Research ALS Today by ALS Association

INSIDE

RESEARCH ALS TODAY

THE ALS ASSOCIATION

VOLUME 14

Yeast Cells Sheila Essey Award Clinical Management Grants Antisense for ALS Journal News

SPRING 2014

Yeast Cells are Rising to the Occasion for ALS Research

or thousands of years, yeast has been an integral part of the lives of humans, providing bakers and brewers with the ability to produce a delicious array of bread, beer and wine. These days, in addition to yeast’s job unlocking culinary mysteries in vineyards and bakeries, it has been working in the laboratory to help unlock some of the mysteries of amyotrophic lateral sclerosis (ALS). The baker’s yeast, known by its scientific name Saccharomyces cerevisiae, is a tiny single-celled organism that remarkably possesses most of the same basic cellular machinery as complicated neurons in our brain. At first it might seem crazy that yeast cells could help us learn anything about ALS, but it is worth pointing out that almost everything we now know about cancer started out from basic studies using yeast to learn how one yeast cell divides into two. Just a few months ago the Nobel Prize in Physiology or Medicine was awarded to a researcher who used yeast cells to figure out how proteins are shipped to different locations within the cell––some of the same principles that underlie how neurons in the brain commu-

nicate with each other. Therefore, let’s see what these simple, yet powerful yeast cells can teach us about ALS. The RNA-binding protein TDP-43 was recently discovered to play a major role in ALS: it is mislocalized to cytoplasmic clumps in the degenerating motor neurons of most ALS patients (except those with SOD1 mutations), and mutations in the TDP-43 gene have been identified as a cause of rare familial and sporadic ALS cases. A yeast model has been developed to study what makes TDP-43 form aggregates in cells and what makes it kill those cells. Just like in neurons, TDP-43 also forms clumps in yeast cells when expressed at high levels. But unlike in neurons, where this Continued on page 6

By Aaron D. Gitler, Ph.D. Department of Genetics Stanford University School of Medicine Stanford, CA

The Many Wonders of Yeast. In addition to baker’s yeast, Saccharomyces cerevisiae, helping to produce bread, beer and wine, it is being used in the laboratory to study aggregation and toxicity of RNA-binding proteins involved in ALS, like TDP-43.

Aaron D. Gitler, Ph.D.

1, 6 2-3 3 4-5, 7 7-8

RESEARCH ALS TODAY

www.alsa.org/research/

researchgrants@alsa-national.org

VOL.14

Partnerships Continue to Lead to New Discoveries This year has already seen numerous exciting publications increasing our understanding of how the most recently identified mutations in C9ORF72 lead to ALS. In addition, new genes and new potential causes underlying the disease are continually being identified. The most recent finding, the gene Matrin3, is also an RNA-binding protein. Many challenges remain, most importantly translating these findings into meaningful therapies. The Association is committed to finding a treatment and a cure through our partnerships with academia and industry and continuing to support a diverse but focused portfolio. Bringing new investigators into the fold through our Milton Safenowitz Post-Doctoral fellowship, encouraging clinician scientists to focus on ALS research, and supporting the TREAT ALS/ NEALS (Northeast ALS Consortium) clinical trials network remain a key focus. Collaboration is important to making an impact in ALS research. The partnerships with industry, academia, government and funding organizations led to the success of bringing the first antisense trial for a neurological disorder into the clinic, which is detailed in an article in this publication. We are also becoming more and more aware that ALS is not one disease and individuals may respond very differently to treatment approaches. Efforts are underway to more effectively stratify ALS patients through biomarker discovery and induced pluripotent stem cell technologies. Model systems, especially the mouse models for ALS, are often focused on as not being useful because they cannot predict clinical trial outcomes in people with ALS. In reality, the issue is much more complicated and failure in clinical trials cannot be blamed on the limitations of mouse models. Indeed, the mouse models have been extremely instructive in helping us tease out the time course of disease, the importance of glial cells in disease and in providing new potential targets to generate novel therapies. Clinical trial failures require us to think more carefully about the trial itself. Did we use the correct dose, did the drug that we are administering reach the target we identified, and if it did, did it affect the pathway we identified in the model systems to be important in disease? For this reason, The ALS Association, working closely with clinical and industry colleagues, is focusing on identifying biomarkers that will help answer these questions. So to go back to those model systems, a wide variety are in use, even simple yeast models as described by Dr. Gitler, and they all have their place in moving the field forward.

Lucie Bruijn, Ph.D., M.B.A. Chief Scientist The ALS Association

Finally I would like to acknowledge the vision and leadership of Dr. Jeremy Shefner, our current Sheila Essey Award recipient. His commitment to clinical research for ALS has been crucial in developing biomarkers for ALS, improving clinical trial design and facilitating numerous clinical trials through the TREAT ALS/NEALS network. As we look forward to the research accomplishments for 2014, we reflect on the needs of those people living with ALS and feel even more committed to improving their lives and finding treatments and a cure for the disease.

