this context, the bioengineered-skin humanized mouse model emerges as a powerful tool. One of the several potential advantages over other genetically modified or xenotrasplantation animal models is the feasibility of performing studies in a human context on homogeneous and large samples. This strategy was recently used to generate a bona fide skin-humanized mouse model for psoriasis (Fig. 3) [28]. We demonstrated that a healthy human skin regenerated by tissue bioengineering, might develops a psoriasiform phenotype if the

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appropriate signals are present, i.e. a wounding stimulus and the appropriate cytokines together with specific lymphocyte subpopulations (Th1/Th17) .These signals play a pivotal role in the development of the psoriatic plaque. This approach has contributed to clarify the immunopathogenesis of psoriasis.

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Clinical Applications Of Bioengineered Skin Skin replacement is common practice in the clinic (i.e. temporary coverage of burn patients with allogeneic cadaver skin grafts) but a rejection of the epidermal layer of the grafted skin is clinically evident within 2-3 weeks post-grafting. Therefore, permanent skin regeneration has only been achieved with autologous ESCs transplantation. Nevertheless, allogeneic skin substitute transplantation is currently used to improve the healing of both acute and chronic wounds, including EB lesions [65], [66], [67].

Permanent replacement of skin losses Bioengineered skin substitutes appeared as artificial alternatives to skin grafts that avoid the pain, potential complications and surface limitations of native skin harvesting. Skin substitute development is based taking into account the following three components: 1) cell source, 2) tissue regeneration-inducing factors, and 3) matrix or scaffold [68]. Many recent reviews have summarized the development and current status of matrices and skin substitutes [69], [70], [71], [72]. Although an ideal skin substitute is still a matter of study, we have

developed

fibroblast-containing

fibrin-based

bioengineered

skin

product

(WO/2002/072800) that does fulfill many of the clinical requirements. Fibrin is the primary and temporary wound healing matrix allowing blood clotting and migration of both, epithelial and mesenchymal cellular elements, which are required for the repair of damaged tissue. The development of a fibrin-based dermal matrix from pure plasma, allowed the generation of fully autologous skin equivalents since keratinocytes, fibroblasts and fibrin may come from the same individual [17]. Furthermore, the fibrin-based bioengineered skin developed by our team has been used successfully, in its autologous version, for permanent skin regeneration in different clinical challenges such as extensive burns, necrotizing fascitis, removal of giant nevi and graft-versus-host disease (Fig. 4), [17], [73], [74].

Temporary dressing for chronic wounds In the case of chronic wounds, the goals of skin substitute therapy have evolved to providing a temporary biologic dressing that improves skin tissue regeneration and wound healing by stimulating

the recipient's

own skin cells. A great improvement in

efficacy/recurrence ratio has been achieved by using this allogeneic bioengineered skin as a temporary cover. In fact, an 80% healing rate was attained by the weekly application of fresh allogeneic bioengineered skin during an average period of 6.6 weeks (Fig.4). Relatively high percentage of ulcer recurrence (25%) is observed since these temporary substitutes do not cure the underlying ischemic or diabetic disease [75].

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Skin bioengineering for Epidermolysis Bullosa Rare diseases are often chronically debilitating or even life-threatening and the impact on the quality of life of affected patients (of whom many are children) and their family members is significant. Cutaneous gene therapy has been one of most intensively studied fields [76]. The first successful gene therapy trial for junctional epidermolysis bullosa (JEB), has been reported [77]. Skin bioengineering in combination with genetically modified human keratinocytes has lead to the long-term correction of RDEB, in pre-clinical assays [78], [21].

Allogeneic bioengineered skin Allogeneic bioengineered skin transplantation has proven to be of clinical value when used as a healing device aiming at tissue repair/regeneration for chronic wounds [79], [80], [81], [82], [83], [84]. Surgical intervention is commonly performed in EB patients, to correct deformities of the hands and feet, but high recurrence and consequently the need for repetitive surgery are common. Standard surgical procedures for the management of hand deformities in DEB include incisional release of contracture and digits follow by autologous partial-thickness skin grafts transplantation to cover secondary wounds. We are currently

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testing the clinical benefits of allogeneic bioengineered skin transplantation on DEB patient donor sites to reduce pain and accelerate healing.

Autologous “revertant” bioengineered skin The concept of somatic revertant mosaicism refers to the occurrence of a natural phenomenon involving spontaneous genetic correction of a first pathogenic mutation in a somatic cell [85]. The frequency of this phenomenon of genetic reversion, thought to be rare for years, appears to be more common than expected [86], [87]. To date, the appearance of revertant mosaicism has already been found in three Spanish RDEB patients. These patients display patches of non-blistering unaffected skin, characterized by the presence of type VII collagen that was almost absent in the non revertant skin. Transplantation of autologous “revertant” bioengineered skin may be a personalized EB therapy for patients with somatic mosaicism and is currently being explored in our laboratory in collaboration with the laboratory of Dr. Marcel Jonkman in the Netherlands. Preliminary pre-clinical results are encouraging. Insight into human epidermal stem cell function and homeostasis helped to establish the base for the development of modern skin tissue engineering. Excitement has been recently brought to the field by the broad range of possibilities offered by hES and iPS cells which includes the capacity to be converted into keratinocytes. Upon further refinements, the use of these cells will certainly improve skin bioengineering for important therapeutic applications such as extensive skin losses or severe genetic blistering disorders. Moreover, we can´t obviate the great progress that involves the development of humanized mouse models in understanding pathogenesis and designing new therapeutic strategies for all types of skin diseases, in a preclinical context.

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52. Bitoun, E., et al., LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome. 2003. Hum Mol Genet 12(19):2417-30. 53. Chavanas, S., et al., Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. 2000. Nat Genet 25(2):141-2. 54. Borgoño, C.A., et al., A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation. 2007. J Biol Chem 282(6):3640-52. 55. Descargues, P., et al., Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and chymotrypsin-like hyperactivity in Netherton syndrome. 2006. J Invest Dermatol 126(7):1622-32. 56. Ishida-Yamamoto, A., et al., LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum. 2005. J Invest Dermatol 124(2):360-6. 57. Di Cesare, A., P. Di Meglio, and F.O. Nestle, A role for Th17 cells in the immunopathogenesis of atopic dermatitis?. 2008. J Invest Dermatol 128(11):2569-71. 58. Danilenko, D.M., Review paper: preclinical models of psoriasis. 2008. Vet Pathol 45(4):563-75. 59. Nestle, F.O. and B.J. Nickoloff, From classical mouse models of psoriasis to a spontaneous xenograft model featuring use of AGR mice. 2005. Ernst Schering Res Found Workshop (50):203-12. 60. Schon, M.P., Animal models of psoriasis - what can we learn from them?. 1999. J Invest Dermatol 112(4):405-10. 61. Shiohara, T., J. Hayakawa, and Y. Mizukawa, Animal models for atopic dermatitis: are they relevant to human disease?. 2004. J Dermatol Sci 36(1):1-9. 62. Zheng, T. and Z. Zhu, Lessons from murine models of atopic dermatitis. 2005. Curr Allergy Asthma Rep 5(4):291-7. 63. Gilhar, A., et al., T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice. 1997. J Invest Dermatol 109(3):283-8. 64. Nickoloff, B.J., et al., Severe combined immunodeficiency mouse and human psoriatic skin chimeras. Validation of a new animal model. 1995. Am J Pathol 146(3):580-8. 65. Eisenberg, M. and D. Llewelyn, Surgical management of hands in children with recessive dystrophic epidermolysis bullosa: use of allogeneic composite cultured skin grafts. 1998. Br J Plast Surg 51(8):608-13. 66. Falabella, A.F., et al., Tissue-engineered skin (Apligraf) in the healing of patients with epidermolysis bullosa wounds. 2000. Arch Dermatol 136(10):1225-30. 67. Fivenson, D.P., et al., Graftskin therapy in epidermolysis bullosa. 2003. J Am Acad Dermatol 48(6):886-92. 68. Langer, R. and J.P. Vacanti, Tissue engineering. 1993. Science 260(5110):920-6. 69. Beele, H., Artificial skin: past, present and future. 2002. Int J Artif Organs 25(3):163-73. 70. Ehrenreich, M. and Z. Ruszczak, Update on tissue-engineered biological dressings. 2006. Tissue Eng 12(9):2407-24. 71. Hansen, S.L., et al., Using skin replacement products to treat burns and wounds. 2001. Adv Skin Wound Care 14(1):37-44; quiz 45-6. 72. Horch, R.E., et al., Tissue engineering of cultured skin substitutes. 2005. J Cell Mol Med 9(3):592-608. 73. Llames, S., et al., Clinical results of an autologous engineered skin. 2006. Cell Tissue Bank 7(1):47-53. 74. Gómez, C., et al., Use of an autologous bioengineered composite skin in extensive burns: Clinical and functional outcomes. A multicentric study. 2011. Burns 37(4):580-9.

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75. Llames, S., et al., Treatment of diabetic foot trophic lesions using a tissue-engineered dermal graft. 2008. Tissue Engineering: Part A 14(5):924. 76. Del Rio, M., F. Larcher, and J. Jorcano, Recent advances in gene therapy with skin cells. 2002. European Reviews 10:369-388. 77. Mavilio, F., et al., Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. 2006. Nat Med 12(12):1397-402. 78. Del Rio, M., et al., Current approaches and perspectives in human keratinocyte-based gene therapies. 2004. Gene Ther 11 Suppl 1:S57-63. 79. Camblor-Santervas, L., et al., Tratamiento de úlceras vasculares crónicas con equivalentes cutáneos obtenidos mediante ingeniería tisular. 2003. Angiología 55(1):21-33. 80. Coto-Segura, P., et al., Letter: efficacy of a self-made artificial skin in the treatment of chronic ulcers. 2007. Dermatol Surg 33(3):392-4. 81. Coto-Segura, P., et al., Potent analgesic effect of tissue-engineered skin in a terminal patient with severe leg ulcer pain. 2008. Dermatol Surg 34(10):1414-6. 82. Eisenbud, D., et al., Skin substitutes and wound healing: Current status and challenges. 2004. Wounds 16:2-17. 83. Herschthal, J. and R.S. Kirsner, Potent topical steroids during pregnancy affect newborn birth weight. 2011. J Invest Dermatol 131(4):808. 84. Limova, M., Active wound coverings: bioengineered skin and dermal substitutes. 2010. Surg Clin North Am 90(6): 237-55. 85. Davis, B.R. and F. Candotti, Genetics. Mosaicism--switch or spectrum?. 2010. Science 330(6000):46-7. 86. Lai-Cheong, J.E., J.A. McGrath, and J. Uitto, Revertant mosaicism in skin: natural gene therapy. 2011. Trends Mol Med 17(3):140-8. 87. May, M., Mutations to the rescue. 2011. Nat Med 17(4):405-7.

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Modeling Parkinson’s disease using induced pluripotent stem cells Adriana Sánchez-Danés1, Antonella Consiglio1,2, Ángel Raya3,4,5 1. Institute for Biomedicine (IBUB), University of Barcelona, Barcelona, Spain 2. Department of Biomedical Science and Biotechnology, University of Brescia, Brescia, Italy 3. Control of Stem Cell Potency Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain 4. Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain 5. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease, with a prevalence of 1% at age 65 and around 5% by age 85 [1]. PD is a progressive and incurable neurodegenerative disorder characterized by motor clinical manifestations, although non-motor affectations are also relevant in advanced stages of the disease [2-4]. The four cardinal motor symptoms include bradykinesia, resting tremor, rigidity and postural instability. Motor clinical manifestations have been associated with the degeneration of dopaminergic neurons (DAn) from the substantia nigra pars compacta, specifically known as A9-subtype ventral mesencephalic DAn (vmDAn), responsible for controlling body movement [reviewed in 5]. Around 90% of PD cases are sporadic, while 10% have familial or genetic origin, mutations in the LRRK2 gene being the most frequent cause of genetic PD. The pathogenic mechanisms leading to PD remain poorly understood, mainly owing to the lack of suitable animal and cellular models of PD initiation and progression. Therefore, there is an urgent need for developing reliable experimental models that recapitulate the key features of PD.

Modeling disease using induced pluripotent stem cells (iPSC) The development in 2007 of human induced pluripotent stem cells (iPSC) enabled a new generation of disease modeling strategies. Yamanaka’s group described the process of induced cell reprogramming, by which human fibroblasts could be reprogrammed to a pluripotent state after ectopic expression of just four transcription factors (OCT4, SOX2, KLF4 and c-MYC) [6]. Since then, multiple iPSC lines have been generated from different

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types of somatic cells and combinations of transcription factors [reviewed in 7]. Human iPSC share many characteristics with human embryonic stem cells (hESC), including similarities in their morphologies, gene expression profiles, proliferation rates, and capacity to differentiate into cell types of the three embryonic germ layers in vitro and in vivo [reviewed in 7]. A key advantage of induced cell reprogramming is the possibility of generating iPSC from patients that carry the precise genetic variants, both known and unknown, which may contribute to the disease. Specifically, iPSC can be generated from patients showing sporadic or familial forms of the disease. It is important to note that iPSC-based disease modeling may not be applicable to some conditions. Certain genetic alterations might affect the efficiency of reprogramming process, thus making iPSC derivation more difficult or altering the nature of the iPSC obtained. In some diseases, correction of the genetic mutation could be required for successful reprogramming, as it has been reported for Fanconi anemia [8]. Reprogramming and generation of iPSC requires cell division, therefore, iPSC from diseases affecting in vitro cell growth would be difficult to generate. As reprogramming erases most epigenetic modifications, modeling epigenetic diseases could also be problematic. Probably, the easiest diseases to model using iPSC would be highly penetrant and cell autonomous diseases or early onset. Although, in principle, multigenic disorders could also be modeled, diseases heavily influenced by environmental factors would be more complicated to model through iPSC-based technology. Modeling human disease using iPSC technology involves two steps. First, the generation of iPSC from representative patients; and second, the differentiation of patientspecific iPSC towards disease-relevant cell type(s). Hence, a critical issue for disease modeling with iPSC is the availability of reliable and reproducible protocols that could efficiently direct pluripotent stem cells towards the specific cell types affected in the disorder of interest. A growing number of patient-specific iPSC lines are being generated and analyzed in the context of human disease modeling [reviewed in 9]. However, only in some cases it has been possible to reproduce the diseased phenotype in vitro. In the case of neurological disorders, only a handful of iPSC-based models have generated convincing phenotypes, providing initial “proof of concept” for this approach: spinal muscular atrophy [10], familial dysautonomia [11], and Rett’s syndrome [12]. It has been postulated that disease phenotypes that appear during development would be easier to model than those of lateonset neurodegenerative diseases such as PD or Alzheimer’s disease [13]. Moreover, it has been hypothesized that neural cell types derived from pluripotent stem cells in vitro may represent early stages of nervous system development. Finally, it has been argued that many neurodegenerative diseases may result form a particular combination of genetic factors with environmental stressors. For these reasons, it was unclear until very recently whether iPSC-based technology could be used to model the most frequent forms of neurodegenerative diseases, which all share the characteristics of late onset, slow progressive course, and complex genetic predisposition.

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3. Modeling PD using iPSC An experimental model of PD should recapitulate most, if not all, the salient features of the disease, including progressive loss of vmDAn and the typical neuropathological inclusions (Lewy bodies and Lewy neurites) in the surviving neurons [reviewed in 4]. To date, several studies have reported the generation of iPSC from patients suffering from sporadic and genetic PD (Table 1). The first reported generation of PD-specific iPSC was from a sporadic PD patient in 2008 [14], although that study did not go beyond the generation of patient-specific iPSC. Over the following year, the Jaenisch’s laboratory showed that iPSC derived from PD patients were able to differentiate towards dopaminergic neurons, but no signs of neurodegeneration or disease-related phenotypes were observed in those cells [15]. Of note, the authors generated reprogramming-gene free iPSC lines from skin fibroblasts of 5 patients of idiopathic PD. Using in vivo experiments it was shown that dopaminergic neurons differentiated from these PD-specific iPSC were able to survive and engraft in the rodent striatum for at least 12 weeks, with a small number of cells co-expressing tyrosine hydroxylase (TH) and G-protein-gated inwardly rectifying K+ channel subunit (GIRK2), a hallmark of vmDAn [16]. Remarkably, injection of iPSC-derived DAn into the brains of 6OHDA-lesioned rats resulted in improvement of the motor symptoms [16]. However, no PDrelated neurodegeneration phenotypes were evident in the injected cells, raising the question as to whether relevant spontaneous phenotypes could be observed in the time frame of in vitro experiments or in vivo assays in short-lived organisms. It has also been reported the derivation of iPSC from one patient with a triplication of

SNCA locus [17]. The SNCA gene, encoding α-synuclein, was the first gene associated to familial PD [18]. Moreover, α-synuclein is the major component of Lewy bodies [reviewed in 5]. DAn derived from these patient-specific iPSC produced double amount of α-synuclein transcript and protein. Again, however, no degeneration was observed in the iPSC-derived neurons. These results were independently confirmed by another group, which generated iPSC from one patient with a triplication in SNCA locus [19], and reported accumulation of αsynuclein, overexpression of oxidative stress markers, and increased sensitivity to peroxideinduced oxidative stress in iPSC-derived DAn. These combined results provide support for the usefulness of iPSC as valuable tools for studying synucleinopathies in vitro. Nguyen and colleagues reported the generation of iPSC lines from one PD patient carrying the G12019S mutation in the LRRK2 gene, the most common cause of genetic PD. However, degeneration of DAn from LRRK2-iPSC was not detected unless the neurons were challenged with stressors [20]. Indeed, LRRK2-iPSC-derived DAn showed caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, or 6-hydroxydopamine. Furthermore, the expression of key oxidative stressresponse genes and α-synuclein was found increased in neurons from LRRK2-iPSC, when compared to those differentiated from control iPSC or hESC; although the differences observed were modest. The fact that those results were based on cell lines generated from only one patient and one control cell line makes further validation warranted.

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iPSC lines generated from mutations in genes associated with mitochondrial function, such as PINK1 and PARKIN have also been reported. However, neurodegeneration phenotypes in iPSC-derived DAn have not observed in any of these studies. The Krainc’s laboratory generated iPSC from patients with three different mutations in the PINK1 gene [21]. They showed that patient-specific iPSC lines were able to differentiate into DAn and that mutant neurons showed impaired recruitment of lentivirally expressed Parkin to mitochondria upon depolarization, increased mitochondrial copy number, and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. They also showed that ectopic expression of wild type PINK1 in mutant neurons corrected these alterations. Similarly, iPSC from two patients with mutations in the PARKIN gene have been generated [22]. In this case, iPSC derived-DAn showed increased spontaneous DA release, decreased DA uptake and increased ROS generation upon treatment with dopamine. Importantly, lentiviral transduction of Parkin was able to rescue these phenotypes. Therefore, these two models represent valuable cellular models to study the mitochondrial role/involvement in PD pathogenesis. Recently, our groups have generated iPSC lines from seven patients with idiopathic PD and four patients carrying G2019S mutation in the LRRK2 gene [23]. In this study, we showed that over long-time culture (2.5 months), vmDAn differentiated from PD-iPSC exhibited morphological alterations, including fewer and shorter neurites (Fig. 1),

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and increase in the number of apoptotic neurons (Fig. 2), signs that were not evident in DAn differentiated from control iPSC. Moreover, we found α-synuclein accumulation in DAn derived from LRRK2-iPSC when cultured for 40 days. Importantly, the appearance of the neurodegeneration phenotypes in DAn differentiated from either idiopathic or LRRK2-associated PD was shown to be the consequence, at least in part, of impaired autophagy in those cells, illustrating the usefulness of iPSC-based disease modeling to recapitulate the pathogenic mechanisms underlying PD progression. Moreover, our study revealed that the increased susceptibility of DAn derived from idiopathic PD patients to degenerate in vitro after long-time culture appears to be encoded in their genome [23].