Jeremy Shefner, M.D., Ph.D. Receives the Sheila Essey Award Each year, The ALS Association and the American Academy of Neurology (AAN) present the Sheila Essey Award for ALS Research to acknowledge and honor an individual who is making significant contributions in research for the cause, treatment, prevention or cure for ALS. The award is made possible through the generosity of the Essey Family Fund, in memory of Sheila Essey, who battled ALS for 10 years and died from the disease in 2004. Past recipients have used the funds to continue ALS research or to support promising young scientists on their research teams. This year, The Association and the AAN are very pleased to give this award to Jeremy Shefner, M.D., Ph.D., whose career-long leadership in ALS clinical research has been instrumental in accelerating the pace and improving the quality of clinical trials for the disease. In 1995, Dr. Shefner co-founded the Northeast ALS Clinical Trials Consortium (NEALS), along with Merit Cudkowicz, M.D., to coordinate ALS clinical trials among nine centers in the northeastern United States. Since then, under the continued leadership of Drs. Shefner and Cudkowicz, NEALS has grown to include more than 100 member sites across North America and elsewhere. In recognition of the vital work done by NEALS, The Association’s TREAT ALS (Translational Research Advancing Treatments for ALS) program has provided support for the past six years. NEALS has coordinated more than 30 trials since its inception and is currently conducting over a dozen studies of new drugs, new symptomatic therapies and new biomarkers for improving future trials. Also, NEALS has become a thriving training ground for new investigators, through its workshops, investigator meetings and mentoring of young clinical researchers. Dr. Shefner himself has been a mentor to dozens of young investigators who have gone on to become leaders themselves in the field of ALS clinical research. Dr. Shefner stepped down as NEALS co-chair at the end of 2013. In his valedictory address to the organization, he said, “Leading NEALS is one of the things in my professional career I am proudest of. It is gratifying to me to see how we have grown.” Dr. Shefner earned his Ph.D. in psychology from the University of Illinois, and his M.D. from Northwestern University. He is now Professor and Chair of Neurology at SUNY Upstate Medical University in Syracuse, N.Y., where he continues an active clinical practice. He has been a member of the AAN since 1989 and authored more than 100 research publications. Continued on page 3

–Lucie Bruijn, Ph.D., M.B.A.

Shefner Receives the Sheila Essey Award

Clinical Management Grants

The aim of the TREAT ALS Clinical Management Grant Program is to improve care and living with ALS with a focus on clinical, psychological and/or social management of ALS. Examples of relevant clinical management studies for this mechanism include, but are not limited to, the following:

Continued from page 2

From early in his career, Dr. Shefner developed expertise in electrodiagnostic medicine and brought that expertise to bear in developing motor unit number estimate (MUNE) as an objective biomarker to track ALS disease progression. MUNE is now in use in a number of ALS clinical trials, and the techniques he developed have become standard for its application in the field. “By developing new measures and refining others, Dr. Shefner has significantly improved the way ALS trials are performed worldwide,” said Orla Hardimann, M.D., Head of Academic Neurology at Trinity College, Dublin.

• Studies that address the gaps in the delivery of care (as outlined in the ALS practice parameters). • Studies that explore and develop telemedicine for the care of individuals with ALS. • Psychological interventions in ALS to address the significant mental health issues facing ALS patients and caregivers. • Nutritional and respiratory intervention. • Studies to address the needs of caregivers for patients with ALS and cognitive/ behavioral impairment. Jeremy Shefner, M.D., Ph.D.

Dr. Shefner has been the principal investigator of numerous ALS trials, most recently of a skeletal muscle activator (Tirasemtiv, Cytokinetics) that increases muscle sensitivity to calcium release. This symptomatic therapy has been shown in initial trials to increase force output in the midrange of muscle activation, potentially aiding activities of daily living for people with ALS. A multinational double-blind phase 2 trial is currently underway, under Dr. Shefner’s leadership. “Dr. Shefner has tirelessly devoted his career to the care of patients and families with ALS, and to developing new treatments for them,” said Dr. Cudkowicz. “It can safely be said that Dr. Shefner’s involvement has been one of the best things to happen to ALS therapeutic research. He is truly an extraordinary leader in every sense of the word.”

• Development of novel endpoints for clinical trials. BUDGET: A maximum of $100,000 per year for a maximum of two years. No indirect costs will be paid for these awards.

APPLICATION RECEIPT DATES Brief study outline Request to submit full applicationSubmission of full application Notification of award

Send grant submissions and any questions to researchgrants@alsa-national.org. Funds begin on receipt of all relevant signatures.

THE ALS ASSOCIATION RESEARCH AWARDS

ALS Epidemiology http://aces.stanford.edu/ForRes.html

Coriell NINDS DNA repository http://ccr.coriell.org/ninds/

SOD1 mutant mice, Jackson Laboratory mouse models http://jaxmice.jax.org/index.html

$2,938,493 16%

Control and SOD1 fibroblasts http://www.ccr.coriell.org/Sections/BrowseCatalog/DiseaseDetail.aspx?PgId=403&omim=ALS40000&coll= ALS Untangled http://www.wfnals.org/alsu.html ALS Association Research Webinars http://www.alsa.org/research/research-webinars.html

16%

Stem Cells

$2,199,511 12%

12% PROJECTS

PROJECTS

Gene cs Gene cs $2,038,872 11%

$2,038,872 11%

Active: March 31, 2014

$1,498,370 8%

Total Awarded: $18,148,979 Total Projects: 98

PROJECTS

Stem Cells $2,199,511 11

Biomarkers $1,498,370

10 PROJECTS

SOD1 mutant rats, Taconic http://www.taconic.com/2148

Jackson Laboratories ALS Mouse Repository http://www.jax.org/news/archives/2010/als_repository.html / http://www.alzforum.org/

Biomarkers

Therapy

Therapy Development Development $2,938,493

Resources ALS mutations database http://alsod.iop.kcl.ac.uk/index.aspx

May 30, 2014 June 29, 2014 September 15, 2014 November 2014

Clinical Research

PROJECTS

Clinical Research 17 $4,327,271

PROJECTS $4,327,271

24%

PROJECTS

Biomarkers Biomarkers Clinical ResearchClinical Research Disease Mechanism Disease Mechanism Genetics Genetics Stem Cells Stem Cells

Disease Mechanism Disease Mechanism Therapy Development Therapy Development $5,146,463 29%

$5,146,463 29%

RESEARCH ALS TODAY

www.alsa.org/research/

researchgrants@alsa-national.org

VOL.14

Antisense for ALS: Important New Treatment Strategy Advances

s the growing number of ALS genes attests, many cases of the disease begin with the expression of a mutant gene. Blocking that expression, therefore, stands out as a potentially definitive therapy, stopping the complex cascade of events leading to motor neuron death before it starts.

ANTISENSE DRUGS TARGET RNA, NOT PROTEINS Gene

mRNA

ANTISENSE DRUGS (Oligonucleotides)

Translation

Antisense oligonucleotide (ASO) therapy has emerged as a highly promising approach for preventing expression of mutant genes in ALS. The ALS Association has taken the lead in supporting development of ASO therapy, beginning with early preclinical work providing proof of principle, and continuing through the first clinical trial of central delivery of ASOs in any neurologic condition. That trial, designed to test the safety and tolerability of the drug and establish the dosing requirements, was recently completed in people with ALS. Plans are underway to launch a treatment trial, possibly in 2015.

No Translation of Disease-Causing Protein

NO DISEASE

Frank Rigo, ISIS

In the diagram to the right, we outline how antisense works and describe the state of progress in therapy development of ASOs for the treatment of two genetic forms of ALS.

What are Antisense Therapeutics? Antisense oligonucleotides are short single-stranded DNA molecules that can be visualized as beads on a string. Once taken up inside of a cell––for example, a nerve cell––they can selectively target the molecular machinery that produces a protein such as superoxide dismutase (SOD1). Antisense does not correct the mutant gene, it is most commonly designed to reduce the harmful effects of a gain-of-function mutation. However, in some situations, it can increase production of a gene product, by overcoming splicing defects. This strategy is currently in development for treatment of spinal muscular atrophy, a pediatric motor neuron disease due to loss of function of the SMN (survival of motor neuron) protein.

Continued on page 5

1985: The ALS Association funds study of inherited motor neuron disease

TIMELINE 1869: French neurologist Jean-Martin Charcot identifies ALS

ASOs used in therapeutic applications are chemically modified to resist degradation by cellular enzymes, thus prolonging their effect. Efficacy is based on exploitation of an ancient defense system that can prevent a virus from hijacking a cell’s molecular machinery (the RNAase pathway), and similar therapeutic strategies are under consideration. RNA interference, for example, using a double-stranded RNA delivered by a viral vector can also degrade the synthesis of a targeted protein, but “the prolonged activity of the RNase pathway from a single dose of ASO may offer significant advantages for a chronic disease such as ALS,” according to Don Cleveland, Ph.D., of the University of California at San Diego.