Probably the most significant finding of this latter work was the identification of spontaneous phenotypes in long-term cultures of DAn from PD patients. It is important to note that the appearance of PD-related phenotypes critically depended on the time span of the cultured neurons. Based on these findings, we hypothesized that DAn cultured for up to 75 days suffered from culture-related stress conditions, which could mimic in vivo aging of patients, and consequently induce the development of PD-related phenotypes in vitro. In support of this view, Soldner and colleagues had argued that the absence of PD-related phenotypes in their studies could be due to the short time span of their cultured neurons (3242 days). It is therefore, likely, that the absence of spontaneous phenotypes in iPSC-derived DAn obtained in other studies, including that of Nguyen and colleagues [20], could be due to their neuron cultures not being “aged” enough. In addition to this, another reason that could account for our success in observing spontaneous phenotypes in PD-specific iPSC-derived DAn, as opposed to previous studies [15-17, 19-22], could be the enrichment of vmDAn in

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our cultures [23, 24], since this subtype of DAn is particularly susceptible to PD-related neurodegeneration (see Table 1).

Concluding remarks The generation of reliable iPSC-based models for late-onset neurodegenerative disorders has been described as a difficult task to achieve [13]. However, recent studies have demonstrated the feasibility of developing PD models based on iPSC from patients of both genetic and idiopathic forms of PD that recapitulate the key features of the disease, including α-synuclein accumulation and dopaminergic degeneration [23]. Importantly, the appearance of spontaneous phenotypes in such models appears to depend on the differentiation towards the specific neuronal subtype affected in the disease (vmDAn), and on the maintenance of dopaminergic neurons for long time in culture, thus mimicking in vitro “aging”. The generation of these genetic and idiopathic PD models opens the door to the elucidation of the pathogenic mechanisms that lead to PD initiation and progression, and to the search for drugs that may prevent or rescue neurodegeneration in PD.

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References 1. de Rijk, M.C., Launer, L.J., Berger, K., Breteler, M.M., Dartigues, J.F., Baldereschi, M., Fratiglioni, L., Lobo, A., Martinez-Lage, J., Trenkwalder, C., and Hofman, A. Prevalence of Parkinson's disease in Europe: A collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology (2000) 54:S21-3. 2. Lees, A.J., Hardy, J., and Revesz, T. Parkinson's disease. Lancet (2009) 373:2055-66. 3. Obeso, J.A., Rodriguez-Oroz, M.C., Goetz, C.G., Marin, C., Kordower, J.H., Rodriguez, M., Hirsch, E.C., Farrer, M., Schapira, A.H., and Halliday, G. Missing pieces in the Parkinson's disease puzzle. Nature medicine (2010) 16:653-61. 4. Schapira, A.H. and Tolosa, E. Molecular and clinical prodrome of Parkinson disease: implications for treatment. Nature reviews. Neurology (2010) 6:309-17. 5. Martin, I., Dawson, V.L., and Dawson, T.M. Recent advances in the genetics of Parkinson's disease. Annu Rev Genomics Hum Genet (2011) 12:301-25. 6. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell (2007) 131:861-72. 7. Stadtfeld, M. and Hochedlinger, K. Induced pluripotency: history, mechanisms, and applications. Genes Dev (2010) 24:2239-63. 8. Raya, A., Rodriguez-Piza, I., Guenechea, G., Vassena, R., Navarro, S., Barrero, M.J., Consiglio, A., Castella, M., Rio, P., Sleep, E., Gonzalez, F., Tiscornia, G., Garreta, E., Aasen, T., Veiga, A., Verma, I.M., Surralles, J., Bueren, J., and Belmonte, J.C. Diseasecorrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature (2009) 460:53-9. 9. Robinton, D.A. and Daley, G.Q. The promise of induced pluripotent stem cells in research and therapy. Nature (2012) 481:295-305. 10. Ebert, A.D., Yu, J., Rose, F.F., Jr., Mattis, V.B., Lorson, C.L., Thomson, J.A., and Svendsen, C.N. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature (2009) 457:277-80. 11. Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A., Tabar, V., Sadelain, M., and Studer, L. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature (2009) 461:402-6. 12. Marchetto, M.C., Carromeu, C., Acab, A., Yu, D., Yeo, G.W., Mu, Y., Chen, G., Gage, F.H., and Muotri, A.R. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell (2010) 143:527-39. 13. Han, S.S., Williams, L.A., and Eggan, K.C. Constructing and deconstructing stem cell models of neurological disease. Neuron (2011) 70:626-44. 14. Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K., and Daley, G.Q. Disease-specific induced pluripotent stem cells. Cell (2008) 134:877-86. 15. Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., Isacson, O., and Jaenisch, R. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell (2009) 136:964-77. 16. Hargus, G., Cooper, O., Deleidi, M., Levy, A., Lee, K., Marlow, E., Yow, A., Soldner, F., Hockemeyer, D., Hallett, P.J., Osborn, T., Jaenisch, R., and Isacson, O. Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc Natl Acad Sci U S A (2010) 107:159216.

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17. Devine, M.J., Ryten, M., Vodicka, P., Thomson, A.J., Burdon, T., Houlden, H., Cavaleri, F., Nagano, M., Drummond, N.J., Taanman, J.W., Schapira, A.H., Gwinn, K., Hardy, J., Lewis, P.A., and Kunath, T. Parkinson's disease induced pluripotent stem cells with triplication of the alpha-synuclein locus. Nat Commun (2011) 2:440. 18. Polymeropoulos, M.H., et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science (1997) 276:2045-7. 19. Byers, B., Cord, B., Nguyen, H.N., Schule, B., Fenno, L., Lee, P.C., Deisseroth, K., Langston, J.W., Pera, R.R., and Palmer, T.D. SNCA triplication Parkinson's patient's iPSCderived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress. PLoS ONE (2011) 6:e26159. 20. Nguyen, H.N., Byers, B., Cord, B., Shcheglovitov, A., Byrne, J., Gujar, P., Kee, K., Schule, B., Dolmetsch, R.E., Langston, W., Palmer, T.D., and Pera, R.R. LRRK2 Mutant iPSC-Derived DA Neurons Demonstrate Increased Susceptibility to Oxidative Stress. Cell stem cell (2011) 8:267-80. 21. Seibler, P., Graziotto, J., Jeong, H., Simunovic, F., Klein, C., and Krainc, D. Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells. J Neurosci (2011) 31:5970-6. 22. Jiang, H., Ren, Y., Yuen, E.Y., Zhong, P., Ghaedi, M., Hu, Z., Azabdaftari, G., Nakaso, K., Yan, Z., and Feng, J. Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells. Nat Commun (2012) 3:668. 23. Sanchez-Danes, A., Richaud-Patin, Y., Carballo-Carbajal, I., Jimenez-Delgado, S., Caig, C., Mora, S., Di Guglielmo, C., Ezquerra, M., Patel, B., Giralt, A., Canals, J.M., Memo, M., Alberch, J., Lopez-Barneo, J., Vila, M., Cuervo, A.M., Tolosa, E., Consiglio, A., and Raya, A. Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease. EMBO Mol Med (2012) 4:380-95. 24. Sanchez-Danes, A., Consiglio, A., Richaud, Y., Rodriguez-Piza, I., Dehay, B., Edel, M., Bove, J., Memo, M., Vila, M., Raya, A., and Izpisua Belmonte, J.C. Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells. Hum Gene Ther (2012) 23:56-69.

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2.3. Neurodegeneration and Cell Biology

Neurodegeneration: A challenge for cell biology in the 21st century Antonia Gutierrez1,3 and Joan X. Comella2,3 1. Dept. of Cell Biology, Genetics and Physiology, Faculty of Sciences, University of Malaga, Spain 2. Cell Signaling and Apoptosis Group, Vall d'Hebron-Institut de Recerca, Barcelona, Spain; 3. Network Biomedical Research Center for Neurodegenerative Diseases (CIBERNED), Spain Address Correspondence to: Antonia Gutierrez. Dept. Biologia Celular. Facultad de Ciencias. Universidad de Málaga. Campus Teatinos 29071. Malaga, Spain. Email: agutierrez@uma.es Progressive dysfunction and loss of selected neuronal populations leading to the progressive failure of defined brain systems is the common feature of neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) among many others [1]. These diseases can be broadly classified as disorders of cognition and memory or movement, and both features can often coexist in a single disease. For these chronic debilitating disorders there is no preventive or curative treatment available at present, in fact current approved therapeutic interventions treat solely the symptoms and do not change the course of the disease. Advances in our understanding of the cellular/molecular mechanisms underlying these diseases are critical for the development of effective treatments. The main risk factor for neurodegenerative diseases is age, therefore the prevalence and burden of these diseases is growing inexorably as the population ages, with a tremendous toll on the patient, family, health system and society as a whole. The most common neurodegenerative disorder, Alzheimer’s disease, represents the major form of dementia and the 4th leading cause of death. Approximately 30 million people worldwide suffer from AD and, in the absence of effective therapy this number is predicted to quadruple by 2050 with increased life expectancy [2]. The average age of onset is 65 years in the most common, sporadic form of AD. The incidence increases with advanced age being 10% in population over the age of 65 and 50% in 85-year-olds or elder. Parkinson's disease is the second most common neurodegenerative disorder after AD, affecting more than 4 million people worldwide. PD is a slowly progressing motor system neurodegeneration characterized by average age of onset of 60 years, and the incidence is at 1% in people at

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the age of 65 and 2% in the population over the age of 80. Millions more suffer with other neurodegenerative disorders, and the number of people affected with these destructive diseases continues to increase every year. The magnitude of this global health problem parallels the dramatic increase in neurodegenerative disease research as evidenced by a PubMed search of the US National Library of Medicine for the keyword 'neurodegenerative disease', which retrieved more than 95,000 citations for the past ten years, including over 32,000 citations on 'Alzheimer', approximately 21,000 citations for 'Parkinson' and 6500 for 'Huntington'. Though substantial progress has been made in understanding the pathogenesis of these devastating disorders, the mechanisms that lead to selective neuronal degeneration are not well established. Understanding the aetiology of major neurodegenerative diseases, as well as identifying ways of early detection has become one of the greatest challenges for basic science and clinical medicine to develop disease-modifying treatment and prevention strategies. Despite varied clinical phenotypes neurodegenerative diseases share certain common features that lead to neuronal dysfunction and death [3;4]. In vitro and in vivo models have proved invaluable in this regard, yielding insights into cell death. Accumulation of abnormally folded proteins, inflammation, oxidative stress, mitochondrial dysfunction, axonal transport deficits,

proteasomal

dysfunction,

altered

autophagy-lysosome

pathways,

calcium

dyshomeostasis and death-receptor activation appear to be common in these disorders [514].

All the diseases are currently viewed as cerebral proteopathies in which aberrant

accumulation either extracellularly or intraneuronally, of abnormally aggregated proteins, is a key causative factor. Thus, highly soluble proteins are gradually aggregated as oligomers and converted into insoluble, filamentous polymers with characteristic crossed-β-pleated sheet structures that accumulate in a disease- and protein-specific manner as fibrillar amyloid deposits in the cytosol or nuclei of affected brain cells or in the extracellular space. In AD both intra- and extracellular accumulation occurs, in the form of neuronal tangles containing phosphorylated tau and plaques consisting of amyloid peptides (for review see [15]). In PD the protein that accumulates is mutated α-synuclein as Lewy bodies in the cytoplasm of neurons (for review see [16]), and in HD the protein is an abnormally expanded form of huntingtin present in intranuclear inclusions (for review see [17]). The accumulation of the damaged proteins might result in neuronal dysfunction and can ultimately cause cell death. Accumulating evidence suggests that acceleration of the removal of toxic accumulations of damaged proteins might be a tractable therapeutic strategy for neurodegenerative disorders [18-21]. The pathways by which most cytosolic and misfolded proteins are degraded are carried out by ubiquitin-proteasome system and autophagylysosome pathway (see review [22]). The ubiquitin–proteasome pathway is mainly responsible for the breakdown and degradation of short-lived proteins of low to medium molecular weight; the narrow proteasome barrel precludes entry of oligomers and highmolecular weight protein aggregates commonly observed in neurodegenerative disorders. These latter proteins and aggregates, as well as cytoplasmic contents such as damaged membranes and organelles, can undergo bulk degradation in an efficient manner via distinct types of autophagy–lysosome pathways. Impairment of either of these systems may lead to

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the accumulation and aggregation of proteins resulting in cellular toxicity and eventual neurodegeneration. The presence of abnormal autophagic-lysosome activity is frequently observed in selective neuronal populations afflicted in common neurodegenerative diseases [23;24], therefore therapeutic interventions that restore normal autophagy-lysosome function might be beneficial in the prevention of neuronal cell death [25]. As there are no naturally occurring animal models for many neurodegenerative diseases, identification of disease-related gene mutations have permitted the development of animal and cell based models that recapitulate some of the cardinal features of specific human pathology and facilitated study of early pathogenic events pathways [26-28]. Evidence is now accumulating that the disruption of connectivity within neural circuits, loss of synapses and deteriorated synaptic plasticity precede death of neurons. Synaptic loss is currently the best neurobiological correlate of cognitive deficits in AD [29-31]. The selective neuronal/synaptic vulnerability in neurodegenerative disease most likely arises from the complex interactions between interconnected cell types. Moreover, in the last few years studies of nonprion neurodegenerative proteinopathies suggest a circuit-based transfer of misfolded protein propagation between synaptically connected regions [32-35]. Loss of prosurvival neurotrophic factors has also been hypothesized to contribute to the selective neuronal vulnerability of several neurodegenerative disorders. Examples include nerve growth factor (NGF) in AD, glial-derived neurotrophic factor (GDNF) in PD, brain-derived neurotrophic (BDNF) factor in HD, and both insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) in ALS [36-43]. Neurotrophic factors have been proposed as potential neuroprotective agents. Nowadays, the main challenge is to find the way to administer these factors chronically and conditionally to the target neurons. In fact, the neuroregenerative capacity that the adult brain might still retain could be stimulated by growth factors or stem-cell therapies [44]. There is also considerable evidence linking disruption of axonal transport and cytoskeleton damage to various neurodegenerative diseases. Deficiencies in axonal transport have been visualized in various models mimicking neurodegenerative disorders (for reviews see [45-48]). Axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. Finally, neurodegeneration has an important inflammatory component associated with microglial activation and reactive astrogliosis. Inflammation is secondary to protein accumulation in neurodegenerative diseases. Evidence for the activation of the brain innate immune response, in particular of microglia, the resident macrophages of the central nervous system, in neurodegenerative diseases have been extensively reported (see reviews [5;49]). Detailed studies at both cellular and molecular levels have revealed complex interactions, demonstrating that activated microglial cells secrete both neurotoxic and neuroprotective molecules. These activated microglial cells can exert a beneficial function (alternative phenotype) such as phagocytosis of extracellular accumulated proteins or promote disease by causing neuronal damage (classic phenotype). Moreover, microglia can display different phenotypes during the progression of the disease pathogenesis [50]. There is no definite answer to the question of whether neuroinflammation is cause or consequence of neurodegeneration, and one of the reasons underlying this impasse is the lack of

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characterization of the functional heterogeneity of microglia, and astrocytes, throughout the disease progression. A better understanding of the neuroinflammatory response will be crucial to either target pathogenic responses, or augment the beneficial effects of immune responses as a strategy to intervene in chronic neurodegenerative diseases. Because of the multitude of aetiologies and molecular mechanisms involved in neuronal degeneration, therapeutic approaches aimed at neuronal/synaptic prevention or restitution in most neurodegenerative diseases will probably require multiple strategies.

Acknowledgments Supported by grants FIS PS09/00099 from Instituto de Salud Carlos III, SAS PI0496/2009 from Junta de AndalucĂa (Spain) and CIBERNED PI2010/08.

References 1. Saxena S, Caroni P (2011) Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration. Neuron 71:35-48 2. Reitz C, Brayne C, Mayeux R (2011) Epidemiology of Alzheimer disease. Nat Rev Neurol 7:137-152 3. Forman MS, Trojanowski JQ, Lee VM (2004) Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat Med 10:1055-1063 4. Skovronsky DM, Lee VM, Trojanowski JQ (2006) Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu Rev Pathol 1:151-170 5. Amor S, Puentes F, Baker D, van d, V (2010) Inflammation in neurodegenerative diseases. Immunology 129:154-169 6. Huang Q, Figueiredo-Pereira ME (2010) Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications. Apoptosis 15:1292-1311 7. Karbowski M, Neutzner A (2012) Neurodegeneration as a consequence of failed mitochondrial maintenance. Acta Neuropathol 123:157-171 8. Khandelwal PJ, Herman AM, Moussa CE (2011) Inflammation in the early stages of neurodegenerative pathology. J Neuroimmunol 238:1-11 9. Lee JA (2012) Neuronal autophagy: a housekeeper or a fighter in neuronal cell survival? Exp Neurobiol 21:1-8 10. Lezi E, Swerdlow RH (2012) Mitochondria in neurodegeneration. Adv Exp Med Biol 942:269-286 11. Lorz C, Mehmet H (2009) The role of death receptors in neural injury. Front Biosci 14:583-595 12. Nijholt DA, De KL, Elfrink HL, Hoozemans JJ, Scheper W (2011) Removing protein aggregates: the role of proteolysis in neurodegeneration. Curr Med Chem 18:2459-2476 13. Perlson E, Maday S, Fu MM, Moughamian AJ, Holzbaur EL (2010) Retrograde axonal transport: pathways to cell death? Trends Neurosci 33:335-344 14. Zundorf G, Reiser G (2011) Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid Redox Signal 14:1275-1288 15. Holtzman DM, Morris JC, Goate AM (2011) Alzheimer's disease: the challenge of the second century. Sci Transl Med 3:77sr1

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16. Rochet JC, Hay BA, Guo M (2012) Molecular insights into Parkinson's disease. Prog Mol Biol Transl Sci 107:125-188 17. Bano D, Zanetti F, Mende Y, Nicotera P (2011) Neurodegenerative processes in Huntington's disease. Cell Death Dis 2:e228 18. Cheung ZH, Ip NY (2011) Autophagy deregulation in neurodegenerative diseases recent advances and future perspectives. J Neurochem 118:317-325 19. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728-741 20. Renna M, Jimenez-Sanchez M, Sarkar S, Rubinsztein DC (2010) Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases. J Biol Chem 285:11061-11067 21. Sridhar S, Botbol Y, Macian F, Cuervo AM (2012) Autophagy and disease: always two sides to a problem. J Pathol 226:255-273 22. Wong E, Cuervo AM (2010) Integration of clearance mechanisms: the proteasome and autophagy. Cold Spring Harb Perspect Biol 2:a006734 23. Banerjee R, Beal MF, Thomas B (2010) Autophagy in neurodegenerative disorders: pathogenic roles and therapeutic implications. Trends Neurosci 33:541-549 24. Son JH, Shim JH, Kim KH, Ha JY, Han JY (2012) Neuronal autophagy and neurodegenerative diseases. Exp Mol Med 44:89-98 25. Harris H, Rubinsztein DC (2012) Control of autophagy as a therapy for neurodegenerative disease. Nat Rev Neurol 8:108-117 26. Chesselet MF, Richter F (2011) Modelling of Parkinson's disease in mice. Lancet Neurol 10:1108-1118 27. Gama Sosa MA, De GR, Elder GA (2012) Modeling human neurodegenerative diseases in transgenic systems. Hum Genet 131:535-563 28. Trancikova A, Ramonet D, Moore DJ (2011) Genetic mouse models of neurodegenerative diseases. Prog Mol Biol Transl Sci 100:419-482 29. Arendt T (2009) Synaptic degeneration in Alzheimer's disease. Acta Neuropathol 118:167-179 30. Ma T, Klann E (2012) Amyloid beta: linking synaptic plasticity failure to memory disruption in Alzheimer's disease. J Neurochem 120 Suppl 1:140-148 31. Nimmrich V, Ebert U (2009) Is Alzheimer's disease a result of presynaptic failure? Synaptic dysfunctions induced by oligomeric beta-amyloid. Rev Neurosci 20:1-12 32. Clavaguera F, Bolmont T, Crowther RA, Abramowski D, Frank S, Probst A, Fraser G, Stalder AK, Beibel M, Staufenbiel M, Jucker M, Goedert M, Tolnay M (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat Cell Biol 11:909-913 33. de CA, Polydoro M, Suarez-Calvet M, William C, Adamowicz DH, Kopeikina KJ, Pitstick R, Sahara N, Ashe KH, Carlson GA, Spires-Jones TL, Hyman BT (2012) Propagation of tau pathology in a model of early Alzheimer's disease. Neuron 73:685-697 34. Desplats P, Lee HJ, Bae EJ, Patrick C, Rockenstein E, Crews L, Spencer B, Masliah E, Lee SJ (2009) Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci U S A 106:13010-13015 35. Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton AL, Vigouret JM, Paganetti P, Walsh DM, Mathews PM, Ghiso J, Staufenbiel M, Walker LC, Jucker M (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313:1781-1784 36. Baquet ZC, Gorski JA, Jones KR (2004) Early striatal dendrite deficits followed by neuron loss with advanced age in the absence of anterograde cortical brain-derived neurotrophic factor. J Neurosci 24:4250-4258