50s: DNA structure solved 50s: Nerve growth factor (NFG) identified–protective, growth promoting factor for nerve cells

1968: SOD1 enzyme identified

70s: Programmed cell death in motor neurons demonstrated

1986: Genes for muscular dystrophy identified

1990: Congress declares the 1990s the “Decade of the Brain”

1989: The ALS Association funds search for a common genetic link to ALS

1990: Growth factor CNTF is found to increase survival of motor neurons

1991: Researchers link familial ALS to Chromosome 21

1860 1950 1960 1970 1980 1990 1991

The ALS Association begins workshops Glutamate transporter shown to be defective in ALS Growth factor BDNF found to increase survival of motor neurons

1992

Antisense for ALS Continued from page 4

Antisense for Mutant SOD1: From Animal Models to Clinical Trial The mutant SOD1 gene accounts for about 20 percent of all familial ALS and up to two percent of all cases of ALS. The pioneering work of ALSAsupported researchers Dr. Cleveland and Richard Smith, M.D., at the Center for Neurologic Study, in conjunction with Isis Pharmaceuticals (Carlsbad, CA), developer of the ASOs, demonstrated the potential of using antisense in a rat model of SOD1-ALS. Treatment delayed the rate of disease progression by 37 percent when administered near the time of symptom onset. Further work went into scaling up the treatment, culminating in a first-in-human clinical trial, supported by The Association and led by Timothy Miller, M.D., Ph.D., of Washington University. In that trial, patients were randomized to receive either placebo or an anti-SOD1 ASO, developed by Isis Pharmaceuticals and delivered by intrathecal infusion over 11.5 hours. Four cohorts of eight patients each (six active treatment, two placebo) were given escalating doses of drug, with safety assessed over a period of 28 days before commencing each new round. The frequency of adverse events was similar between active-treatment and placebo groups and were mainly Timothy Miller, M.D., Ph.D. Washington University related to the procedure. There were no dose-limiting toxic effects or safety or tolerability concerns. This initial trial did not include a measure of efficacy. Further development of the treatment strategy is underway, with assessment of newer oligonucleotide backbones and RNA targets.

Richard Smith, M.D. Center for Neurologic Study

Antisense for Mutant C9ORF72: A Major Target for ALS Antisense therapy may have a bigger impact in the instance of C9ORF72mediated ALS since it is responsible for approximately 40 percent of familial ALS and up to six percent of sporadic disease. Expansion of a repeated hexanucleotide region in the gene creates RNA transcripts containing hundreds or thousands of GGGGCC units. There are several hypothesized mechanisms of disease. The expanded RNA folds into three-dimensional structures that are thought to trap transcription factors, altering cell metabolism. The expansion is also translated in a non-standard fashion into aggregation-prone dipeptide “repeat-associated non ATG (RAN) translation products,” which may themselves trap other cell molecules. The mutation also reduces the quantity of normal C9ORF72 protein, whose function is unknown. Continued on page 7

Don Cleveland, Ph.D. Ludwig Institute University of California, San Diego

A transgenic rat is designed; efforts start on fly model

TIMELINE cont. SOD1 gene mutation (chromosome 21) discovered in familial ALS Trials using glutamate blocker riluzole begin

Animal studies combining CNTF and BDNF demonstrate decreased motor neuron loss GDNF rescues degenerating motor neurons during development in an in vitro experiment

FDA approves riluzole

Toxic properties of the SOD1 enzyme discovered and linked to familial ALS

RNAi discovered by Craig Mello and Andrew Fire

The ALS Association co-sponsors workshop on high-throughput drug screening with NINDS NINDS issues first ever RFA (request for applications) specifically for ALS research

Attention turns to support cells of nerve tissue to find role in ALS Inflammation and programmed cell death gather research interest ALS2 gene (alsin protein) linked to juvenile ALS The ALS Association/NINDS collaborative effort begins screening drugs

The ALS Association holds scientific workshop on “Environmental Factors and Genetic Susceptibility” Aggressive search for new ALS genes funded by The ALS Association Scientists complete map of mouse genome Agency of Toxic Substances and Disease Registries awards five grants focused on ALS Department of Defense approves funding for ALS-specific research

1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

RESEARCH ALS TODAY

www.alsa.org/research/

researchgrants@alsa-national.org

VOL.14

astonishing pace of new ALS disease gene discoveries “ The in the last three years has invigorated ALS research...”

Yeast Cells for ALS Research Continued from page 1

aggregation process might take years or decades, in yeast TDP-43 forms clumps in a matter of a few hours, allowing experiments to be performed very rapidly. These studies have led to two important findings. First, a small region towards the end of the protein was shown to be critical for TDP-43 aggregation and toxicity. Second, yeast cells revealed that ALS-causing mutations accelerate the rate of TDP-43 clumping, providing a mechanism by which they might cause disease. Importantly, others have confirmed these findings in multiple model systems, including mouse models, underscoring the power of the yeast system for providing disease-relevant insight. The best feature of the yeast model system is the ability to perform unbiased genetic screens. We don’t know why TDP-43 aggregates in yeast (or in neurons) or why it is toxic, but we can ask the yeast cells to tell us the answer. We do this by performing a genetic screen, where we search for yeast genes that can suppress or exacerbate TDP-43 aggregation and toxicity. In other words, can we find a yeast gene that when deleted or overexpressed allows the yeast cells to grow better even in the face of TDP43 aggregates? The yeast genome is very

well characterized and amenable to genetic manipulation. Methods are available to rapidly over express or knock out almost every gene. These experiments are inexpensive and fast––a single student in the laboratory can test every single yeast gene for an effect on TDP-43 in less than one month. One gene that was discovered in a yeast screen for genes that could affect TDP-43 toxicity was the relative of a human gene called ataxin 2, which is known for its role in the neurodegenerative disease spinocerebellar ataxia 2, or SCA2. Increasing ataxin 2 levels worsened TDP-43 toxicity and decreasing ataxin 2 levels actually reduced toxicity. The ataxin 2 protein contains a repeated tract of the amino acid glutamine. Long expansions in the number of glutamines cause the SCA2 disease. Spurred on by the studies in yeast with TDP-43, the number of glutamines in this protein was examined in ALS patients and it was found that there was an increase in the length of the tract––not quite at the SCA2 disease level but longer than normal. These “intermediate-length” glutamine expansions in ataxin 2 are significantly associated with increased risk for ALS. Since these expansions are found in approximately five percent of ALS patients, they are now considered one of the more common genetic risk factors

TIMELINE TIMELINE cont. cont. cont. Study shows surrounding support cells play key role in ALS Study shows that human embryonic stem cells can be stimulated to produce motor neurons Gulf War study shows that vets deployed to Persian Gulf in 1991 developed ALS at twice the rate of those not deployed there IGF-1 gene therapy study proves beneficial in mice with ALS VEGF gene abnormalities shown to be potential factor in ALS The ALS Association collaborates with U.S. Department of Veterans Affairs to enroll all vets with ALS in registry Early tests of ceftriaxone appear to increase survival in mice with ALS Combination of creatine and minocycline prove more effective together in mouse model than either drug alone

Study implicates smoking as likely risk factor in sporadic ALS Study releases evidence that mitochondrial malfunction may play an important role in ALS Study funded by The ALS Association to find biomarkers in cerebrospinal fluid and blood

for ALS. Importantly, multiple independent laboratories from around the world have confirmed this association between ataxin 2 polyQ expansions and ALS. The discovery of a common genetic risk factor for ALS starting from a simple yeast screen highlights the utility of this model system and approach for gaining important insight with direct relevance to human disease. In addition to yeast genetic screens uncovering disease mechanisms and new disease genes and risk factors, they have also unexpectedly revealed some novel concepts for therapeutic targets for ALS. One of the most effective genes at suppressing TDP-43 toxicity that emerged from the yeast screens was a gene called Dbr1, which encodes an enzyme that helps trim out sequences of genes called introns. In the absence of this enzyme, loops of introns accumulated in the cell and acted as a kind of decoy to sequester away TDP-43 aggregates from interfering with other important cellular RNAs and RNA-binding proteins. There is also a human version of this enzyme, which functions in the same way as the yeast one, raising the prospects of harnessing the enzymatic activity of Dbr1 as a new therapeutic target for combating ALS. What’s next for yeast models in ALS research? The astonishing pace of new ALS disease gene discoveries in the last three

Ceftriaxone increases levels of the glutamate transporter GLT1 in a mouse model of ALS First international workshop on frontotemporal dementia discusses link to ALS Stem cells engineered to make GDNF survive when transplanted into rats modeling ALS Early data suggests that mutant SOD1 may be secreted by and may activate microglia Launch of TREAT ALS initiative (Translational Research Advancing Therapies for ALS) to accelerate clinical trials in ALS VEGF increases survival in a rat model of ALS while improving motor

years has invigorated ALS research with many new and promising avenues for future investigation. Simple experimental model systems, like yeast, will help to define how these new genes contribute to disease and how they might be targeted for therapies. For example, the discovery of mutations in the C9ORF72 gene as the most common cause of ALS represents a paradigm shift and intense effort is focused on understanding how C9ORF72 mutations cause disease. Yeast cells do not contain a gene related to C9ORF72 but they might be able to help out in other ways. The ALS-causing mutations in C9ORF72 produce fragments of RNA that accumulate in neurons and might be toxic. Perplexingly, non-standard translation products are also generated from the mutant C9ORF72 gene, and they themselves form aggregates that accumulate in the brain. Can yeast cells be engineered to express mutant C9ORF72 and test if these toxic RNAs and/or translation products accumulate? If so, rapid unbiased genetic and small molecule screens can be performed to discover both how they are produced, as well as methods for blocking their formation. The top leads from these screens can be tested in the numerous cellular and animal models for C9ORF72-ALS currently under development. Stay tuned.