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37. Canals JM, Pineda JR, Torres-Peraza JF, Bosch M, Martin-Ibanez R, Munoz MT, Mengod G, Ernfors P, Alberch J (2004) Brain-derived neurotrophic factor regulates the onset and severity of motor dysfunction associated with enkephalinergic neuronal degeneration in Huntington's disease. J Neurosci 24:7727-7739 38. Gauthier LR, Charrin BC, Borrell-Pages M, Dompierre JP, Rangone H, Cordelieres FP, De MJ, MacDonald ME, Lessmann V, Humbert S, Saudou F (2004) Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 118:127-138 39. Mickiewicz AL, Kordower JH (2011) GDNF family ligands: a potential future for Parkinson's disease therapy. CNS Neurol Disord Drug Targets 10:703-711 40. Rangasamy SB, Soderstrom K, Bakay RA, Kordower JH (2010) Neurotrophic factor therapy for Parkinson's disease. Prog Brain Res 184:237-264 41. Sakowski SA, Schuyler AD, Feldman EL (2009) Insulin-like growth factor-I for the treatment of amyotrophic lateral sclerosis. Amyotroph Lateral Scler 10:63-73 42. Wyatt TJ, Keirstead HS (2010) Stem cell-derived neurotrophic support for the neuromuscular junction in spinal muscular atrophy. Expert Opin Biol Ther 10:1587-1594 43. Zuccato C, Cattaneo E (2009) Brain-derived neurotrophic factor in neurodegenerative diseases. Nat Rev Neurol 5:311-322 44. Enciu AM, Nicolescu MI, Manole CG, Muresanu DF, Popescu LM, Popescu BO (2011) Neuroregeneration in neurodegenerative disorders. BMC Neurol 11:75 45. De Vos KJ, Grierson AJ, Ackerley S, Miller CC (2008) Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci 31:151-173 46. Goldstein LS (2012) Axonal transport and neurodegenerative disease: Can we see the elephant? Prog Neurobiol 47. Lingor P, Koch JC, Tonges L, Bahr M (2012) Axonal degeneration as a therapeutic target in the CNS. Cell Tissue Res 48. Morfini GA, Burns M, Binder LI, Kanaan NM, LaPointe N, Bosco DA, Brown RH, Jr., Brown H, Tiwari A, Hayward L, Edgar J, Nave KA, Garberrn J, Atagi Y, Song Y, Pigino G, Brady ST (2009) Axonal transport defects in neurodegenerative diseases. J Neurosci 29:12776-12786 49. Prinz M, Priller J, Sisodia SS, Ransohoff RM (2011) Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat Neurosci 14:1227-1235 50. Jimenez S, Baglietto-Vargas D, Caballero C, Moreno-Gonzalez I, Torres M, SanchezVaro R, Ruano D, Vizuete M, Gutierrez A, Vitorica J (2008) Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer's disease: agedependent switch in the microglial phenotype from alternative to classic. J Neurosci 28:11650-11661

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Neuron Biology In Health And Disease José Carlos Dávila1,2 1. Depto. Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Spain 2. CIBERNED, Spain Neurons are unique among animal cells. Although the basic cellular organization of neurons is similar to that of other cells, neurons have distinctive features that reflect their specialization for intercellular communication. These include a complex cellular morphology, the specific localization of membrane proteins for electrical signaling, or the presence of small structures specialized for communication. Of all these distinctive features, perhaps the most extraordinary is its shape. Most neurons have a very complex morphology with numerous cellular extensions, often of great length and branching. This elaborate neuronal morphology reflects functional compartmentalization within the neuron. The dendrites, processes that originate from the cell body, may possess so many ramifications they represent a real dendritic tree. On the membrane of the dendritic branches, or its small appendages, the dendritic spines, other neurons make functional contact by means the so called presynaptic boutons (Fig. 1). For this reason the dendritic compartment is considered the "receiver" part of the neuron.

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Another extension that emerges from the cell body is the axon. Virtually all neurons have a single axon, which may also be branched, and may extend from hundreds of micrometers to more than 1 meter in length in some mammalian neuronal types. The axon is the portion of the neuron specialized in "drive" signals over long distances and, like the dendrites has a distinctive protein cytoskeleton (Fig. 2), whose elements are essential for its functional integrity. Since axons lack protein synthesis machinery, the maintenance of them depends largely on the cell body, the metabolic center of the neuron where most of the processes for protein synthesis and degradation occur.

Finally, the portion of the neuron specialized in the intercellular communication is the so called presynaptic bouton or terminal, and it is located at the end of the axonal branches or all along of axons. The presynaptic terminal is adjacent to a specialized portion of the membrane of another cell, the postsynaptic specialization, forming together a functional junction called synapsis, where the "transfer" of information from one cell to another occurs. At classical synapses, the presynaptic terminal releases a chemical transmitter stored in synaptic vesicles which diffuses across a narrow synaptic cleft and activates receptors on

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the postsynaptic membrane of the target cell. The target may be a dendrite, cell body, or axon of another neuron, or a specialized region of a muscle or secretory cell. Although the neuron is an extremely complex cell, both structurally and functionally, when we think about the functions performed by the nervous system it must be considered that these functions, even the simplest one, result from the coordinated activity of networks of interconnected neurons (neural circuits) composed of hundreds or thousands units. Thus, neurons never work isolated, but forming specialized assemblies that process specific types of information, and whose activity is the substrate of our sensations, perceptions and behavior. Therefore, the proper function of the neural circuits, and hence of its constituent elements, determines the normal course of brain functions. Neuronal loss or dysfunction in a specific circuit will cause neurological and functional deficits. The severity of these deficits increases with the severity of the neuronal/circuit damage. Many human neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease or Huntington's disease, course with selective loss of particular groups of neurons in specific locations. While recent years have considerably advanced our understanding in the pathogenesis of neurological diseases, still very little is known about the molecular mechanisms underlying neuronal death. Neuronal loss is an irreversible process, and most current studies focused on early signs of neuronal dysfunction trying to find therapeutic targets to halt the irreparable damage that means the death of neurons. Undoubtedly, some of these potential targets are the synapses.

The synapse is the fundamental unit for storing and transferring information in the brain As

discussed

above, the

essential function

of neurons

the

intercellular

communication, and this process occurs primarily at synapses. Synapses are constituted by presynaptic and postsynaptic elements, separated by a space, the synaptic cleft (Fig. 3). In the so-called chemical synapses, the presynaptic portion is characterized by synaptic vesicles, small organelles of approx. 40 nm in diameter, containing the neurotransmitter. Each

presynaptic

terminal

contains

numerous

synaptic

vesicles

filled

with

neurotransmitter, which will be released into the synaptic space by exocytosis once a nerve impulse reaches the presynaptic membrane. Once released, the neurotransmitter will interact with specific receptors in the postsynaptic membrane, causing changes in membrane potential, which will eventually become action potentials in the postsynaptic neuron thus spreading the signal.

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The synaptic release of neurotransmitter is a complex process that involves several steps, including vesicle docking, priming, and fusion of the synaptic vesicle membrane with the plasma membrane. This latter process is mediated by calcium influx through voltagegated calcium channels at the membrane of the presynaptic terminal. Calcium triggers the exocytosis through a protein machinery, including SNARE proteins (an acronym for soluble N-ethylmaleimide

sensitive

factor

attachment

protein

(SNAP)

receptor),

such

synaptobrevin, syntaxin or SNP-25 and other proteins such as Munc 18, that regulates the assembly and function of the SNARE complex (see [1], for a review). As expected, this complex multi-step process may be intrinsically modulated by changing, for instance, the probability of release and/or the number of synaptic vesicles that fuse in response to action potentials. In general, synaptic transmission is a finely tuned process that also can be modulated to allow lasting changes in signal strength. This ability of synapses to change the signal strength is called synaptic plasticity, and can be either an increase (potentiation) as a decrease (depression) in the signal strength. Synaptic plasticity underlies important brain functions such as learning and memory.

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There is increasing evidence that defects in synaptic transmission and / or synaptic plasticity may contribute to the pathology of neurodegenerative diseases [2, 3]. These defects can affect the presynaptic compartment, the postsynaptic compartment or both. Due to the central role of dendritic spines in the phenomena of synaptic plasticity in the hippocampus [4], it is considered that these structures are the first synaptic elements affected during the early cognitive decline that occurs in the progression of Alzheimer's disease

[5],

characterized

hippocampus-dependent

episodic

memory

loss.

Postsynaptic alterations are suggested by several studies, including post-mortem analysis of Alzheimer's patients, demonstrating a significant reduction in the density of dendritic spines in hippocampal neurons [6], or studies in transgenic models of the disease, which also show age-dependent reductions of the density of dendritic spines [7]. While most studies have focused on postsynaptic changes as potential substrates of Alzheimer's disease, especially in excitatory synapses for their well characterized role in synaptic plasticity, learning and memory [3, 8], other studies have pointed to the presynaptic compartments. Thus, alterations or mutations in specific presynaptic proteins have been implicated in a number of neurological diseases (reviewed in [9]).

Proper presynaptic axoplasmic flow

function

requires

regular

Regarding the role of the presynaptic compartment in neurological diseases, we should not forget that the presynaptic boutons are dynamic structures, able to change not only in strength but also in size (either increase or decrease). This dynamism requires a proper balance in the process of protein synthesis and degradation. Since presynaptic boutons have virtually no machinery for protein synthesis or degradation, these processes depend largely of the continuous flow between the bouton and the somatodendritic compartment where most of the metabolic processes take place. Therefore, most of the presynaptic proteins, including proteins involved in the synthesis and release of neurotransmitters are synthesized in the cell body and delivered to the presynaptic bouton through vesicular intermediates. Similarly, degradation of presynaptic proteins occurs primarily in the cell body, using one of the two major pathways for degradation of proteins in neurons: the endosomallysosomal pathway (for removal of membrane-associated proteins) and the ubiquitinproteasome system (UPS). Components of the latter system have been detected in presynaptic boutons, suggesting that the UPS also mediates protein degradation in the presynaptic compartment itself. By contrast, most lysosomes are in the cell body, then presynaptic proteins destined for lysosomal degradation must be retrogradely transported from the boutons to the cell body via endosomal intermediates. An efficient axonal transport, therefore, is critical for the maintenance of synaptic function, including the processes of presynaptic proteins renewal. Anterograde axonal transport takes place from the cell body to the presynaptic terminals while retrograde transport from the terminals to the cell body. The forward movement is responsible for the

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trafficking of organelles such as vesicles or mitochondria, and proteins from its site of biogenesis, the soma to the presynaptic terminals. The retrograde movement, on the other hand, allows the return of the organelles and proteins to the cell body for degradation. Organelles such as vesicles or mitochondria, and proteins are transported by axoplasmic flow through the axon. This transport occurs along microtubule tracks, which provides structural support. The continuous flow of organelles and proteins along the axons depends on the action of motor proteins (molecular motors) that interact on one side with the microtubule cytoskeleton tracks, and on the other with the cargo, allowing the displacement of this in either direction. There are two main types of motor proteins: kinesins and dyneins. The kinesins are involved in axonal transport from the soma to the presynaptic terminals, i.e. the anterograde transport, while dyneins are involved in transport from the presynaptic terminals to the soma, i.e. the retrograde transport. Of the different types of known kinesins, the best characterized and the first to be discovered is kinesin-1. This protein, originally discovered in the squid giant axon in 1985, transports vesicles and other cargos toward the plus end (plus end-directed) of microtubules and therefore is involved in the fast anterograde transport. Kinesin-1 binds to mitochondria, lysosomes, and receptor and adapter proteins. Kinesin-1 is essential for transport of vesicles into the presynaptic terminal and, therefore, important in synaptic transmission. The cytoplasmic dynein, on the other hand, is a microtubule-based motor protein that mediates the transport of vesicles toward the minus end (minus end-directed) of microtubules via interactions with dynactin, another microtubule-associated protein. Besides an essential role in the retrograde axonal transport, this protein has also been implicated in presynaptic processes [10]. An impaired axoplasmic transport may affect synaptic function by engaging the processes of exchange of organelles / proteins between the cell body and the presynaptic terminals. In addition, disruption of axonal transport may cause an abnormal accumulation of organelles and proteins in axons and cell bodies of neurons, eventually resulting in neuronal dysfunction and death. Some neurodegenerative diseases like Huntington's, Parkinson's or Alzheimer's are characterized by intracellular accumulation of protein aggregates [11]. The alterations in the axoplasmic transport may have at least two different origins. On one hand, it may be affected the microtubule cytoskeleton, which provides structural support for axonal transport, and on the other hand it may be affected the motor proteins, kinesin and dynein. A major histopathological feature of Alzheimer's disease is the presence of intracellular fibrillary deposits of hyperphosphorylated tau (neurofibrillary tangles). Tau is associated with microtubule stabilizing them. However, when tau is hyperphosphorylated, microtubules become destabilized causing axonal transport disruption. It has been suggested that changes in the function of tau may impair axonal transport mechanisms causing the disease [12, 13]. A series of recent studies have indicated that changes in the levels of kinesin or dynein, or its transport capabilities, produce a reduction in synaptic vesicle transport (reviewed in [14]), which could affect the integrity of the synapse, leading to synaptic loss, a typical feature of Alzheimer and other dementias [15, 16, 17].

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Finally, it has also been shown that dynein dysfunction disrupts the bidirectional traffic of vesicles and the synaptic vesicle docking, which could alter normal neuronal activity, contributing together with other risk factors to diseases such as Alzheimer [10].

References 1. Südhof TC, Rizo J. Synaptic vesicle exocytosis. 2012. Cold Spring Harb Perspect Biol 2011; 3:a005637. 2. Südhof TC. Neuroligins and neurexins link synaptic function to cognitive disease. 2008. Nature 455: 903-911. 3. van Spronsen M, Hoogenraad CC. Synapse pathology in psychiatric and neurologic disease. 2010. Curr Neurol Neurosci Rep 10: 207-214. 4. Bourne JN, Harris KM. Balancing structure and function at hippocampal dendritic spines. 2008. Ann Rev Neurosci 31: 47-67. 5. Scheff SW, Price DA, Schmitt FA, DeKosky ST, Mufson EJ. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. 2007. Neurology 68: 1501-1508. 6. Ferrer I, Guionnet N, Cruz-Sánchez F, Tuñón T. Neuronal alterations in patients with dementia: a Golgi study on biopsy samples. 1990. Neurosci Lett 114: 11-16. 7. Lanz TA, Carter DB, Merchant KM. Dendritic spine loss in the hippocampus of young PDAPP and Tg2576 mice and its prevention by the ApoE2 genotype. 2003. Neurobiol Dis 13: 246-253. 8. Citri A, Malenka RC. Synaptic plasticity: multiple forms, functions, and mechanisms. 2008. Neuropsychopharmacology 33: 18-41. 9. Waites CL, Garner CC. Presynaptic function in health and disease. 2011. TINS. 34: 366337. 10. Kimura N, Okabayashi S, Ono F. Dynein dysfunction disrupts intracellular vesicle trafficking bidirectionally and perturbs synaptic vesicle docking via endocytic disturbances. A potential mechanism underlying age-dependent impairment of cognitive function. 2012. Am J Pathol 180, 550-561. 11. Coleman M. Axon degeneration mechanisms: commonality amid diversity. 2005. Nat Rev Neurosci 6: 889-898. 12. Goldstein LS. Do disorders of movement cause movement disorders and dementia? 2003. Neuron 40: 415-425. 13. De Vos KJ, Grierson AJ, Ackerley S, Miller CC. Role of axonal transport in neurodegenerative diseases. 2008. Annu Rev Neurosci 31: 151-173. 14. Morel M, Héraud C, Nicaise C, Suain V, Brion J-P. Levels of kinesin light chain and dynein intermediate chain are reduced in the frontal cortex in Alzheimer’s disease: implications for axoplasmic transport. 2012. Acta Neuropathol 123:71-84. 15. Hamos JE, DeGennaro LJ, Drachman DA. Synaptic loss in Alzheimer’s disease and other dementias. 1989. Neurology 39, 355-361. 16. Masliah E, Mallory M, Alford M et al. Altered expression of synaptic proteins occurs early during progression of Alzheimer’s disease. 2001. Neurology 56:127-129. 17. Arendt T. Synaptic degeneration in Alzheimer′s disease. 2009. Acta Neuropathol 118: 167-179.

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Brain derived neurotrophic factor: Neuronal dysfunction versus cell death in Huntington’s Disease Jordi Alberch, Josep M Canals, Silvia Ginés and Esther Pérez-Navarro Dept of Cellular Biology, Immunology and Neuroscience. Medical School. IDIBAPS. CIBERNED. University of Barcelona. Casanova 143, 08036 Barcelona. Spain. alberch@ub.edu Neurodegenerative disorders are characterized by the death of specific neuronal populations. For years biomedical research has been focused to identify the mechanisms involved in neuronal death and the development of treatments to halt neurodegenerative processes. Even so, it is unlikely that cells in a diseased brain will perform adequately until the moment they expire. Current research demonstrates that neuronal and synaptic dysfunction precedes cell death by many years and occurs long before, or in the absence of cell death in several neurodegenerative disorders. Hence, it is important to understand the early molecular and cellular dysfunction that takes place in the neurodegenerative disorders. These new insights will be useful in the diagnosis and treatment of these neurological diseases. Huntington’s disease (HD) is a genetic disorder caused by an expansion with a trinucleotide poly(CAG) tract in exon-1 of the huntingtin gene, inducing a prominent degeneration of striatal neurons and, to lesser extent, of cortical pyramidal neurons. This neurodegenerative disorder is characterized by late-onset motor, cognitive, behavioral, and psychological dysfunction (1). There is currently no cure. Genetic mouse models have greatly enhanced our understanding of HD (2). Investigations of synaptic transmission and plasticity in mouse models of HD demonstrate neuronal dysfunction before the onset of classical disease indicators (3,4). Therefore, pharmacological interventions in HD must be also focused to target early and putative pathophysiological disturbances that can reverse neuronal dysfunction and delay progression to neurodegeneration (5). In the search of modulators of neuronal dysfunction and survival, Brain Derived Neurotrophic Factor (BDNF) has emerged as a key molecular target for drug development in neurological disorders (6). However, only in HD, BDNF has been linked with the genetic defects (7). Several studies have shown that mutant huntingtin is affecting synthesis (7), trafficking (8), and release (8) of BDNF. Accordingly, BDNF levels are decreased in HD patients and animal models (7). This neurotrophin has potent effect in supporting survival and maintenance of neurons in the striatum and cortex (9,10). The functional effect of BDNF on mutant huntingtin toxicity has been confirmed using HD mouse models with different levels of BDNF. The down-regulation of BDNF in R6/1 mice induces an early onset and

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more severe cognitive and motor symptoms (11), with neuronal loss and decrease expression of enkephalin, which is the neuronal population more affected in HD (11). On the other hand, BDNF overexpression in the striatum and cortex in a HD model substantially ameliorated motor dysfunction, reversed brain weight loss, restored TkB signaling in the striatum, and reduced the formation of mutant huntingtin aggregates (12). All these findings have generated considerable excitement about the possibility of using a â&#x20AC;&#x153;BDNF therapyâ&#x20AC;? for HD. Although a number of issues such as age-responsiveness to BDNF, and the dosage and the method of delivery remain to be established.