ALS patient samples collected to NINDS ALS Repository Repository samples allow genome analysis for sporadic ALS First TREAT ALS clinical trials funded First TREAT ALS clinical trials begun TDP-43 discovered as a common link in FTD, ALS Chromosome 9 region intense focus for FTD

Stem cell study shows SOD1 mutant support cells can kill any motor neuron ALS U.S. registry efforts gaining ground in Congress Fish model of ALS: Progress reported SOD1 in altered form common to both sporadic and inherited ALS Engineered stem cells making GDNF help motor neurons survive in SOD1 mutant rats First genome screening data published based on NINDS ALS Repository

2003 2004 2005 2006 2007

JOURNAL NEWS

Antisense for ALS Continued from page 5

Motor Neurons in a Dish: A State-of-the-Art Review

“The identification of the C9ORF72 gene mutation, and the likelihood that it causes a toxic gain of function, make it a promising candidate for treatment with antisense,” according to Lucie Bruijn, Ph.D., M.B.A., Chief Scientist for The ALS Association. The Association moved immediately to fund several research groups equipped to explore this therapeutic possibility.

Understanding ALS requires studying the disease in multiple models, from individual cells to whole organisms. Among these, one of the most important is the study of individual motor neurons. The difficulty of growing large numbers of motor neurons has historically hindered such research. In a recently published state-of-the-art review, Brandi Davis-Dusenberry, Kevin Eggan and colleagues describe in detail how basic research in neuronal development provided the key insights that allow scientists to convert non-neuronal cells into neurons. They also outline the importance of quality control and use of cross-laboratory best practices in standardizing the motor neuron supply. Dr. Davis-Dusenberry is an ALS Association Milton Safenowitz Fellow.

Results from these studies have begun to emerge. Working in patient-derived induced pluripotent stem cells (iPSC) converted to motor neurons, several groups have shown that ASOs can reduce C9ORF72 pathology, including aggregation of RNA, aberrant binding of transcription factors, dysregulation of expression of other genes, susceptibility to glutamate excitoxicity, and neuronal firing abnormalities. Not every abnormality responded to treatment—RAN translation products remained unaffected, and newly discovered RNA aggregates made from the opposite strand of DNA (CCCCGG repeats) remained. However, the results are promising, and The ALS Association is facilitating the development of the technology with the aim of beginning a clinical trial as soon as it is safe and practicable.

Development of Motor Neurons in the Embryo During early embryogenesis, a mixture of inhibitory and inductive signals triggers development of neural cells from ectodermal precursors. Key among these are inhibitors of bone morphogenetic protein, fibroblast growth factors and epidermal growth factors. Additional signals establish the rostrocaudal and dorsoventral axes. Retinoic acid is principally responsible for caudalization of spinal cord neurons, leading to differentiation of primitive brain structures. Combinatoric expression of multiple transcription factors, including homeobox genes, causes motor neurons to arise from a subset of ventral spinal cord precursors. Further elaboration of this process gives rise to multiple anatomic (cervical, brachial, thoracic and lumbar) and functional (fast twitch, slow twitch and intrafusal) subtypes of motor neurons. “The body of work describing the molecular underpinnings of motor neuron specification during development has enabled recapitulation of these signals in vitro for the ex vivo generation of motor neurons,” the authors point out, allowing generation of cells in numbers that would be impossible to obtain through direct culturing of embryonic tissue. “Continued integration of findings from developmental studies in model organisms and in vitro-derived motor neurons will allow a greater understanding of motor neuron specification, and as a consequence, further improve strategies to recapitulate motor neuron development in vitro.”

ASOs are also being developed for use in spinal muscular atrophy, Huntington’s disease, Azheimer’s disease (targeting tau protein), and myotonic dystrophy. References Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, et al. Antisense oligonucleotide therapy for neurodegenerative disease. J Clin Invest. Aug 2006;116(8):2290-6. Epub 2006 Jul 27.

Motor Neurons from Stem Cells and Other Cells These methods are now in wide use to generate abundant motor neurons from pluripotent stem cells (PSCs). PSCs can be isolated from preimplantation blastocysts, or created from fibroblasts (so-called induced PSCs, or iPSCs). Conversion of PSCs into motor neurons in vitro follows the in vivo developmental pathway, with timely addition of BMP inhibitors, growth factors, retinoic acid and other transcriptional regulators.

Miller TM, Pestronk A, David W, Rothstein J, Simpson E, Appel SH, et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomized, first-in-man study. Lancet Neurol. May 2013;12(5):435-42. Sareen D, O’Rourke JG, Meera P, Muhammad AK, Grant S, Simpkinson M, et al. Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72 Repeat Expansion. Sci Transl Med. Oct 23, 2013;5(208):208ra149.