BDNF age-specific responsiveness of cortical, striatal and hippocampal neurons Since HD is a progressive disorder, the responsiveness of neurons to BDNF during maturation and aging must be considered for using this neurotrophin as a therapeutic agent. It has been reported that BDNF levels decrease with aging (13,14). Changes in neuronal plasticity have been observed in BDNF heterozygote mice, but with different sensitivity depending on the brain area and age. BDNF+/- mice experience difficulties in the acquisition of new motor skills, but not in the acquisition of new declarative / explicit memories. However, these disabilities are only observed in middle-aged (30 weeks) but not in young BDNF+/- mice (12 weeks). A reduction in the density and size of the dendritic spines of the motor cortex, but not in the hippocampus is detected in the motor cortex and dorsal hippocampus of 30-week old BDNF+/- mice. Accordingly, several synaptic markers as PSD95 and GluR1 are reduced only in the motor cortex of 30-week old BDNF+/- mice. These differences between brain areas could be explained by a compensatory up-regulation of the TrkB receptor in the hippocampus, but not in the cerebral cortex, of middle-aged BDNF+/mice. Interestingly in the striatum of BDNF +/- mice neither synaptic markers nor TrkB receptor levels are modified (15,16). BDNF knockout mice die during the first two postnatal weeks. Thus, conditional or region-specific knockout models have been generated to understand the function of BDNF in older ages. The lack of cortical BDNF using forebrain-specific BDNF mutant mice causes reduced volume of the striatum but not of the hippocampus in old BDNF-deficient mice. Furthermore the loss of striatal neurons is only observed in old but not young adult (17). These mice display aspects of behavioral and anatomical abnormalities seen in mouse models of HD (17). Similarly, studies using another conditional mouse mutant lacking BDNF throughout the central nervous system show that BDNF is not essential for prolonged postnatal survival, but the behavior of such mutant animals is markedly altered (18). However, BDNF is required for the postnatal growth of the striatum, but not the hippocampus, regulating dendritic complexity and spine density (18). All these data provide important information about region-specific and age-dependent changes in neuronal plasticity to be considered to develop therapeutic strategies using BDNF to modulate neuronal dysfunction.

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Experimental therapeutic strategies for delivering BDNF in Huntington’s disease Several approaches have been used to deliver BDNF in the central nervous system. Intrastriatal injection of BDNF with minipumps has potent neuroprotective effect. BDNF increased the expression of enkephalin in the striatum of mice overexpressing exon 1 of human mutant huntingtin (11). Other systems have also been tested in animal models, such as gene therapy and BDNF-releasing grafts (6). However, any of these approaches have been completely successful, because of the poor pharmacokinetics properties of BDNF, as short in vivo half-life, low blood-brain barrier penetrability and limited diffusion (6). Therefore, new systems must be searched to get better diffusion and conditional delivery. Recently we have generated a transgenic mouse that over-expressed BDNF under the promoter of GFAP (pGFAP-BDNF; 19). Thus, these mice only over-expressed BDNF under pathological conditions. pGFAP-BDNF mice show striatal neuroprotection against acute administration of quinolinate, the excitotoxic model of HD (19). Furthermore, this mouse can be a source to isolate astrocytes for grafting in models of HD and other neurodegenerative disorders. pGFAP-BDNF-grafted astrocytes are neuroprotective against excitotoxicity, and induced larger behavioral improvements than wild type grafted astrocytes even 60 days after transplantation (19). To generate a “BDNF up-regulation” model of HD, these mice were crossed-mated with R6/2 mice (R6/2:pGFAP-BDNF; (20). In these R6/2:pGFAP-BDNF animals, the decrease in striatal BDNF levels induced by mutant huntingtin is prevented in comparison to R6/2 animals at 12 weeks of age. The recovery of the neurotrophin levels in R6/2:pGFAP-BDNF mice correlates with an improvement in several motor coordination tasks. Although the total number of cells are not affected, the down-regulation of DARPP-32 and enkephalin protein levels in R6/2 mice is improved in R6/2:pGFAP-BDNF mice. An improvement in corticostriatal connectivity in these mice is also observed. Interestingly, the over-expression of BDNF prevents the decrease of cortico-striatal presynaptic (VGLUT1) and postsynaptic (PSD-95) markers in the R6/2:pGFAP-BDNF striatum (20). These positive changes in synaptic markers correlates with an absence of basal synaptic transmission dysfunction and a greater resistance to synaptic fatigue in R6/2:pGFAP-BDNF mice than in R6/2 mice (20). Therefore, the conditional release of BDNF under GFAP promoter can prevent the toxic effect of mutant huntingtin.

Conclusions BDNF plays an important role modulating synaptic plasticity alterations during early stages, but also regulates neuronal death during the symptomatic stage in HD. However, it must be considered that the responsiveness of neurons of different brain areas is changing during maturation and aging. This fact can explain the different vulnerability of selective neuronal populations modulating the onset and severity of neurodegenerative disorders.

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Since BDNF can be a good candidate for HD treatment, the main problem is to identify methods to deliver BDNF in brain areas affected. BDNF release under the promoter of GFAP is a new tool that provides a conditional system to deliver BDNF only under pathological conditions. This system is able to block the pathological effects of mutant huntingtin, and is an interesting approach to develop cell therapy strategies for HD.

Acknowledgements This work was supported by grants from Ministerio de Ciencia e Innovación (SAF201129507, J.A.; SAF2009-07077, S.G.; SAF2009-07774 and PLE2009-0089, to J.M.C.), Instituto de Salud Carlos III: PI10/01072 to E.P., CIBERNED and Red de Terapia Celular (RD06/0010/0006).

References 1. The Huntington's disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. 1993. Cell 72:971–983. 2. Southwell AL, Patterson PH. Gene therapy in mouse models of huntington disease. 2011 Neuroscientist17(2):153-62. 3. Cummings DM, Milnerwood AJ, Dallérac GM, Waights V, Brown JY, Vatsavayai SC, Hirst MC, Murphy KP. Aberrant cortical synaptic plasticity and dopaminergic dysfunction in a mouse model of Huntington’s disease. 2006. Hum Mol Genet 15:2856–2868. 4. Milnerwood AJ, Cummings DM, Dallérac GM, Brown JY, Vatsavayai SC, Hirst MC, Rezaie P, Murphy KP.et al. Early development of aberrant synaptic plasticity in a mouse model of Huntington’s disease. 2006. Hum Mol Genet 15:1690–1703. 5. Milnerwood AJ, Raymond LA. Early synaptic pathophysiology in neurodegeneration: insights from Huntington's disease. 2010. Trends Neurosci 33(11):513-23. 6. Zuccato C, Cattaneo E. Brain-derived neurotrophic factor in neurodegenerative diseases. 2009. Nat Rev Neurol 5(6):311-22. 7. Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, Conti L, MacDonald ME, Friedlander RM, Silani V, Hayden MR, Timmusk T, Sipione S, Cattaneo E. 2001. Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease. Science. 293:493-8. 8. Del Toro D, Canals JM, Ginés S, Kojima M, Egea G, Alberch J. Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism. 2006. J Neurosci 26(49):12748-57. 9. Gavaldà N, Pérez-Navarro E, Gratacòs E, Comella JX, Alberch J. Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons. 2004. Mol Cell Neurosci 25(3):460-8. 10. Gavaldà N, Pérez-Navarro E, García-Martínez JM, Marco S, Benito A, Alberch J. Bax deficiency promotes an up-regulation of Bim(EL) and Bak during striatal and cortical postnatal development, and after excitotoxic injury. 2008. Mol Cell Neurosci 37(4):663-72. 11. Canals JM, Pineda JR, Torres-Peraza JF, Bosch M, Martín-Ibañez R, Muñoz MT, Mengod G, Ernfors P, Alberch J. Brain-derived neurotrophic factor regulates the onset and severity of motor dysfunction associated with enkephalinergic neuronal degeneration in Huntington's disease. 2004. J Neurosci 24(35):7727-39.

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12. Gharami K, Xie Y, An JJ, Tonegawa S, Xu B. Brain-derived neurotrophic factor overexpression in the forebrain ameliorates Huntington's disease phenotypes in mice. 2008. J Neurochem 105(2):369-79. 13. Hattiangady B, Rao MS, Shetty GA, Shetty AK. Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus. 2005. Exp Neurol 195(2):353-71. 14. Hayashi M, Mistunaga F, Ohira K, Shimizu K. Changes in BDNF-immunoreactive structures in the hippocampal formation of the aged macaque monkey. 2001. Brain Res 918:191-196. 15. Torres-Peraza JF, Giralt A, García-Martínez JM, Pedrosa E, Canals JM, Alberch J. Disruption of striatal glutamatergic transmission induced by mutant huntingtin involves remodeling of both postsynaptic density and NMDA receptor signaling. 2008. Neurobiol Dis 29(3):409-21. 16. Ginés S, Bosch M, Marco S, Gavaldà N, Díaz-Hernández M, Lucas JJ, Canals JM, Alberch J. Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain. 2006. Eur J Neurosci 23(3):649-58. 17. Baquet ZC, Gorski JA, Jones KR. Early striatal dendrite deficits followed by neuron loss with advanced age in the absence of anterograde cortical brain-derived neurotrophic factor. 2004. J Neurosci 24(17):4250-8. 18. Rauskolb S, Zagrebelsky M, Dreznjak A, Deogracias R, Matsumoto T, Wiese S, Erne B, Sendtner M, Schaeren-Wiemers N, Korte M, Barde YA. Global deprivation of brain-derived neurotrophic factor in the CNS reveals an area-specific requirement for dendritic growth. 2010. J Neurosci 30(5):1739-49. 19. Giralt A, Friedman HC, Caneda-Ferrón B, Urbán N, Moreno E, Rubio N, Blanco J, Peterson A, Canals JM, Alberch J. BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease. 2010. Gene Ther 17(10):1294-308. 20. Giralt A, Carretón O, Lao-Peregrin C, Martín ED, Alberch J. Conditional BDNF release under pathological conditions improves Huntington's disease pathology by delaying neuronal dysfunction. 2011. Mol Neurodegener 6(1):71.

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Oligomeric Amyloid-Beta And Neuronal Dysfunction In Alzheimer’s Disease Antonia Gutierrez1,3 and Javier Vitorica2,3 1. Dept. of Cell Biology, Genetics and Physiology, Faculty of Sciences, University of Malaga, Spain 2. Dept Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville / IBIS, Spain 3. Network Biomedical Research Center for Neurodegenerative Diseases (CIBERNED), Spain. Address Correspondence to: Antonia Gutierrez. Dept. Biologia Celular. Facultad de Ciencias. Universidad de Málaga. Campus Teatinos 29071. Malaga, Spain. Email: agutierrez@uma.es Alzheimer's disease (AD) is the most common form of dementia among people over 65 years. At the tissue level is characterized by 1) accumulation of aggregated proteins as extracellular deposits of beta-amyloid peptide (Abeta) and as intraneuronal neurofibrillary tangles composed of hyperphosphorylated tau protein, 2) loss of synapses, and 3) selective neuronal death in higher brain regions responsible for memory and learning processes (for review see [1]). Currently, AD is incurable and the few available drugs only marginally affect the symptoms. The lack of an effective therapy is mainly due to the absence of appropriate animal models and therapeutic targets. An effective treatment should prevent or delay the loss of synapses and the progressive neuronal death. Simply with a delayed onset of symptoms would improve significantly the quality of life of patients and greatly reduce health care costs and care. Synaptic loss is actually the best neurobiological correlate of cognitive deficits in early stages of AD. This slowly progressing disorder apparently is preceded by a clinically silent period of several years or even decades (for review see [2]). Similarly, synaptic degeneration might progress from an initially reversible condition to stages irreversibly associated with synapse loss. Brain amyloid load does not correlate well with synaptic loss, neuronal death or cognitive dysfunction [3]. In the brain of patients and transgenic models, Abeta peptides (a 39 to 43 amino acids produced by proteolytic processing of amyloid precursor protein, APP, by sequential action of enzymes beta- and gamma-secretase) can be found as monomers or oligomers [1]. Abeta aggregation is a poorly understood process, resulting in the formation of multiple species, dimers, trimers, tetramers, hexamers and other complex over 15-20 or more monomers called ADDLs (Abeta-derived diffusible ligands) [4]. The soluble Abeta oligomeric forms are potent neurotoxins and are now considered the causative agents of synaptic and cognitive dysfunction in the early stages of the disease

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[5;6]. Both natural and synthetic oligomers produced decrease in synaptic plasticity [6-9], memory [8;10;11], and further loss of synapses [12;13]. Transgenic mouse models, overexpressing mutated forms of human APP, are widely used to study AD pathogenesis. However, none of them displayed all pathologic signs of the disease. The most relevant discrepancies between AD and the transgenic models is a late, if any, neuronal degeneration, even in the presence of large AÎ˛ accumulation since early ages. We have characterized the pathological progression of a double PS1/APP transgenic model (PS1M146L/APP751SL) that develop early amyloidosis in vulnerable brain regions [14-20]. This model exhibited extracellular Abeta deposits throughout the hippocampus from a very early age (Fig. 1A). The number and size of the amyloid deposits progressively increased with age (Fig. 1B).

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The most relevant feature of our model is that, unlike many other transgenic mice, there is a selective neurodegeneration of specific subsets of interneurons and, most importantly, pyramidal neurons in hippocampus and entorhinal cortex, following a regional and temporal pattern [14;16-20]. Neurodegeneration of principal neurons in the entorhinal cortex of this PS1/APP model occurs earlier than in hippocampus [18], as also observed in AD brains. This pyramidal neuronal loss was found to be associated with a neurotoxic inflammatory response induced by soluble oligomeric Abeta peptides [16]. We observed a close spatial and temporal parallelism between Abeta deposits and microglial activation (Fig. 2A). In response to any kind of brain damage or injury, microglial cells become activated and undergo morphological as well as functional transformations

Resident microglial cells in the healthy brain are thought to rest in a dormant state with typical small cell bodies bearing a few ramified thin processes, whereas activation is associated with morphological alterations such as enlargement of the cell body and retraction and swelling of microglial processes (see Fig. 2B and C). At early ages (4-6 months) in our AD model, the apparition of Abeta plaques determined the microglia activation to an alternative phenotype (YM1-positive) with, apparently, a neuroprotective role with Abeta phagocytic capabilities. At older ages (18 months), the accumulation of extracellular oligomeric Abeta produced marked widespread inter-plaque microglial activation toward a classic phenotype and the production of cytotoxic factors (TNF-alpha and related factors) that could, probably, be responsible for the principal cell death. This dual role role (neuroprotective/neurotoxic) of microglial activation during the time course of the Abeta-

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associated neuroinflammatory response could explain the scarce or delayed neuronal loss in most AD models, despite extracellular Abeta accumulation. The reasons that determined the age-dependent increase in the Abeta oligomers content remained unknown. We have also recently demonstrated in our PS1/APP model that the presence of multiple soluble Abeta oligomeric forms at advanced ages blocked the neurotrophin (including soluble APPα) mediated PI3K/Akt/GSK-3β pro-survival pathway, and then contributing to the observed late neuronal vulnerability [17]. On the other hand, in young transgenic mice, sAPPα activated, at least through IGF-1 receptor and insulin receptor, the PI3K/Akt pathway, phosphorylated the GSK-3β activity and, in consequence, exerted a neuroprotective action. Therefore, the activation of this pathway, classically assigned as prosurvival, could influence on the limited tau phosphorylation and neuronal degeneration observed in most APP transgenic models. The effect of Abeta oligomers might be mediated by interaction with the different receptors. A key pathological feature of AD that APP-based transgenic mouse lines nicely reproduce is the formation of neuritic plaques with clusters of dystrophic neurites. All the fibrillar amyloid deposits in the hippocampus of our transgenic mice were identified as human-like neuritic plaques with dystrophic neurites (Fig. 1C and D; Fig. 3A). Though neuritic dystrophy may apply to both dendritic and axonal morphological changes, in our model the predominant axonal nature of the dystrophies was demonstrated [20]. Axonal dystrophies may result in dysfunction of synaptic terminals which could represent an early manifestation of axonal damage that precedes the appearance of synaptic loss.

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Impaired axonal transport and autophagy process are linked to the pathogenic processes of AD and the formation of dystrophies surrounding Abeta plaques [20;21]. Several studies showed that extracellular Abeta deposition induced the formation of axonal abnormalities, contributing to the impairments in the axonal transport [22]. In this sense, it has been reported that oligomeric Abeta, disrupts the microtubule cytoskeleton and caused neuritic dystrophy through tau hyperphosphorylation [23]. On the other hand, several lines of investigation support the notion that defective autophagy process, a cellular catabolic mechanism essential for degradation of aggregated proteins and organelles, significantly contributes to AD pathogenesis [24-27]. Numerous autophagy vacuoles accumulate within dystrophic neurites in AD brains and AD models [28-30]. In our PS1/APP model the axonal dystrophies were seen to have a large accumulation of a great variety of vesicles in the process of autophagy (Fig. 3B). We have recently reported the involvement of natural and synthetic Abeta oligomers in the activation of GSK-3β and tau phosphorylation. Thus, it is tempting to speculate that Abeta oligomers, acting through a yet unidentified mediator, caused the interruption of axonal transport, accumulation of vesicles and axonal dystrophy. The extracellular or intracellular origin of these soluble amyloid oligomers has not yet been well defined. In relation to this, we have seen that Abeta peptides (Abeta42 the most abundant) accumulated in PS1/APP synaptosomes. In addition, and taken together, the close temporal and spatial association between amyloid plaques and dystrophic neurites (Fig. 1C), the presence of A11-positive Abeta oligomers in the plaque periphery (Fig. 1D) and the Abeta oligomers in the soluble fractions suggested that amyloid plaques also might be a source of the oligomers that could induce neuritic damage, synaptic loss, microglial activation and neuronal death. Thought it is strongly believed that diffusible Abeta oligomers play a principal role inducing local neuronal injury and dysfunction, many other factors, such as, tau, vascular alterations, glial responses, inflammation and oxidative stress, among many others, may have important co-pathogenic roles. There is an urgent need to elucidate the link between Aβ oligomers and these factors, with special interest tau pathology, to provide novel avenues for therapeutic intervention to treat this devastating neurodegenerative disease.

Acknowledgements This work was supported by grants FIS PS09/00099 (to AG), PS09/00151 (to JV) from Instituto de Salud Carlos III (Spain), and by grants CTS-4795 (to JV) and SAS PI-0496/2009 (to AG) from Junta de Andalucía (Spain). We are very grateful to R. Sanchez-Varo, D. Baglietto-Vargas, I. Moreno-Gonzalez, E. Sanchez-Mejias, L. Trujillo-Estrada, S. Jimenez and M. Torres for their excellent technical work.

References 1. Holtzman DM, Morris JC, Goate AM (2011) Alzheimer's disease: the challenge of the second century. Sci Transl Med 3:77sr1

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2. Arendt T (2009) Synaptic degeneration in Alzheimer's disease. Acta Neuropathol 118:167-179 3. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572-580 4. Larson ME, Lesne SE (2012) Soluble Abeta oligomer production and toxicity. J Neurochem 120 Suppl 1:125-139 5. Klein WL, Krafft GA, Finch CE (2001) Targeting small Abeta oligomers: the solution to an Alzheimer's disease conundrum? Trends Neurosci 24:219-224 6. Selkoe DJ (2002) Alzheimer's disease is a synaptic failure. Science 298:789-791 7. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 95:6448-6453 8. Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ (2008) Amyloidbeta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 14:837-842 9. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416:535-539 10. Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH (2005) Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci 8:79-84 11. Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH (2006) A specific amyloid-beta protein assembly in the brain impairs memory. Nature 440:352-357 12. Lacor PN, Buniel MC, Furlow PW, Clemente AS, Velasco PT, Wood M, Viola KL, Klein WL (2007) Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease. J Neurosci 27:796807 13. Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL (2007) Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27:2866-2875 14. Baglietto-Vargas D, Moreno-Gonzalez I, Sanchez-Varo R, Jimenez S, Trujillo-Estrada L, Sanchez-Mejias E, Torres M, Romero-Acebal M, Ruano D, Vizuete M, Vitorica J, Gutierrez A (2010) Calretinin interneurons are early targets of extracellular amyloid-beta pathology in PS1/AbetaPP Alzheimer mice hippocampus. J Alzheimers Dis 21:119-132 15. Caballero C, Jimenez S, Moreno-Gonzalez I, Baglietto-Vargas D, Sanchez-Varo R, Gavilan MP, Ramos B, Del Rio JC, Vizuete M, Gutierrez A, Ruano D, Vitorica J (2007) Interindividual variability in the expression of the mutated form of hPS1M146L determined the production of Abeta peptides in the PS1xAPP transgenic mice. J Neurosci Res 85:787-797 16. Jimenez S, Baglietto-Vargas D, Caballero C, Moreno-Gonzalez I, Torres M, SanchezVaro R, Ruano D, Vizuete M, Gutierrez A, Vitorica J (2008) Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer's disease: agedependent switch in the microglial phenotype from alternative to classic. J Neurosci 28:11650-11661 17. Jimenez S, Torres M, Vizuete M, Sanchez-Varo R, Sanchez-Mejias E, Trujillo-Estrada L, Carmona-Cuenca I, Caballero C, Ruano D, Gutierrez A, Vitorica J (2011) Age-dependent

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accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model. J Biol Chem 286:18414-18425 18. Moreno-Gonzalez I, Baglietto-Vargas D, Sanchez-Varo R, Jimenez S, Trujillo-Estrada L, Sanchez-Mejias E, Del Rio JC, Torres M, Romero-Acebal M, Ruano D, Vizuete M, Vitorica J, Gutierrez A (2009) Extracellular amyloid-beta and cytotoxic glial activation induce significant entorhinal neuron loss in young PS1(M146L)/APP(751SL) mice. J Alzheimers Dis 18:755776 19. Ramos B, Baglietto-Vargas D, Del Rio JC, Moreno-Gonzalez I, Santa-Maria C, Jimenez S, Caballero C, Lopez-Tellez JF, Khan ZU, Ruano D, Gutierrez A, Vitorica J (2006) Early neuropathology of somatostatin/NPY GABAergic cells in the hippocampus of a PS1xAPP transgenic model of Alzheimer's disease. Neurobiol Aging 27:1658-1672 20. Sanchez-Varo R, Trujillo-Estrada L, Sanchez-Mejias E, Torres M, Baglietto-Vargas D, Moreno-Gonzalez I, De C, V, Jimenez S, Ruano D, Vizuete M, Davila JC, Garcia-Verdugo JM, Jimenez AJ, Vitorica J, Gutierrez A (2012) Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer's mice hippocampus. Acta Neuropathol 123:53-70 21. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM (2005) Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol 64:113-122 22. Higuchi M, Iwata N, Matsuba Y, Takano J, Suemoto T, Maeda J, Ji B, Ono M, Staufenbiel M, Suhara T, Saido TC (2012) Mechanistic involvement of the calpaincalpastatin system in Alzheimer neuropathology. FASEB J 26:1204-1217 23. Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ (2011) Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci U S A 108:5819-5824 24. Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. J Neurosci 28:6926-6937 25. Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D, Cuervo AM, Nixon RA (2010) Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimerrelated PS1 mutations. Cell 141:1146-1158 26. Nixon RA (2007) Autophagy, amyloidogenesis and Alzheimer disease. J Cell Sci 120:4081-4091 27. Nixon RA, Yang DS (2011) Autophagy failure in Alzheimer's disease-locating the primary defect. Neurobiol Dis 28. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM (2005) Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol 64:113-122 29. Nixon RA (2007) Autophagy, amyloidogenesis and Alzheimer disease. J Cell Sci 120:4081-4091 30. Yu WH, Kumar A, Peterhoff C, Shapiro KL, Uchiyama Y, Lamb BT, Cuervo AM, Nixon RA (2004) Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease. Int J Biochem Cell Biol 36:2531-2540

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Stem Cells And Adult Neurogenesis In The Human Brain Mª Salomé Sirerol-Piquer and José Manuel GarcíaVerdugo Department of Comparative Neurobiology. Cavanilles Institute. University of Valencia. CIBERNED. Spain.

Abstract Neural stem cells (NSCs) persist in the adult mammalian brain and exhibit the two fundamental properties of stem cells: self-renewal and pluripotency, generating neurons, astrocytes and oligodendrocytes. NSCs are mainly restricted to two proliferative areas: the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. These two brain proliferative areas persist among mammalian species from rodent to human, although their organization remains slightly different between different species. Neuroblasts born in the adult rodent and non-human primates SVZ migrate from the SVZ to the olfactory bulb (OB), where they differentiate into local interneurons. Curiously, this migration to the OB is not observed in adult human. However it is present during development in fetal and infant brain samples, disappearing at 18 months of age. Surprisingly, early postnatally a novel migratory pathway to the prefrontal cortex has recently been described.

SVZ structure from rodent to humans The SVZ is located close to the striatum and the lateral walls of the lateral ventricles and represents the main source of NSCs in the adult mammalian brain. Classically, the rodent SVZ has been described as a discontinuous layer composed of four cell types, arranged up to four cell layers (1, 2). Type E cells (ependymal cells) line the cavity of the lateral ventricle, presenting cubical morphology and multiple long cilia in contact with the ventricle. Cilia can be detected by acetil-tubulin and γ-tubulin immunofluorescent staining (Figure 1A-D). Type B cells (astrocytes), subdivided in B1 and B2 subtypes. Type B1 cells are the NSCs, contact the ventricular cavity where they send a small primary cilium and divide at a low rate generating the transient amplifying cells (type C cells). Type B2 cells do not contact the ventricle. Type C cells are highly proliferative cells and give rise immature neuroblasts (type A cells) cells, which migrate towards the OB (1, 2). Recent studies with SVZ wholemounts staining and confocal microscopy revealed the existence of a novel cell type: type E2 cells (biciliated cells), a subtype of type E cell, which appears dispersed in the wall of the lateral ventricles. Type E2 cells present two long cilia in contact with the ventricle (Figure 1A-C) (3). Moreover the number of type B1 cells was

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nearly one-third (31%) of the cells contacting the ventricle, much more than expected, and appeared clustered and surrounded by ependymal cells in a specific pattern, resembling a pinwheel (Figure 1D) (3). Finally, analysis beneath the ventricular surface and ependymal layer showed the presence of a long basal process in type B1 cells, which terminated in an endfoot on a blood vessel (Figure 1E) (3-5). All these observations paved the way to propose a new 3-dimensional model of the adult SVZ neurogenic niche, revealing its pinwheel organization, the presence of the biciliated cells, as well as, the presence of the apical short primary cilium, contacting the ventricular surface and the long basal process contacting the blood vessel, in type B1 cells (Figure 1F) (3, 6).

In non-human primates and humans, the SVZ organization remains slightly different to that observed in rodents. While in rodents the SVZ consists of a thin layer, in the others appears organized in three layers; the ependymal layer (layer I), the gap layer (hypocellular

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or layer II), composed by the basal ependymal expansions and astrocytic processes and the astrocytic ribbon layer (layer III), which is composed mainly by astrocytes cell bodies, although the human gap layer appears thicker to that observed in non-human primates (710). Type C and type A cells are completely absent in adult human SVZ and no neuroblasts migrating chains are observed (Figure 2) (9, 11).

Migration to the OB from rodent to human In rodents, large chains of migrating neuroblast ensheated by astrocytes (gliotubes) migrate tangentially from the SVZ to the OB through the rostral migratory stream (RMS) and differentiate into granular and perigranular neurons (Figure 3A). In Macaca monkeys

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migrating neuroblast chains from the RMS converge into the olfactory tract (OT) (Figure 3B). Initially, the OT appears triangular shaped, but progressively and advancing rostrally it becomes more elongated and the number of neuroblast and the size of migrating chains also appears diminished (Figure 3C-H) (7, 8).

There is a progressive decrease in the

number of migrating chains and neuroblast from rodent to non-human primates, and finally, in adult human samples, no migrating neuroblast chains have been observed in the RMS and in the OT. Curiously, a large blood vessel lined by endothelial cells and smooth muscle can be seen in the olfactory tract core (Figure 3I-J) (12). Although migrating neuroblast chains are not present in adult human samples, it is possible to observe isolated rare immature neuroblasts, which may be sporadically migrating to the OB (Figure 3K).

However, the fetal and early post-natal SVZ, RMS and OT contain an extensive corridor of migrating immature neurons, which decreases progressively disappearing at 18 moth of

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age (Figure 4) (13, 14). In the first six month of life, the structure of the SVZ in infants differs considerably from that observed in adults. The astrocytic ribbon and gap layer are not evident and, as seen in the fetal human brain, cells with elongated radial processes (type B cells) line the lateral ventricular wall. Adjacent to these radial glia cells a dense network of elongated and unipolar and bipolar cells, most of them expressing doublecortin and Î˛-III tubulin (type A cells). Progressively, between 6 to 18 months of age, the SVZ decreases drastically the number of radial glial cells and is depleted of this dense network of immature migratory neurons, which corresponds with the disappearance of the migration through the RMS and OT to the OB in humans (14).

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Early postnatal migration to the prefrontal cortex in human samples Surprisingly, paediatric human SVZ neurogenesis also seems to serve regions other than the OB. Serial coronal reconstruction of the frontal lobe has revealed and additional migratory stream of DCX+ cells (neuroblasts) branching off the proximal limb of the RMS and ending in the ventromedial prefrontal cortex (VMPFC). This medial migratory stream (MMS) has been observed in human specimens aged 4-6 months but not 8-18 months (Figure 4). This pathway could represent a novel source of interneurons in the developing human prefrontal cortex, important for learning, memory and post-natal plasticity (14).

References 1. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. Cellular composition and threedimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 1997;17:5046-5061. 2. Garcia-Verdugo JM, Doetsch F, Wichterle H, Lim DA, Alvarez-Buylla A. Architecture and cell types of the adult subventricular zone: in search of the stem cells. J Neurobiol 1998;36:234-248. 3. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 2008;3:265-278. 4. Shen Q, Wang Y, Kokovay E, et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 2008;3:289-300. 5. Tavazoie M, Van der Veken L, Silva-Vargas V, et al. A specialized vascular niche for adult neural stem cells. Cell Stem Cell 2008;3:279-288. 6. Currle DS, Gilbertson RJ. The niche revealed. Cell Stem Cell 2008;3:234-236. 7. Gil-Perotin S, Alvarez-Buylla A, Garcia-Verdugo JM. Identification and characterization of neural progenitor cells in the adult mammalian brain. Adv Anat Embryol Cell Biol 2009;203:1-101, ix. 8. Gil-Perotin S, Duran-Moreno M, Belzunegui S, Luquin MR, Garcia-Verdugo JM. Ultrastructure of the subventricular zone in Macaca fascicularis and evidence of a mouselike migratory stream. J Comp Neurol 2009;514:533-554. 9. Quinones-Hinojosa A, Sanai N, Soriano-Navarro M, et al. Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells. J Comp Neurol 2006;494:415-434. 10. Sanai N, Tramontin AD, Quinones-Hinojosa A, et al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 2004;427:740-744. 11. Quinones-Hinojosa A, Chaichana K. The human subventricular zone: a source of new cells and a potential source of brain tumors. Exp Neurol 2007;205:313-324. 12. Sanai N, Berger MS, Garcia-Verdugo JM, Alvarez-Buylla A. Comment on "Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension". Science 2007;318:393; author reply 393. 13. Guerrero-Cazares H, Gonzalez-Perez O, Soriano-Navarro M, Zamora-Berridi G, GarciaVerdugo JM, Quinones-Hinojosa A. Cytoarchitecture of the lateral ganglionic eminence and rostral extension of the lateral ventricle in the human fetal brain. J Comp Neurol 2011;519:1165-1180.

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14. Sanai N, Nguyen T, Ihrie RA, et al. Corridors of migrating neurons in the human brain and their decline during infancy. Nature 2011;478:382-386.

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2.4. Nanotechnology opportunities

and

Cell

Biology:

challenges

and

Functional Magnetic Nanoparticles For Life Sciences Clara Marquina1,2 , Alejandro Pérez-Luque3, Gerardo F. Goya2,4, Rodrigo Fernández-Pacheco5, Laura Asin4, Jesús M. De La Fuente4, Maria-Carmen Risueño6, Pilar S. Testillano6 and Manuel R. Ibarra2,4,5 1. Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC- Universidad de Zaragoza, Zaragoza (SPAIN) 2. Departamento de Física de la Materia Condensada,Universidad de Zaragoza, Zaragoza (SPAIN) 3. IFAPA, Centro Alameda del Obispo, Área de Mejora y Biotecnología, PO Box 3092, Córdoba, 14004 (SPAIN) 4. Instituto de Nanociencia de Aragón (INA) (Universidad de Zaragoza) Zaragoza, (SPAIN) 5. Laboratorio Microscopias Avanzadas (LMA) (Universidad de Zaragoza) Zaragoza (SPAIN) 6. Centro de Investigaciones Biológicas, CSIC Madrid (SPAIN) The scope of this contribution is to establish a parallelism between the application of nanotechnology in animal and plant kingdoms. We focus our study on the increasing interest for the application of magnetic nanoparticles in life sciences. A clear example is the use of magnetic nanoparticles for selective drug or chemicals release with clear application in therapy or in agriculture. There exist a broad scientific scenario concerning how new inorganic functional nanostructured materials interact with elemental biological units, in particulars with cells. In the field of medicine new therapy and diagnosis agents are being investigated with the goals of a selective drug delivery and an early diagnosis. Although incipient and promising applications in the clinic are encouraging the new field of nanobiomedicine, this discipline still is in the infancy. However, the scientific interest is rapidly increasing and nowadays nanotechnology is considered as the main candidate for an effective treatment of cancer and neurodegenerative diseases. (1-5) Other emerging activity in this field concerns the application of nanoparticles in plants (6-8). One can image the great impact in agriculture, ecology and health food. There exists an overwhelming lack of information regarding the interaction of functional nanoparticles with

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plants, which is a topic of major interest in environmental issues, phytosanitary and food industries. In this contribution an overview on the interdisciplinary investigations carried out in collaboration among several research groups is given. The reported results involve the use iron or iron oxides nanoparticles encapsulated in a carbon matrix (FexOy@C) (Fig. 1). The magnetic behavior and the biocompatibility of these core-shell nanoparticles provide an excellent frame of work for their use in life sciences. In MRI, the magnetic behavior of the nanoparticles has a strong influence on the local internal fields acting on the tissues where the magnetic nanoparticles are allocated, modifying the resonance of the H protons in the tissueâ&#x20AC;&#x2122;s water; this is relevant for enhancing the MRI contrast. Organ targeting by magnetic nanoparticles can be favored by steady magnetic fields and further local heating by application of radiofrequency electromagnetic fields (magnetic hyperthermia) allows a selective treatment of cancer tumor. Some drawbacks for the systemic application of nanoparticles inside the organism are the Reticule Endothelial-System (RES) capture and physiological barriers as the Brain Blood Barrier (BBB).

These could be overcome by functionalization of the nanoparticles surface. Other emerging field in nanobiomedicine to avoid the macrophage capture is based in the use of vaccines based in dendritic cells uploaded with magnetic nanoparticles (See Fig. 2). This procedure allows the targeting of living magnetic nanovectors, as vaccines, to the tumor place, allowing a theragnostic based on the magnetic functionality.

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In a parallel way as in nanobiomedicine, we have undertaken a series of studies of the application of the above mentioned nanoparticles in the plant kingdom, in order to investigate the translocation and cell penetration when the nanoparticles are systemically injected in living plants (see Fig. 3). We observed that nanoparticles move upwards through

the xylem vessels following the transpiration stream and also downwards by source-sink pressure gradient perhaps through the phloem. We also found evidence of the radial transport out of the vascular system after 48 h, probably by transcytosis mechanisms. From our studies we have demonstrated that plants can tolerate the core-shell nanoparticles, and that they can be used for localized treatments by applying a magnetic field (Fig. 3). The nanoparticles in the plants form aggregates that are clearly visible as dark region in the images. Then the regions before and after the place where the magnet was located are displayed in Fig. 4 a clear different contrast reveals the effect of the local effect of the magnetic field provided by the permanent magnet. In addition to the systemic applications of bioferrofluid (nanoparticles water based suspension) into the plants we achieved a successful uptake of the nanoparticles

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by the plant roots; we monitored the translocation of such nanoparticles into the aerial

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part of the plant belonging to the four crop

species (Fig. 5) after 24 and 48 hours of

exposure to the bioferrofluid. The black deposit corresponding to the nanoparticles was clearly visible in the xylem vessels after 24 hours (8). It implies that the nanoparticles had quickly moved towards the aerial part of the plants following the transpiration stream.

In conclusion the application of magnetic nanoparticles en life science opens new expectation in human clinic in early diagnosis and new therapies. These advances can be translated to the plant kingdom in which we can learn about mechanism for nanoagent effect on the everyday life which is nowadays a great challenge in nanotechnology

References 1. Fernández-Pacheco R. et al “Magnetic Nanoparticles for Local Drug Delivery Using Magnetic Implants”;James Weifu Lee and Robert S. Foote (eds.), Micro and Nano Technologies in Bioanalysis, (2009) Methods in Molecular Biology, vol. 544 559 2. R. Fernández-Pacheco et al., “Carbon Coated Magnetic Nanoparticles For Local Drug Delivery Using Magnetic Implants” (2005) Nanobiotechnology, DOI: 10.1385/Nano:1:3:300. 3. Goya G.F. et al. “Magnetic Nanoparticles for Cancer Therapy”. Current Nanoscience, 2008, 4, 1-16 1. 1573-4137/08 Bentham Science Publishers Ltd. 4. G.F. Goya, et al. “Dendritic cell uptake of iron-based magnetic nanoparticles”,(2008) Cell Biology International, 32, 1001-1005.

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5. Marcos-Campos I. et al. “Cell death induced by the application of alternating magnetic fields to nanoparticle-loaded dendritic cells”. (2011) Nanotechnology, 22 (20), art. 205101. 6. González-Melendi P, et al. “Nanoparticles as smart treatment-delivery systems in plants: Assessment of different techniques of microscopy for their visualization in plant tissues”. (2008) Ann Bot 101:187-195. 7. Corredor E, et al. “Nanoparticle penetration and transport in living pumpkin plants: in situ subcellular identification”. (2009) BMC Plant Biol 9,45. 8. Cifuentes et al. Journal of Nanobiotechnology (2010), 8, 26 (http://www.jnanobiotechnology.com/content/8/1/26)

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Tumor targeting of drug-loaded magnetic nanoparticles by an external magnetic field for in vivo cytokine delivery in cancer immunotherapy Raquel Mejías and Domingo F. Barber Department of Immunology & Oncology, Centro Nacional de Biotecnología (CNBCSIC), Darwin 3, Cantoblanco 28049, Madrid, Spain.

Correspondence to

dfbarber@cnb.csic.es

Abstract Modulation of the anti-tumor immune response by administration of cytokines, chemokines or antibodies is one of the strategies used in cancer immunotherapy. It is nonetheless difficult to achieve adequate therapeutic dosages without generating toxicity to other tissues. The use of magnetic nanoparticles, which can be guided to the site of action by application of an external magnetic gradient, or of magnetic implants, is one of the most promising strategies for localized drug delivery. We will evaluate magnetically guided drug delivery systems.

Use of cytokines in tumor immunotherapy Classical cancer treatment is based on surgery, radiotherapy and chemotherapy. These procedures affect both tumor and healthy tissue, which has led to a search for new therapies that affect tumors specifically. Tumor immunotherapy attempts to enhance the immune system’s natural ability to detect and eliminate tumor cells. The immune response relies on the balance of cytokines produced by two T helper (Th) cell subsets, Th1 and Th2. The type 1 response, regulated by cytokines such as interleukin-2 (IL-2), IL-12 and interferon-gamma (IFN-γ), is the most effective against tumors and is essential for their in

vivo eradication.