The field of stem cell biology has been moving at a lightning pace, and techniques for generating motor neurons have developed accordingly. Most recently, advances in understanding the signals controlling differentiation have led to “direct lineage conversion,” in which fibroblasts are converted directly to neurons without first becoming stem cells. In the mouse, this has even been performed in vivo, converting cardiac fibroblasts into cardiomyocytes to improve cardiovascular function. “It will be exciting to determine if a similar approach can be adopted in the nervous system to repair injuries and/or reverse neurodegenerative disease,” the authors note.

Donnelly CJ, Zhang PW, Pham JT, Heusler AR, Mistry NA, Vidensky S, et al. RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention. Neuron. Oct 16, 2013;80(2):415-28. Lagier-Tourenne C, Baughn M, Rigo F, Sun S, Liu P, Li HR, et al. Targeted degradation of sense and antisense C9ORF72 RNA foci as therapy for amyotrophic lateral sclerosis and frontotemporal dementia. Proc Nat Acad Sci USA. Nov 19, 2013;110(47):E4530-9.

The authors propose four characteristics for evaluating motor neurons created through these methods. Cells should display motor neuron-specific markers, or even better, a full suite of gene changes evaluated with genome-wide transcription profiling. Cells should be electrically active, mimicking the firing activity of bona fide motor neurons in contact with a muscle. When cultured with muscle, they should form

TIMELINE cont. Stem cells generated from ALS patients Discovery of DPP6 in two genomewide association studies in ALS Mutations in TDP-43 linked to familial and sporadic ALS Induced Pluripotent Stem Cell Technology opens up new avenues for ALS

Identification of new gene linked to familial ALS, Fused in Sarcoma (FUS) on Chromosome 16 FDA approval of SOD1 antisense and stem cell trials in U.S.

First patients enrolled for antisense and stem cell trials in U.S.

2008

2009

2010

Continued on page 8

Ubiquilin-2 discovery; C9ORF72 discovery

March: The ALS Association hosts 2nd Drug Discovery Workshop for ALS September: Researchers find genetic region influencing age at which people develop ALS

February: Identification of C9RAN translated peptide March: First human antisense trial published–– approach safe in people with ALS November: Progress in understanding effects of C9ORF72 gene in ALS

March: Mutations in Matrin 3 identified linked to ALS

2011

2012

2013

2014

RESEARCH ALS TODAY

www.alsa.org/research/

researchgrants@alsa-national.org

VOL.14

JOURNAL NEWS Continued from page 7

Motor Neurons in a Dish: A State-of-the-Art Review cont.

so-called ribonucleoprotein granules, which are transported bidirectionally between the cell nucleus and the tip of the axon. However, granules conneuromuscular junctions, the special synapses at which the neuron contacts taining mutant TDP-43 protein were less likely to be moved away from the the muscle and controls contraction. Finally, they should be able to survive nucleus and more likely to be moved toward it. This change in average mografting into the spinal cord of a model animal, although this is so technically tion was seen in several disease models, including in neurons derived from challenging that it may not be testable in all situations. ALS patient skin cells. It is not yet known how this alteration in transport The authors conclude, “We are optimistic that continued efforts to collab- contributes to ALS, or how aggregation and transport deficiencies may be oratively establish best practices for motor neuron production, culture and linked. evaluation in vitro will provide the keys to unlock novel therapeutic strate- Alami NH, Smith RB, Carrasco MA, et al. Axonal Transport of TDP-43 mRNA Granules Is gies for devastating neurological disorders, including ALS.” Impaired by ALS-Causing Mutations. Neuron. Feb 5, 2014;81(3):536-43. Davis-Dusenbery BN, Williams LA, Klim JR, Eggan K. How to make spinal motor neurons. Development. Feb 2014;141(3):491-501.