The anti-tumor efficiency of several cytokines has been evaluated in

preclinical and clinical trials, including IL-2, IL-12, IL-4, IFN-α , IFN-β, IFN-γ, IL-15, GM-CSF, IL-24 and TNF-α, alone or in combination (reviewed in [1, 2]).

The results have been

variable, and depend mainly on the route of administration (Table 1).

Systemic

administration of cytokines has several problems. The elevated levels of cytokine needed produce serious side effects such as hypotension, increased vascular permeability, cardiac and respiratory failure, renal and hepatic toxicity, fever, coagulopathies and lethargy, which can be lethal in severe cases [3]; even so, the cytokine concentration at the site of action is usually far below the levels necessary to activate the immune response. Moreover, the increase in cytokine concentration is transient, since these molecules are rapidly eliminated

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by renal and hepatic clearance, and time is thus not sufficient for mobilization and activation of the immune system [2].

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Systems of cytokine distribution and/or release in immunotherapy One strategy that sidesteps some of the above problems is the use of gene therapy, an experimental technique involving the use of vectors to deliver therapeutic DNA into target cells. To increase cytokine dosage locally and sustainably in the tumor environment, several types of viral vectors carrying cytokine genes have been injected into tumors. Alternatively, tumor cells isolated from the patient have been transfected in vitro with plasmids bearing cytokine genes and reinjected into the tumors. Results of these approaches have been extremely variable, depending on virus efficiency, the type of viral vector used, and the efficiency of in vitro cell transfection ([1, 2]). Another important problem with these strategies is that the vector must be injected into or near the tumor mass, which can be difficult to achieve depending on the anatomical location of the tumor. In addition, repeated tumor injection can lead to infection and fistula formation [20]. There is also evidence that although these vectors are injected locally, they can spread throughout the body [21], potentially leading to adverse effects on other tissues [22]. Recent results using polymeric microspheres and gels to deliver cytokines into tumors have been promising [2, 8].

These compounds provide more homogeneous cytokine

release and, as they consist of biocompatible materials, do not generate toxic reactions. As in the case of genetic vectors, however, they must be injected locally, and tend to diffuse. Results so far have not been as favorable as anticipated, due mainly to the difficulty in achieving a therapeutically beneficial dose without generating excessive tissue toxicity [2]. New drug delivery systems are therefore being developed to allow local delivery and retention at the site of action as long as possible, with minimal side effects. Some of these systems are based on magnetic nanoparticles, which can be guided and retained in the area of interest using external magnetic gradients; they represent one of the most active research fields at present (reviewed in [23]).

Nanoparticle-based drug delivery systems The effect of some drugs is occasionally compromised by the physicochemical properties of these compounds; they might show reduced solubility or biodistribution, be rapidly removed or generate adverse reactions when administered [24]. One means of improving these properties is to bind the drug to a carrier [25]. Nanoparticles, which are defined as submicronic-sized solid and stable colloidal particles, were introduced in biomedicine for this purpose and were first used as chemotherapeutic drugs; later, other biomedical applications were developed (Table 2).

Nanoparticle-based drug delivery

systems have notable advantages over other systems, including the ability to reach most areas in the body due to their small size. In addition, they can be directed to areas of interest using diverse strategies; this reduces the amount of drug needed to achieve a therapeutically relevant dose in that region and decrease drug levels in other tissues, thus minimizing possible side effects.

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Factors that affect the efficiency of nanoparticle-based drug delivery systems Passive or active strategies can be used to direct nanoparticles to their site of action [27]. Passive strategies are the result of nanoparticle extravasation to the target region. Nanoparticles accumulate more easily in inflamed or infected regions, or in tumor masses, due a process known as enhanced permeability and retention (EPR [28]). Nanoparticle concentration in these areas is caused by increased vascular permeability and decreased lymphatic drainage [29]. Active strategies are based on the exclusive expression of specific proteins in target tissues, or specific physicochemical characteristics of nanoparticles such as their size, hydrophobicity or hydrophilicity, and their response to factors such as temperature, pH, electrical charge or magnetism [30]. Of these features, the most decisive in determining nanoparticle fate in the body is particle size, given the differential permeability of each blood vessel type (Figure 1). Blood circulation time of nanoparticles is also sizedependent [31].

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Use of nanoparticles in tumor therapy Among the new therapeutic strategies developed to increase anti-tumor specificity, some of the most promising are based on nanoparticles composed of different materials (reviewed in [33-36]). The EPR effect is particularly important in tumors, since their blood vessels are larger and have a discontinuous endothelium with fenestrations [37], making them more permeable than those in normal tissues [38]. Venous return is slower in tumors, and tumors show reduced lymphatic drainage compared to normal, or even to inflamed or infected tissue [39]. This is the case for most tumors, although vascular organization can depend on tumor type, growth rate and microenvironment [40]. Numerous studies demonstrate passive accumulation of nanoparticles in solid tumors after intravenous administration [41-44]. Several preclinical and clinical trials showed that binding different anti-tumor drugs to nanoparticles of various types and materials increases drug effectiveness compared to administration of the same dose of soluble drug [45-48]. Passive strategies can nonetheless be hampered by the generation of elevated interstitial pressure in tumors, which tends to be higher in the central region and decreases towards the periphery, causing fluid movement from the center of the tumor mass and hindering prolonged drug permanence at its site of action [49].

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To improve the effectiveness of nanoparticle-based localized drug delivery systems, various strategies have been developed to direct nanoparticles into the region of interest. Some possibilities include union of nanoparticles to ligands or antibodies that target a specific tissue type, or use of specific particle characteristics and their response to certain factors. One design of interest is the use of magnetic nanoparticles, which can be guided by application of a magnetic field gradient, such that the particles are directed to a specific location, where they can be immobilized for the time required.

Magnetic nanoparticles The use of magnetic nanoparticles as drug carriers guided by an external magnetic field gradient was first proposed in 1960 by Freeman (reviewed in[26, 27]). This idea is based on competition between the forces exerted on the particles by the blood flow and a transversally applied magnetic field. When the latter exceeds the former, nanoparticles are retained in the area to which the field is applied (Figure 2). For use in biomedicine, especially for in vivo applications, nanoparticles must have certain characteristics that are determined by the magnetic core and the coating.

6.1 Magnetic core

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The magnetic material must meet certain basic requirements [26], the first of which is superparamagnetic behavior. In magnetic materials, fluctuations in the direction of magnetization affect constituent atoms individually. In superparamagnetic materials, formed by small-sized particles with a single magnetic domain, these fluctuations affect the entire particle, and their magnetic behavior is reversible.

This is especially important for

biomedical use, as when the magnetic field is not applied, the material has no residual magnetization (Figure 3); this prevents attraction and agglomeration between particles and avoids potential problems such as embolization of blood capillaries.

Biocompatibility of the magnetic core is also necessary. Iron-cored nanoparticles are processed by cells via the biochemical pathways of iron metabolism [50, 51], whereas other nanoparticle types with non-biocompatible cores must be coated to allow their excretion [52]. Finally, they must have high magnetization, so that movement can be controlled by application of an external magnetic field.

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6.2 Nanoparticle coating Nanoparticles must be coated with a biocompatible material to avoid toxicity and immunogenicity and to increase blood circulation time. The coating can also provide the ability to bind certain compounds or a surface electric charge at physiological pH, allowing stabilization and preventing aggregate formation by the repulsion between same-sign charges.

Using magnetic nanoparticles as delivery systems for anti-tumor drugs One of the first studies that used these particles as anti-tumor drug carriers was published in 1996, in murine models of renal and colon cancer. Mice were treated with magnetic nanoparticles coated with anhydroglucose and loaded with epirubicin, with a hydrodynamic size between 50 and 150 nm; injection into the afferent tumor arteries led to embolization of these vessels and rapid regression in tumor size [53].

The authors

conducted a small clinical trial in 14 patients with advanced solid tumors using the same type of nanoparticles, which delayed tumor growth in some cases [54]. Since then, several nanoparticle types with different coatings have been developed for use as drug delivery systems, with varying results. Injection of starch polymer-coated magnetic nanoparticles loaded with mitoxantrone into rabbits with squamous cell carcinoma led to complete tumor remission [55-58]. In these studies, magnetic nanoparticles with a 100 nm hydrodynamic size were administered intra-arterially in the afferent tumor vessels, ensuring their delivery to the tumor tissue. This route of administration nonetheless shows several complications depending on tumor type, as accessibility can be restricted by location.

Folate-coated

magnetic nanoparticles were used as a doxycycline delivery system in rats and rabbits with liver cancer.

In this case, nanoparticles were directed to the area of interest through

magnetic field application and by folate interaction with its receptor expressed on tumor cells, and resulted in a significant reduction in tumor size [59]. Although there are several complications, the results of these studies encourage further development of new anti-tumor drug delivery systems.

Dimercaptosuccinic acid (DMSA)-coated magnetite nanoparticles for magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy We recently proposed the use of magnetic nanoparticles as a localized IFN-Îł delivery system in murine cancer models [60, 61]. The cytokine was adsorbed to the surface of different types of magnetic nanoparticles by electrostatic interaction. Since IFN-Îł has a positive charge at physiological pH, we used negatively charged nanoparticles. The results indicate that factors such as pH, ionic strength of the medium, and type of nanoparticle

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coating affect drug adsorption. Most efficient IFN-γ adsorption was obtained with DMSAcoated nanoparticles [60]. The development of new procedures in biomedicine involves the need to identify potential risks arising from their use. In the case of nanotechnology, this is particularly important because of the high surface/mass ratio of nanoparticles, which renders them particularly reactive.

Different nanoparticle types can have toxic effects by altering cell

adhesion and division or affecting metabolism, with a decrease in viability [62-64]. These effects depend on nanoparticle uptake by distinct cell types, which depends in turn on nanoparticle sample characteristics such as chemical composition, size and shape, the nature of the coating, surface charge, crystallinity, state of aggregation, hydrophobicity or hydrophilicity, and degree of oxidation and degradation [23, 65, 66]. Several studies showed that DMSA-coated magnetic nanoparticles produce no genotoxic effects, significantly decrease cell viability in human fibroblasts or HeLa cells [64, 67], and affect HeLa cytoarchitecture [64]. We analyzed possible direct and indirect toxic effects of DMSA-coated magnetic nanoparticles in vitro in the murine pancreatic ductal adenocarcinoma cell line Pan02, and found no adverse effects on cell metabolism, alterations in cell shape, cytoskeletal organization or cell division, although nanoparticles were internalized by these cells [61]. For in vivo applications, study of magnetic nanoparticle biocompatibility and biodistribution is essential. Nanoparticle biodistribution depends on features such as surface properties, size, shape, and concentration [68-72]. Studies that analyzed DMSA-coated magnetic nanoparticle biodistribution found that preferential accumulation in the lung [73]; this could lead to pathological processes due to transendothelial migration of immune cells to the lung parenchyma, resulting in acute inflammation and tissue damage [74-76]. Our analysis of DMSA-coated magnetic nanoparticle distribution in C57BL/6 mice yielded similar results, with particle accumulation in lung tissue and in organs where iron removal takes place such as liver and spleen [61, 77]. Since particle surface charge and size are basic aspects that determine their destination in the body, however, biodistribution could vary after binding of a drug to the particle surface. We therefore analyzed biodistribution of IFN-γadsorbed DMSA-coated magnetic nanoparticles. Our results indicated that biodistribution changes when IFN-γ is adsorbed to the nanoparticle surface, decreasing lung accumulation and reducing the risk of tissue damage, while increasing particle accumulation in liver and spleen. Nanoparticles were also detected at higher concentrations in blood, probably due to partial neutralization of the surface charge, which increases blood circulation time [61]. After injection of DMSA-coated magnetic nanoparticles, alone or IFN-γ-adsorbed, mice were observed daily throughout the treatment period for signs of systemic toxicity (weight loss, disheveled hair, respiratory or gastrointestinal symptoms, immobility or death); no signs were detected. Other studies show that application of an external magnetic field can vary magnetic nanoparticle biodistribution [78]; this strategy has been used to concentrate nanoparticles in tumors [53, 55, 56, 79-82]. Our tests used magnets with a field strength of 0.4 Tesla, which was sufficient to attract IFN-γ -adsorbed DMSA-coated magnetic nanoparticles to the site of action in two murine cancer models, one generated by subcutaneous injection of Pan02

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cells, and the other induced chemically by subcutaneous injection of 3-methylcholanthrene (3-MCA). In both models, nanoparticle accumulation in tumors was accompanied by an increase in IFN-γ levels, indicating that the system effectively distributed the drug to the tumor [61].

Nonetheless, IFN-γ effects were not the same in the two models.

IFN-γ

accumulation within tumors led to increased immune cell infiltration, particularly of T cells in 3-MCA-induced tumors and of NK cells in Pan02 tumors.

Macrophage number was

increased in both tumor types. We also analyzed the anti-angiogenic effect of IFN-γ, and found a significant decrease in degree of vascularization (blood vessel number and size) in both models. IFN-γ exerted anti-proliferative and proapoptotic effects on Pan02 and 3-MCAinduced tumor cells, as determined by immunohistochemical detection of annexin V in tumor sections. All these effects together resulted in a significant reduction in tumor size after treatment compared to controls [61]. These findings indicate that DMSA-coated magnetic nanoparticles have potential widespread use for drug delivery. In summary, nanotechnology opens up exciting alternatives for treatment of different types of neoplastic diseases; these include local drug delivery, as we have seen, or the use of nanoliposomes or nanovesicles, which permit introduction of therapeutic agents into cells. An appropriate combination of these strategies could enable the design of individualized tumor therapies.

Acknowledgements We thank C Mark for editorial assistance. RM receives a FPU pre-doctoral fellowship from the Spanish Ministry of Science and Innovation (MICINN). This work was partially supported by grants from the MICINN (SAF-2008-00471 and SAF-2011-23639 to DFB), and the ISCIII-Spanish Ministry for Health & Social Policy (ISCIII-MSPS) Cooperative Research Thematic Network program (RETICS) Research Network in Inflammation and Rheumatic Diseases (RIER RD08/0075/0015 to DFB).

References 1. Chada S, Ramesh R, Mhashilkar AM. Cytokine- and chemokine-based gene therapy for cancer. 2003. Curr Opin Mol Ther 5(5):463-74. 2. Li CY, Huang Q, Kung HF. Cytokine and immuno-gene therapy for solid tumors. 2005. Cell Mol Immunol 2(2):81-91. 3. Vial T, Descotes J. Immune-mediated side-effects of cytokines in humans. 1995. Toxicology 105(1):31-57. 4. White MV, Igarashi Y, Emery BE, Lotze MT, Kaliner MA. Effects of in vivo administration of interleukin-2 (IL-2) and IL-4, alone and in combination, on ex vivo human basophil histamine release. 1992. Blood 79(6):1491-5. 5. Rochlitz CF, Jantscheff P, Bongartz G, Dietrich PY, Quiquerez AL, Schatz C, Mehtali M, Courtney M, Tartour E, Dorval T, Fridman WH, Herrmann, R. Gene therapy with cytokinetransfected xenogeneic cells in metastatic tumors. 1998. Adv Exp Med Biol 451:531-7. 6. Atkins MB, Kunkel L, Sznol M, Rosenberg SA. High-dose recombinant interleukin-2 therapy in patients with metastatic melanoma: long-term survival update. 2000. Cancer J Sci Am 6 Suppl 1:S11-4.

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7. Fisher RI, Rosenberg SA, Fyfe G. Long-term survival update for high-dose recombinant interleukin-2 in patients with renal cell carcinoma. 2000. Cancer J Sci Am 6 Suppl 1:S55-7. 8. Arora A, Su G, Mathiowitz E, Reineke J, Chang AE, Sabel MS. Neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity. 2006. J Surg Oncol 94(5):403-12. 9. Lotze MT, Rubin JT, Carty S, Edington H, Ferson P, Landreneau R, Pippin B, Posner M, Rosenfelder D, Watson C, Carlos T, Kirkwood J, Lembersky B, Logan T, Rosenstein M, Rybak ME, Whiteside T, Elder E, Moen RC, Jacob W, Chen Y, Pinkus RL, Bryant J. Gene therapy of cancer: a pilot study of IL-4-gene-modified fibroblasts admixed with autologous tumor to elicit an immune response. 1994. Hum Gene Ther 5(1):41-55. 10. Whitehead RP, Unger JM, Goodwin JW, Walker MJ, Thompson JA, Flaherty LE, Sondak VK. Phase II trial of recombinant human interleukin-4 in patients with disseminated malignant melanoma: a Southwest Oncology Group study. 1998 J Immunother 21(6):440-6. 11. Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, Sosman JA, Dutcher JP, Vogelzang NJ, Ryan JL. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. 1997. Blood 90(7):25418. 12. Sun Y, Jurgovsky K, Moller P, Alijagic S, Dorbic T, Georgieva J, Wittig B, Schadendorf D. Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study. 1998. Gene Ther 5(4):481-90. 13. Kang WK, Park C, Yoon HL, Kim WS, Yoon SS, Lee MH, Park K, Kim K, Jeong HS, Kim JA, Nam SJ, Yang JH, Son YI, Baek CH, Han J, Ree HJ, Lee ES, Kim SH, Kim DW, Ahn YC, Huh SJ, Choe YH, Lee JH, Park MH, Kong GS, Park EY, Kang YK, Bang YJ, Paik NS, Lee SN, Kim SH, Kim S, Robbins PD, Tahara H, Lotze MT, Park CH. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. 2001. Hum Gene Ther 12(6):671-84. 14. Sertoli MR, Bernengo MG, Ardizzoni A, Brunetti I, Falcone A, Vidili MG, Cusimano MP, Appino A, Doveil G, Fortini C, Rosso, R. Phase II trial of recombinant alpha-2b interferon in the treatment of metastatic skin melanoma. 1989. Oncology 46(2):96-8. 15. Falkson CI, Falkson G, Falkson HC. Improved results with the addition of interferon alfa2b to dacarbazine in the treatment of patients with metastatic malignant melanoma. 1991. J Clin Oncol 9(8):1403-8. 16. Jett JR, Maksymiuk AW, Su JQ, Mailliard JA, Krook JE, Tschetter LK, Kardinal CG, Twito DI, Levitt R, Gerstner JB. Phase III trial of recombinant interferon gamma in complete responders with small-cell lung cancer. 1994. J Clin Oncol 12(11):2321-6. 17. Abdel-Wahab Z, Weltz C, Hester D, Pickett N, Vervaert C, Barber JR, Jolly D, Seigler HF. A Phase I clinical trial of immunotherapy with interferon-gamma gene-modified autologous melanoma cells: monitoring the humoral immune response. 1997. Cancer 80(3):401-12. 18. Kircheis R, Ostermann E, Wolschek MF, Lichtenberger C, Magin-Lachmann C, Wightman L, Kursa M, Wagner E. Tumor-targeted gene delivery of tumor necrosis factoralpha induces tumor necrosis and tumor regression without systemic toxicity. 2002. Cancer Gene Ther 9(8):673-80. 19. Tani K, Azuma M, Nakazaki Y, Oyaizu N, Hase H, Ohata J, Takahashi K, OiwaMonna M, Hanazawa K, Wakumoto Y, Kawai K, Noguchi M, Soda Y, Kunisaki R, Watari K, Takahashi S, Machida U, Satoh N, Tojo A, Maekawa T, Eriguchi M, Tomikawa S, Tahara H, Inoue Y, Yoshikawa H, Yamada Y, Iwamoto A, Hamada H, Yamashita N, Okumura K, Kakizoe T, Akaza H, Fujime M, Clift S, Ando D, Mulligan R, Asano S. Phase I study of autologous tumor vaccines transduced with the GM-CSF gene in four patients with stage IV

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55. Alexiou C, Arnold W, Klein RJ, Parak FG, Hulin P, Bergemann C, Erhardt W, Wagenpfeil S, Lubbe AS. Locoregional cancer treatment with magnetic drug targeting. 2000. Cancer Res 60(23):6641-8. 56. Alexiou C, Jurgons R, Schmid RJ, Bergemann C, Henke J, Erhardt W, Huenges E, Parak F. Magnetic drug targeting--biodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment. 2003. J Drug Target 11(3):139-49. 57. Alexiou C, Jurgons R, Seliger C, Brunke O, Iro H, Odenbach S. Delivery of superparamagnetic nanoparticles for local chemotherapy after intraarterial infusion and magnetic drug targeting. 2007. Anticancer Res 27(4A):2019-22. 58. Lyer S, Tietze R, Jurgons R, Struffert T, Engelhorn T, Schreiber E, Dorfler A, Alexiou C. Visualisation of tumour regression after local chemotherapy with magnetic nanoparticles - a pilot study. 2010. Anticancer Res 30(5):1553-7. 59. Maeng JH, Lee DH, Jung KH, Bae YH, Park IS, Jeong S, Jeon YS, Shim CK, Kim W, Kim J, Lee J, Lee YM, Kim JH, Kim WH, Hong SS. Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer. 2010. Biomaterials 31(18):4995-5006. 60. Mejias R, Costo R, Roca AG, Arias CF, Veintemillas-Verdaguer S, Gonzalez-Carreno T, del Puerto Morales M, Serna CJ, Manes S, Barber DF. Cytokine adsorption/release on uniform magnetic nanoparticles for localized drug delivery. 2008. J Control Release 130(2):168-74. 61. Mejias R, Perez-Yague S, Gutierrez L, Cabrera LI, Spada R, Acedo P, Serna CJ, Lazaro FJ, Villanueva A, Morales MP, Barber DF. Dimercaptosuccinic acid-coated magnetite nanoparticles for magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy. 2011. Biomaterials 32(11):2938-52. 62. Lipski AM, Pino CJ, Haselton FR, Chen IW, Shastri VP. The effect of silica nanoparticlemodified surfaces on cell morphology, cytoskeletal organization and function. 2008. Biomaterials 29(28):3836-46. 63. Pernodet N, Fang X, Sun Y, Bakhtina A, Ramakrishnan A, Sokolov J, Ulman A, Rafailovich M. Adverse effects of citrate/gold nanoparticles on human dermal fibroblasts. 2006. Small 2(6):766-73. 64. Villanueva A, Canete M, Roca AG, Calero M, Veintemillas-Verdaguer S, Serna CJ, Morales MP, Miranda R. The influence of surface functionalization on the enhanced internalization of magnetic nanoparticles in cancer cells. 2009. Nanotechnology 20(11):115103. 65. Hillaireau H, Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. 2009. Cell Mol Life Sci 66(17):2873-96. 66. Nel AE, Madler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V, Thompson M. Understanding biophysicochemical interactions at the nano-bio interface. 2009. Nat Mater 8(7):543-57. 67. Auffan M, Decome L, Rose J, Orsiere T, De Meo M, Briois V, Chaneac C, Olivi L, BergeLefranc JL, Botta A, Wiesner MR, Bottero JY. In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: A physicochemical and cyto-genotoxical study. 2006. Environ Sci Technol 40(14):4367-73. 68. Chouly C, Pouliquen D, Lucet I, Jeune JJ, Jallet P. Development of superparamagnetic nanoparticles for MRI: effect of particle size, charge and surface nature on biodistribution. 1996. J Microencapsul 13(3):245-55. 69. Jain TK, Reddy MK, Morales MA, Leslie-Pelecky DL, Labhasetwar V. Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats. 2008. Mol Pharm 5(2):316-27.

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70. Kunath K, von Harpe A, Petersen H, Fischer D, Voigt K, Kissel T, Bickel U. The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice. 2002. Pharm Res 19(6):810-7. 71. Owens DE, 3rd, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. 2006. Int J Pharm 307(1):93-102. 72. Panagi Z, Beletsi A, Evangelatos G, Livaniou E, Ithakissios DS, Avgoustakis K. Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA-mPEG nanoparticles. 2001. Int J Pharm 221(1-2):143-52. 73. Chaves SB, Lacava LM, Lacava ZGM, Silva O, Pelegrini F, Buske N, Gansau C, Morais PC, Azevedo RB. Light microscopy and magnetic resonance characterization of a DMSAcoated magnetic fluid in mice. 2002. IEEE Trans Magn 38(5):3231-3. 74. Garcia M, Miranda Parca M, Chaves SB, Silva LP, Santos AD, Lacava ZGM, Morais PC, Azevedo RB. Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA. 2005. J Magn Magn Mat 293(1):277-82. 75. Valois CR, Braz JM, Nunes ES, Vinolo MA, Lima EC, Curi R, Kuebler WM, Azevedo RB. The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta2 integrin-dependent pathway. 2010. Biomaterials 31(2):366-74. 76. Valois CR, Nunes ES, Jaeger RG, Lima EC, Morais PC, Azevedo RB: Expression patterns of cell adhesion molecules in mice's lung after administration of meso-2,3dimercaptosuccinic acid-coated maghemite nanoparticles. 2009. J Nanosci Nanotechnol 9(5):2846-55. 77. Gutierrez L, MejĂas R, Barber DF, Veintemillas-Verdaguer S, Serna CJ, Lazaro FJ, Morales MP. Ac magnetic susceptibility study of in vivo nanoparticle biodistribution. 2011. J Phys D: Appl Phys 44(25):255002. 78. Wu T, Hua M, Chen J, Wei K, Jung S, Chang Y, Jou M, Ma Y. Effects of external magnetic field on biodistribution of nanoparticles: A histological study. 2007. J Magn Magn Mat 311(1):372-5. 79. Alexiou C, Schmid RJ, Jurgons R, Kremer M, Wanner G, Bergemann C, Huenges E, Nawroth T, Arnold W, Parak FG. Targeting cancer cells: magnetic nanoparticles as drug carriers. 2006. Eur Biophys J 35(5):446-50. 80. Alexiou C, Schmidt A, Klein RJ, Hulin P, Bergemann C, Arnold W. Magnetic drug targeting: biodistribution and dependency on magnetic field strength. 2002. J Magn Magn Mat 252:363-6. 81. Chertok B, David AE, Yang VC. Polyethyleneimine-modified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration. 2010. Biomaterials 31(24):6317-24. 82. Fernandez Pacheco F, Marquina C, Valdivia J, Gutierrez M, Romero M, Cornudella R, Laborda A, Viloria A, Higuera T, Garcia A, Garcia de Jalon JA, Ibarra MR. Magnetic nanoparticles for local drug delivery using magnetic implants. 2007. J Magn Magn Mat 311(1):318-22.

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Rational design and development of nanotechnology-based vasoactive intestinal peptide (VIP) applications in health research Rebecca Klippstein1,2 Soledad Lopez-Enriquez1,2 and David Pozo1,2,3* 1. CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine (CSIC-University of Seville-UPO), Americo Vespucio s/n. Parque Cientifico y Tecnologico Cartuja 93, E-41092, Seville, Spain 2. Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Sanchez Pizjuan, 4. E-41009, Seville, Spain 3. BIONAND-Andalusian Center for Nanomedicine and Biotechnology Av. Severo Ochoa, 34, Parque Tecnologico de Andalucia, E-29590 Malaga, Spain *Address correspondence to this author at the Department of Cell Signalling. Andalusian Center for Molecular Biology and Regenerative Medicine. CABIMER Bld. Americo Vespucio s/n. E410092, Seville. Spain; Tel: 34-95-4467841; Fax: +34-95-4461664; E-mail: david.pozo@cabimer.es

Introduction Engineering nanoconjugates constitute an extensive field of research due to its translational potential for biomedical applications. This is one of many examples of the emergence of nanoscience and nanotechnology as cutting-edge research areas that are being rapidly developed. The efforts are mainly related to the application of nanoengineering methods and materials to develop new diagnostic platforms and effective therapies for human diseases. Presently, there is a growing interest in different nanosystems and their biological features as nanocarriers of chemotherapeutic agents and other compounds known as “smart drug delivery” products. Drug delivery nano-systems are being studied to allow more selective and potent treatments as well as to improve the efficacy/toxicity ratio of our current and future therapeutic arsenal. The technical designs and the experimental approaches are aimed at formulating therapeutic agents in biocompatible nanocomposites such as noble metals, organic and inorganic nanoparticles, nanocapsules, and a variety of micellar systems.

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In this sense, engineered nanoparticles can potentially be designed to carry out multiple specific functions at once or in a predefined sequence, and as such have a unique advantage over other complimentary technologies and methods. These conceptual advances can represent a lifeline for some of the limitations shown by translational neuropeptide research despite overwhelming evidence for key physiological roles (1, 2). According to the adopted criteria of the International Neuropeptide Society/Society for Biologically Active Peptides, a neuropeptide is a small protein-like molecule -regardless of whether it is secreted by neurons or nonneural cells that expresses the same genetic information and undergoes identical processes of synthesis and transport, and binds to similar families of receptors, in order to act on specific target cells.

Rationale for VIP applications Among the neuropeptides, the 28-amino acid VIP is a molecule that has evolved from being considered a mere neuropeptide/hormone into a novel agent for modifying immune function and, possibly as a cytokine-like molecule (3, 4). Originally identified by Said and Mutt in the late 60â&#x20AC;&#x2122;s, VIP was originally isolated as a vasodilator and hypotensive peptide (5, 6). Subsequently, its biochemistry was elucidated and within the first decade its signature features as a neuropeptide/neurotransmitter became consolidated: it is currently known to act as a neuromodulator in many organs and tissues, including heart, lung, thyroid gland, kidney, immune system, urinary tract and genital organs (7). Sustained interest in therapeutic applications of vasoactive intestinal peptide (VIP) include areas related to neuroprotection (8-10), inflammation and autoimmune disorders (11-18), or asthma (19, 20). Many efforts have been made to obtain highly potent VIP analogues (21) focusing on the improvement of stability to create drug candidates for the treatment of several diseases including asthma (22), neurodegenerative diseases (8), impotence (23), septic shock (24, 25), diabetes (26) or as a tumor imaging agent (27). However, none of these analogues has yet reached the clinical stage. Major drawbacks include internal degradation and loss, as well as a difficult balanced equilibrium between potential side effects and low availability of the peptide at the disease site when systemic peptide doses have to be necessarily increased. In this sense, peptide encapsulation and/or functionalization using nanoparticles are important applications that could solve the limitation of the therapeutic use of peptides owing to their short half-lives caused by enzymatic degradation, catalytic antibodies, and spontaneous hydrolysis in biological fluids. Studies of the effects of VIP on several biological functions provide a powerful rationale for the assessment of VIP as a novel therapeutic approach for the synthesis of novel nanosystems that could improve the efficacy of the treatment. The fact that VIP is so attractive for therapeutic use by itself leads to the study of nano-applications such as a peptide delivery system that may solve the problem of drug break-down by digestive acids and enzymes before they reach their targets (28). The half-life of VIP needs to be substantially prolonged in biological fluids to be employed in therapeutics with increased effectiveness, as VIPbased drug design is hampered by the instability of the peptide and has limited bioavailability. Therefore, nanoparticle encapsulation protects the peptide contained from

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being broken down too early and has a slow release mechanism that allows a gradual delivery of its contents. It can be predicted that the future of drug delivery involves smart systems that maintain the drug at a desired therapeutic level in the body and avoid the need of frequent administration. Additionally, it would be desirable to use a nanoparticle that acts only on a unique receptor or biological site of interest with the possibility of being loaded with a drug at different concentrations. For this reason, VIP has been studied as a method of transport to target cells. A variety of primary human tumors such as breast, prostate, urinary bladder, colon, pancreas and lung cancer, among others, express large numbers of high-affinity receptors for VIP (29, 30) which may represent the molecular basis for nanoparticle applications in cancer. One of the main reasons for the use of peptides and peptide receptors in cancer is the possibility of its targeting. VIP could actively target different cancer cells leading to the delivery of drug compounds or imaging agents to cancer cells that overexpress VIP receptors (31). Moreover, the VIP-NPs-mediated high sensitivity and specificity to detect cancer cells offer the potential for early diagnosis and treatment. This is important as the earlier the diagnosis, the less the cost of patient care, and what is the most important, the higher the chances of treatment success that is normally lower in disease at an advanced stage. Receptor scintigraphy using radiolabeled peptides for the localization of tumors and their metastases as well as for radiotherapy is used with a clinical impact at the diagnostic and therapeutic level, emerging as a serious treatment option. Therefore, VIP-functionalized nanoparticles can be used as a drug delivery system to a specific cancer tissue as well as for in vivo cell labeling and image acquisition. It has been reported that nanoparticles, such as superparamagnetic iron oxide nanoparticles, could be an option as contrast agents for targeted magnetic resonance imaging, which offers a high potential for diagnosis (32, 33).

VIP-based nanoparticles Drug delivery plays a crucial role in the improvement of therapeutic agents since many drugs have unfavourable drawbacks if applied directly. Therefore, developing nanoparticles which can work as carriers for a controlled delivery of a peptide is a promising option to improve its availability by reducing its side effects and enhancing its efficacy. Several studies have been developed to establish whether the encapsulation of VIP into nanoparticles enhances its effects or whether its attachment to a surface leads to an appropriate nanoparticle transport to its target. Different nanoparticles have been used to encapsulate VIP, such as liposomes (34, 35), biodegradable protamine-oligonucleotide nanoparticles (36) or poly (ethylene glycol)-poly (lactic acid) nanoparticles (37). Depending on the location where the peptide needs to be delivered, the nature of the nanoparticle and/or surface ligands is different (Fig. 1). Liposomes are widely used for drug delivery due to their unique properties (38). They are biodegradable, typically made from natural molecules and non-immunogenic, which makes them even more attractive in view of their ability to encapsulate a variety of molecules, including VIP. Stark and co-workers demonstrated that the encapsulation of VIP into

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liposomes protects the peptide from proteolytic degradation while maintaining its biological activity (35). Furthermore it has been reported that VIP liposome encapsulation enhances its vasoactive effects on systemic arterial blood pressure (34) while another study showed the delayed release of VIP when administered into liposomes within hyaluronic acid gel for uveitis (39).

However, although liposomes can be highly effective as VIP carriers in some cases, this is not the case of VIP transport to the brain. For this reason, other options have been engineered, such as glucose targeted niosomes â&#x20AC;&#x201C;non ionic surfactant-based liposomesallowing an efficient delivery of intact VIP to the brain by crossing the blood-brain barrier following intravenous administration (40) or poly (ethyleneglycol)-poly (lactic acid)

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nanoparticles modified with wheat germ agglutinin that enhanced VIP transport to the brain by intranasal administration (37). Other biodegradable nanoparticles have been used as a drug delivery system to enhance protection against proteolytic cleavage as well as cellular uptake; this is the case of protamine-oligonucleotide nanoparticles, which achieve an appropriate binding efficiency of VIP as well as nanoparticle stability (36). As we have seen, the use of biodegradable nanoparticles for encapsulating VIP offers the advantage of sustained release through its diffusion into the nanoparticle matrix and subsequent degradation. This permits a higher or lower release depending on the composition of the nanoparticle with the possibility of designing specific nano-systems according to the treatment and the therapeutic agent. The advantage of VIP encapsulation relies on the facts of peptide protection and sustained release, thus maintaining its activity and increasing its effectiveness for treatments. However, there are other nano-applications that use VIP as surface ligands and not as a cargo (Fig. 1). This approach takes advantage of the fact that VIP can be used for targeting human cancers and has clinical impact at both diagnostic and therapeutic levels. An example of these nano-systems could be phospholipid nanomicelles grafted with VIP, which have been reported to actively target breast cancer and act as a water-insoluble anticancer drug carrier (41, 42). Pioneer research teams led by Rubinstein and Onyuksel also characterized VIP grafted liposome for targeted breast cancer imaging and could demonstrate that there was significantly more accumulation of VIP liposomes than similar liposomes without VIP, indicating a successful use of VIP receptors for active molecular targeting, rather than passive targeting (43). Other nanoparticles have been used to enhance the therapeutic/diagnostic potential of VIP with the valuable properties of metal nanoparticles in the field of biosensing and medical imaging owing to their plasmonic properties (44). This is the case of silverprotected VIP nanoparticles that retain the functional activity of VIP assayed by inhibition of microglia activation under inflammatory conditions (45). A major issue for the development of clinical applications based on engineered nanoparticles pertains to toxicology, pharmacokinetic and biodistribution studies (46, 47). In this sense, there are few data on VIP-engineered nanoparticles, and mainly limited to animal models, which are not a perfect predictor of outcome in humans. Dagar and collaborators have demonstrated in breast tumor-bearing rats that VIP covalently attached to sterically stabilized liposomes encapsulating a radionuclide did not alter the pharmacokinetic parameters of the liposomes, as half-lives data calculated from blood radioactivity indicated (48). Therefore, VIP could be used to decorate labeled, long-circulating small liposomes to develop new targeted imaging agents for enhance, and thus, early detection of several cancer types. Although biodistribution studies using liposomes have proved increased brain delivery of VIP (37, 40), the authors do not perform comprehensive pharmacokinetic studies, and only indirect data indicate the existence of VIP-engineered nanoparticles clearance by cerebrospinal fluid draining (40). Precise pharmacokinetic analyses of the entire plasma profile, including absorption, distribution and elimination should be included in coming

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studies, and whenever possible, comparison with VIP-engineered nanoparticles intravenous routes of administration should be made.

Technologies involve in VIP-particle fabrication A variety of techniques are described in the literature for the preparation of peptide encapsulated nanoparticles or peptide functionalized nanoparticles, but few of them make direct reference to VIP. As explained above, VIP can be encapsulated as well as attached to the surface of molecules in order to act like a drug itself or to direct another compound to a specific site. There is an invention that uses these two features of VIP to merge them in a single nanoparticle (49). The invention describes a method of delivering VIP to the surface and intracellular compartment of a target tissue by producing a liposome where VIP is located both on and inside the liposome, i.e. VIP is exposed to the outside solvent and to the luminal space of the liposome. Other inventions encapsulate VIP but with the aim of a controlled release. This is the case of an invention that describes a water-soluble peptide encapsulation method in order to prepare polymeric microespheres or nanospheres that encapsulate VIP with high efficiency (50). The author describes a method for reducing the aqueous solubility of the drug, without sacrificing its potency, which is useful in case of high peptide loading and heterogeneous distribution of the drug particles, leading to a nonpredictable release profile. This offers an improved encapsulation method when predictable release profiles are needed. In other applications VIP is used as a surface ligand for targeted delivery. In this case, VIP needs to be efficiently attached to the nanoparticle surface and for this purpose liposomes are commonly used. They offer the possibility of attaching a ligand to the lipid head group or to the distal end of the poly-ethylene-glycerol (PEG) chain on PEG-grafted liposomes and can evade the reticuloendothelial system as well as have prolonged circulation time in blood. Moreover, other agents such as cytotoxic agents can be encapsulated in the liposome and provide an anti-tumor activity effect. A group of inventors describe a peptide-spacer-lipid conjugate that can be incorporated into a liposome as a targeting moiety for liposomal drug delivery to specific cells (51). The impact of this invention will probably be notable in fields of laboratory medicine due to the variety of therapeutic agents which can be used for incorporating into liposomes, for example anti-angiogenic or anti-bacterial compounds. This technology might be useful for new strategies to combat protozoan parasites based on VIP nanoparticles taking into account the recent findings on the effects of neuropeptides in T.

brucei infections (52). Nanoparticles

with

very

different properties

have

also

been

used

for VIP

functionalization. This is the case of silver nanoparticles which are known to have unique optical, electrical and magnetic properties, and to interact with virus, bacteria and the immune system (53). Our group has lately shown how to attach VIP to the surface of silver nanoparticles in two possible orientations, providing a tool to distinguish dependent or independent VIP-mediated receptor effects (54). Most recently, Akhtari and Engel, have

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patented a method to synthesize adrenocorticotropin hormone-conjugated dextran-coated and epoxy surface-modified iron oxide magnetonanoparticles (10-15nm diameter) that could be used to obtain VIP-magnetic nanoparticles

(55). These magnetically responsive

nanoparticles include ferrites of general composition MeOxFe2O3 wherein Me is a bivalent metal such as Co, Au, Mn, or Fe (55). These VIP-magnetic nanoparticles have the potential to be used as new contrast agents for MRI-based diagnostic and monitor development of affected tissues or for magnetic particle hyperthermia applications in cancer therapy.

Perspectives The majority of the challenges facing the development of VIP-engineered nanoparticles relate to their unique distribution characteristics, physical chemistry, manufacturing processes and drug product characterization. Researchers moving to this field should take into consideration that the slightest changes in particle size can substantially alter the biodistribution, renal excretion and pharmacodynamics of VIP nanoparticles. Therefore, to ensure potential consistent VIP-based nanoparticles applications into the clinic, factors such as particle size, surface charge and surface chemistry will require robust characterization with overlapping assays. Although still far from VIP-enabled nanoproducts in the market, the areas of healthcare where VIP nanotechnology can make theirs greatest contributions are cancer research and molecular imaging. Fortunately, changes will not come overnight and provided you welcome open innovation in this emerging field.

Acknowledgements We gratefully acknowledge the financial support provided by the Spanish Ministry of Health (PS09-2252); the Andalusian Ministry of Health (PI-2008-0068), the Andalusian Ministry of Economy, Science and Innovation (Proyecto Excelencia CTS-6928) and the PAIDI Program from the Andalusian Government (CTS-677). RK holds a research contract (CTS-6928) and S. L-E holds a Juan de la Cierva (JCI-2010-08042) postdoctoral fellowship from the Spanish Ministry of Economy and Competitiveness.

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6. Said S, Mutt V. Polypeptide with broad biological activity: isolation from small intestine. 1970. Science 169:1217-1218. 7. Henning RJ, Sawmiller DR. Vasoactive intestinal peptide: cardiovascular effects. 2001 Cardiovasc Res 49(1):27-37. 8. Gozes I. VIP, From gene to behavior and back: summarizing my 25 years of research. 2008. J Mol Neurosci 36(1-3):115-124. 9. Gozes I, Furman S. VIP and drug design. 2003. Curr Pharm Des 9(6):483-494. 10. Gozes I, Bachar M, Bardea A, Davidson A, Rubinraut S, Fridkin M. Protection against developmental deficiencies by a lipophilic VIP analogue. 1998. Neurochem Res 23(5):689693. 11. Abad C, Martinez C, Juarranz MG, Arranz A, Leceta J, Delgado M, Gomariz RP. Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease. 2003. Gastroenterol 124(4):961-971. 12. Keino H, Kezuka T, Takeuchi M, Yamakawa N, Hattori T, Usui M. Prevention of experimental autoimmune uveoretinitis by vasoactive intestinal peptide. 2004. Arch Ophthalmol 122(8):1179-1184. 13. Pozo D, Anderson P, Gonzalez-Rey E. Induction of Alloantigen-Specific Human T Regulatory Cells by Vasoactive Intestinal Peptide. 2009. J Immunol 183:4346-4359. 14. Delgado M, Robledo G, Rueda B, Varela N, O'Valle F, Hernandez-Cortes P, Caro M, Orozco G, Gonzalez-Rey E, Martin J. Genetic association of vasoactive intestinal peptide receptor with rheumatoid arthritis: altered expression and signal in immune cells. 2008. Arthritis Rheum 58(4):1010-1019. 15. Anderson P, Delgado M. Endogenous anti-Inflammatory neuropeptides and proresolving lipid mediators: a new therapeutic approach for immune disorders. 2008. J Cell Mol Med 12:1830-1847. 16. Hamidi SA, Prabhakar S, Said SI. Enhancement of pulmonary vascular remodelling and inflammatory genes with VIP gene deletion. 2008. Eur Respir J 31(1):135-139. 17. Gonzalez-Rey E, Chorny A, Delgado M. Regulation of immune tolerance by antiinflammatory neuropeptides. 2007. Nat Rev Immunol 7(1):52-63. 18. Gomariz RP, Gutierrez-Canas I, Arranz A, Carrion M, Juarranz Y, Leceta J, Martinez C. Peptides targeting Toll-like receptor signalling pathways for novel immune therapeutics. 2010. Curr Pharm Des 16(9):1063-1080. 19. Said SI, Hamidi SA, Gonzalez Bosc L. Asthma and pulmonary arterial hypertension: do they share a key mechanism of pathogenesis? 2010. Eur Respir J 35(4):730-734. 20. Onoue S, Yamada S, Yajima T. Bioactive analogues and drug delivery systems of vasoactive intestinal peptide (VIP) for the treatment of asthma/COPD. 2007. Peptides 28(9):1640-1650. 21. Igarashi H, Ito T, Mantey SA, Pradhan TK, Hou W, Coy DH, Jensen RT. Development of simplified vasoactive intestinal peptide analogs with receptor selectivity and stability for human vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptors. 2005. J Pharmacol Exp Ther 315(1):370-381. 22. Bolin DR, Michalewsky J, Wasserman MA, O'Donnell M. Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma. 1995. Biopolymers 37(2):57-66. 23. Gozes I, Reshef A, Salah D, Rubinraut S, Fridkin M. Stearyl-norleucine-vasoactive intestinal peptide (VIP): a novel VIP analog for noninvasive impotence treatment. 1994. Endocrinology 134(5):2121-2125. 24. Delgado M, Ganea D. Inhibition of endotoxin-induced macrophage chemokine production by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo. 2001. J Immunol 167(2):966-975.

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25. Lv B, Tang Y, Chen F, Xiao X. Vasoactive Intestinal Peptide and pituary adenylate cyclase-activating polypeptide inhibit tissue factor expression in monocyte in vitro and in vivo. 2009. Shock 31(2):185-191. 26. Yung SL, Dela Cruz F, Hamren S, Zhu J, Tsutsumi M, Bloom JW, Caudle M, Roczniak S, Todd T, Lemoine L, MacDougall M, Shanafelt AB, Pan CQ. Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide. 2003. J Biol Chem 278(12):10273-10281. 27. Kothari K, Prasad S, Korde A, Mukherjee A, Mathur A, Jaggi M, Venkatesh M, Pillai AM, Mukherjee R, Ramamoorthy N. 99mTc(CO)3-VIP analogues: preparation and evaluation as tumor imaging agent. 2007. Appl Radiat Isot 65(4):382-386. 28. Domschke S, Domschke W, Bloom SR, Mitznegg P, Mitchell SJ, Lux G, Strunz U. Vasoactive intestinal peptide in man: pharmacokinetics, metabolic and circulatory effects. 1978. Gut 19(11):1049-1053. 29. Reubi JC. In vitro identification of VIP receptors in human tumors: potential clinical implications. 1996. Ann N Y Acad Sci 805:753-759. 30. Reubi JC, Laderach U, Waser B, Gebbers JO, Robberecht P, Laissue JA. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin. 2000. Cancer Res 60(11):3105-3112. 31. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. 2003. Endocr Rev 24(4):389-427. 32. Liao Z, Wang H, Lv R, Zhao P, Sun X, Wang S, Su W, Niu R, Chang J. Polymeric liposomes-coated superparamagnetic iron oxide nanoparticles as contrast agent for targeted magnetic resonance imaging of cancer cells. 2011. Langmuir 27(6):3100â&#x20AC;&#x201C;3105. 33. Schlorf T, Meincke M, Kossel E, Gluer CC, Jansen O, Mentlein R. Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging. 2010. Int J Mol Sci;12(1):12-23. 34. Gao XP, Noda Y, Rubinstein I, Paul S. Vasoactive intestinal peptide encapsulated in liposomes: effects on systemic arterial blood pressure. 1994. Life Sci 54(15):PL247-252. 35. Stark B, Andreae F, Mosgoeller W, Edetsberger M, Gaubitzer E, Koehler G, Prassl R. Liposomal vasoactive intestinal peptide for lung application: protection from proteolytic degradation. 2008. Eur J Pharm Biopharm 70(1):153-164. 36. Wernig K, Griesbacher M, Andreae F, Hajos F, Wagner J, Mosgoeller W, Zimmer A. Depot formulation of vasoactive intestinal peptide by protamine-based biodegradable nanoparticles. 2008. J Control Rel 130(2):192-198. 37. Gao X, Wu B, Zhang Q, Chen J, Zhu J, Zhang W, Rong Z, Chen H, Jiang X. Brain delivery of vasoactive intestinal peptide enhanced with the nanoparticles conjugated with wheat germ agglutinin following intranasal administration. 2007. J Control Rel 121:156-167. 38. Lasic DD. Novel applications of liposomes. 1998. Trends Biotechnol 16(7):307-321. 39. Lajavardi L, Camelo S, Agnely F, Luo W, Goldenberg B, Naud MC, Behar-Cohen F, de Kozak Y, Bochot A. New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis. 2009. J Control Rel 139(1):22-30. 40. Dufes C, Gaillard F, Uchegbu IF, Schatzlein AG, Olivier JC, Muller JM. Glucose-targeted niosomes deliver vasoactive intestinal peptide (VIP) to the brain. 2004. Int J Pharm 285(12):77-85. 41. Rubinstein I, Soos I, Onyuksel H. Intracellular delivery of VIP-grafted sterically stabilized phospholipid mixed nanomicelles in human breast cancer cells. 2008. Chem Biol Interact 171(2):190-194. 42. Onyuksel H, Jeon E, Rubinstein I. Nanomicellar paclitaxel increases cytotoxicity of multidrug resistant breast cancer cells. 2009. Cancer Lett 274(2):327-330.

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43. Dagar S, Krishnadas A, Rubinstein I, Blend MJ, Onyuksel H. VIP grafted sterically stabilized liposomes for targeted imaging of breast cancer: in vivo studies. 2003. J Control Rel 91(1-2):123-133. 44. Caro C, Castillo PM, Klippstein R, Pozo D, Zaderenko AP. Silver nanoparticles: sensing and imaging applications. In: Pozo D, editor. Silver nanoparticles. Vienna: InTech Books; 2010. p. 201-223. 45. Fernandez-Montesinos R, Castillo PM, Klippstein R, Gonzalez-Rey E, Mejias JA, Zaderenko AP, Pozo D. Chemical synthesis and characterization of silver-protected vasoactive intestinal peptide nanoparticles. 2009. Nanomed 4(8):919-930. 46. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. 2008. Nanomed 3(5):703-717. 47. Malam Y, Lim EJ, Seifalian AM. Current trends in the application of nanoparticles in drug delivery. 2011. Curr Med Chem 18(7):1067-1078. 48. Rubinstein I, Patel M, Ikezaki H, Dagar S, Önyüksel H. Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin. 1999. Peptides 20:1497-1501. 49. Paul S, Yasuko N, Rubinstein I. US005770570A. 1998. 50. Ignatious FX. US006270700B1. 2001. 51. Wu SK, Chang TG, Tseng GL, Cheng LJ, Shih KS. US 20070106064A1. 2007. 52. Delgado M, Anderson P, Garcia-Salcedo JA, Caro M, Gonzalez-Rey E. Neuropeptides kill African trypanosomes by targeting intracellular compartments and inducing autophagiclike cell death. 2009. Cell Death Differ 16(3):406-416. 53. Klippstein R, Fernandez-Montesinos R, Castillo PM, Zaderenko AP, Pozo D. Silver nanoparticles interactions with the immune system: implications for health and disease In: Pozo D, editor. Silver Nanoparticles. Vienna InTech Books; 2010. p. 309-324. 54. Pozo D, Castillo PM, Zaderenko AP, Caro CA, Fernandez-Montesinos R, Delgado M, Gonzalez-Rey E, García PP, Mejias JA. WO/2009/095516. 2009. 55. Akhtari M, Engel J, inventors; Use of functionalized magnetic nanoparticles in cancer detection and treatment patent US20110044911A. 2011.

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3. COROLLARY Towards a Cell Evo-Devo Manuel Marí-Beffa Departamento de Biología Celular, Genética y Fisiología Facultad de Ciencias, Universidad de Málaga. Ciber-bbn. 29071-Málaga. Spain

During the last century, science has developed the appraisal of new views of the Theory of Organic Evolution. Since the publication of the book “On the Origin of Species” by Charles Darwin (1), many new scientific results and views have been incorporated to the natural selection theory. Gradually, the chromosomal theory of heredity, the population genetics, a correct systematics, an emerging molecular cytogenetics, the importance of the development of organisms and the results obtained by the “omics” science (2) This analysis has given rise to a modern synthetic version of the evolution theory named Evo-Devo (Evolutionary developmental biology). The theoretical basis of this theory proposes two different stages: evolutionary innovation and selection. As evolutionary innovation, evolution operates upon organic development generating embryonic embryonic novelties (previously "mutants") (previously named as "varieties" or “mutants”). As selection, both embryonic and adult stages have to adapt to the ecological environment for a survival. Under this author view, Cell Biology still has to play a very important role in describing how the organic homeostasis, also during development, is both innovating and adapting. Under the current version of Evo-Devo, genetics and molecular biology are playing a major role. As a result of their effort, a number of changes in systematics and phylogeny are being proposed. Beside classical anatomical characters (i.e. cotyledons to define monocots or dicots), nucleotide and amino acid sequences are now being used to better explain the evolutionary events (i.e. Hox genes, or related genes, are used to define animals or specific clades such as lofotrochozoa or ecdysozoa). However, under the consolidating Developmental Biology, developmental genes are also being studied under a Cell Biology perspective. Developmental biologists now use in situ hybridization, inmunocitochemistry, but also transgenic, mutant animals or clonal analysis to explain gene function under the scope of cellular activities. Intracellular trafficking, signal transduction or cell movements are now basic for a proper understanding of development. So under the current view , it is not only important to know how cdcs control cell proliferation but how cdcs are controlled by developmental genes and organizing cells in a harmonic growth. New transcriptomics and functional proteomics are thus further introducing Cell Biology in the emerging Evo-Devo and a new perspective arise in the scientific future. In the XIV Congress of the Spanish Society of Cell Biology, a number of very important communications have been presented trying to explain, on purpose or not, this cellular view of Evo-Devo Beyond anatomy, genetics or molecular biology, cell biology might also be invoked to play an important role. Some articles on cell death, autophagy , stem cell and their niche activities , formation of cell organelles, i.e. Golgi apparatus and its intracellular traffic role, cell ageing or extracellular matrix composition have been shown in a variety of model species, can be read in different articles of this book for a more profound comparative analysis.

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Among the objectives of this enormous task of Cell Biology in Evo-Devo, this author proposes two:

1.- Search of helping data to clarify taxonomic doubts, as the embryonic synapomorphies in Evo-Devo suggested above. As an example, Durán et al., 2011 (4) propose expression and functional data on teleostean fin collagens. In itself, this study is a regular cell biology study of collagens, but these data can also be applied to better understand ceratotrichia and actinotrichia evolution between sharks and ray-finned fishes. Under this approach, cell biology may provide new cell characters, as actinotrichia-forming cells, to explain evolution during terrestrial conquering by sea vertebrates. Under this author´s view, this new explanation completely modifies the scope. 2.- Comparative cell biology of basic cell activities such as cell proliferation apoptosis, or cell activities affected in pathologies . As a simple example of this task let this author remind the reader of the concepts LUCA last universal common ancestor, LECA last eukaryotic common ancestor or last common ancestor of all animals or plants where biochemistry, genetists, molecular biologists, evolutionary biologists and hopefully cell biologists are meeting each other. As a good example, Golgi apparatus and signalling studies could be better understood if an evolutionary case is outlined as a general theoretical framework. An appropriate evolutionary event would arise if we compare single-cell versus multiple-cell organisms in the search for an explanation of the evolutionary origin of a potential last common ancestor of multi-cell organisms. Under this view, modifications in intracellular trafficking and Golgi apparatus might be related to both the onset of the latter cell concepts or the evolution of multi-cell organisms. The success of this “reunification” of Cell Biology and the rest of biological disciplines must permit the use of cell biology data by Zoologists, Botanists or Mycologists. At this stage, the research becomes routine and faint, just trying to find a good evolutionary example where our data can be applied. But this task is profoundly rewarding. This task refreshes our memory, provides general sense to our research and boosts interdisciplinarity of Cell Biology under Evo-Devo umbrella. Basically, Evo-Devo theory might explain both single cell evolution and single-cell to multi-cell transition under the scope of Modern Cell Biology, and many good examples have been presented in this Congress. This author hopes that this new interdisciplinarity would promote further interest among cell biologists. Indeed, this author is collaborating with ecologists (5), paleo-histologists (Mondejar et al., in preparation), biochemists (6) or evolutionary genetists (Reding et al., in preparation) applying these disciplines to a single evolutionary event in fishes, where cell biology studies can be used. We are also applying these ideas to evolutionary biology of plants collaborating with botanists, biochemists, genetists and human geographers (7). If anyone aims to understand the underlying principles please read Spanish evolutionary biologists Pere Alberch (8). Our deceased scientist was able to explain Evo-Devo in cellular terms relating cell biology, histology or organography with genetics and evolution. Even medical cell biologists are being invited to explain, under both a medical and evolutionary scope, fundamental concepts such as health or organic homeostasis to better understand what is constant (in LUCA, LECA, etc.) and what is variable (e.g. between fishes and tetrapods) during health evolution.

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Finally, under a teaching perspective, we are also adapting our programs to this new view, we are clearly explaining that Cell Biology, Cytology, Histology and Organography are inter-related by Embryology, the latter subject understood as Organogenesis and Histogenesis. This is a classical view in many Universities but we are focusing our task to adapt the cell biology knowledge of our students to the rest of their learning activities. We are aiming to provide examples to boost an activity of conceptual interconnection between most of biological disciplines to finally reach to an evolutionary explanation of organs and tissues. This is our initial commitment with this possible Cell Evo-Devo, to first understand organic and tissue homeostasis and then to explain innovation and adaptation of them along particular evolutionary events.

References 1. Darwin C. Origen de las Especies. 1985. Ediciones Akal, S. A., Los Berrocales del Jarama, Madrid. 2. Mardis ER. Next-generation DNA sequencing methods. 2008. Annu Rev Genomics Hum Genet 9:387-402. 3. Aburto MR, Magariños M and Isabel Varela-Nieto I. Differential Regulation of autophagy, proliferation and cell survival during otic neurogenesis. Chapter 1.4 in this book. 4. Durán I, Marí-Beffa M, Santamaría JA, Becerra J, Santos-Ruiz L. Actinotrichia collagens and their role in fin formation. 2011. Dev Biol 354(1):160-72. 5. Zauner H, Begemann G, Marí-Beffa M, Meyer A. Differential regulation of msx genes in the development of the gonopodium, an intromittent organ, and of the "sword," a sexually selected trait of swordtail fishes (Xiphophorus). 2003. Evol Dev 5(5):466-77. 6. García-Caballero M, Marí-Beffa M, Medina MA, Quesada AR. Dimethylfumarate Inhibits Angiogenesis In Vitro and In Vivo: A Possible Role for Its Antipsoriatic Effect? 2011. J Invest Dermatol 131(6):1347-55. 7. Recio Criado M, Jiménez Lara JA, Nieto Caldera JM, Asensi Marfíl A, Heredia Bayona A, Silva Sánchez P, Pimentel Burgos A, España Ramírez L, Bañares España E, Ruiz Durán G, Mancebo Paños M, Pacheco Rodríguez MJ, Monroy López L, Morales Perales RA, Vázquez Zayas C, Quirós Ortega ME, Sánchez Villodres MA, Saura Rivas J, Sánchez Pérez A, Lara Romero I, Sánchez Postigo N, Marí-Beffa M. Plantas del Jardín Botánico de la Universidad de Málaga. Gimnospermas. 2012. Servicio de Publicaciones. Universidad de Málaga, Málaga. 8. Rasskin Gutman D. Pere Alberch. The creative trajectory of an evo-devo biologist. 2009. Fora de Colecció. Universida de Valencia, Valencia.

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