Loss of TDP-43 Causes Disease in Animal Model

Partial silencing of TDP-43 in astrocytes causes a progressive neurodegenerative disease, according to a new study in mice, suggesting that loss of TDP-43 through aggregation may contribute to the ALS disease process. “If New Technique Generates ALS-Derived Muscle for Study further experiments confirm the role of loss of TDP-43, treatments that preMuscle progenitor cells can be efficiently produced from either embryonic vent TDP-43 aggregation, or replace its lost function, could be a valuable or induced pluripotent stem cells in cell culture, using a new combination of treatment approach,” Dr. Bruijn said. growth factors, according to new research. Researchers transformed iPSCs from two different people with familial forms of ALS; one caused by muta- Yang C, Wang H, Qiao T, et al. Partial loss of TDP-43 function causes phenotypes of tions in the SOD1 gene and the other by mutations in the VAPB gene. The amyotrophic lateral sclerosis. Proc Natl Acad Sci USA. Mar 10, 2014. technique may prove useful in studying the interaction between muscle and motor neurons. Loss of muscle-neuron contact is an early event in ALS TDP-43 Must Risk Misfolding to Function pathogenesis. TDP-43 protein must convert between alternative folded states as part of its normal RNA-binding function, and in so doing may misfold, according to Hosoyama T, McGivern JV, Van Dyke JM, Ebert AD, Suzuki M. Derivation of Myogenic new work, an insight that may help tailor therapies designed to prevent misProgenitors Directly From Human Pluripotent Stem Cells Using a Sphere-Based Culture. folding. In this study researchers found that during the conversion between Stem Cells Transl Med. Mar 21, 2014 (epub). these states, TDP-43 entered an intermediate state in which it was at a high New ALS Gene Strengthens Focus on RNA-Processing risk of becoming misfolded and losing its function, the first step in forming A new ALS-causing gene, MATR3, is a rare cause of familial ALS, but may aggregates. The likelihood of misfolding was increased by cellular stresses provide important clues to disease pathogenesis. According to the study known to be associated with ALS. announcing its discovery, the MATR3 protein, called matrin, interacts with Mackness BC, Tran MT, McClain SP, Matthews CR, Zitzewitz JA. Folding of the RNA TDP-43, also a cause of ALS. TDP-43 aggregates inside cells in most people Recognition Motif (RRM) Domains of the ALS-Linked Protein TDP-43 Reveals an with ALS, and mutant matrin was found to co-aggregate with it in the mus- Intermediate State. J Biol Chem. Feb 4, 2014. cles of one person studied. Like TDP-43, matrin binds to DNA and to RNA Preventing Stress Granule Formation May be Therapeutic and is normally involved in processing messenger RNA. “This discovery strengthens our belief that RNA-processing is central to the Inhibiting phosphorylation of a stress granule regulatory protein countered ALS disease process, and it provides another clue that TDP-43 is one of the the effect of TDP-43 mutation in both flies and mammalian neurons bearing hubs of that process,” said Lucie Bruijn, Ph.D., M.B.A., Chief Scientist for TDP-43 mutations, according to a new study. Mutation promotes formation of stress granules, structures that sequester mRNAs. The authors found that The ALS Association. mutation increased phosphorylation of eIF2α, which promotes granule forJohnson JO, Pioro EP, Boehringer A, et al. Mutations in the Matrin 3 gene cause familial amy- mation, and that inhibiting eIF2α phosphorylation mitigated TDP-43 toxicity. otrophic lateral sclerosis. Nature Neuroscience. Advance online publication March 30, 2014. “These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds TDP-43 is Critical for Axonal Transport of mRNAs that mitigate this process may represent a novel therapeutic approach,” The ALS-associated protein TDP-43 binds to thousands of different messen- they concluded. ger RNAs, and aggregates of TDP-43 are found in almost all people with ALS. Kim HJ, Raphael AR, Ladow ES, McGurk L, et al. Therapeutic modulation of eIF2α Now, researchers have shown that the protein plays a role in axonal trans- phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. port of mRNA. The authors found that normal TDP-43 is incorporated into Nat Genet. Feb, 2014;46(2):152-60.

Highlights from Recent ALS Research

National Office

1275 K Street NW, Suite 205 Washington, DC 20005 alsa.org

the diverse pathologic consequences when the expansion is transcribed into RNA. Here, researchers found that the expansion folded into so-called G-quadruplexes, in which multiple guanines hydrogen-bonded to form a three-dimensional loop, with the two cytosines linking successive guanine segments. The structures reduced the amount of protein that could be made from the gene, and trapped multiple cell proteins that are involved in controlling gene expression. “This important study gives us an unprecedented look at the effects of the C9ORF72 mutation,” said Dr. Bruijn. “It is now imperative that we follow up these observations with further work to clarify how these changes affect disease onset or progression. These insights will be critical in designing therapies to interrupt these processes.” Haeusler AR, Donnelly CJ, Periz G, et al. C9ORF72 nucleotide repeat structures initiate molecular cascades of disease. Nature. Mar 13, 2014;507(7491):195-200.

Focus on Astrocyte Toxicity in Motor Neuron Death Two new studies strengthen the case that astrocytes play a key role in promoting motor neuron death. Meyer et al. developed a “rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes).” In this model, they showed that coculture of iastrocytes from familial ALS patients (SOD1 or C9ORF72), or from sporadic ALS patients, were toxic to motor neurons. Re et al. showed that astrocyteinduced motor neuron death occurred through a process of necroptosis, a type of programmed cell death involving specific signaling pathways. Meyer K, Ferraiuolo L, Miranda CJ, et al. Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proc Natl Acad Sci USA. Jan 14, 2014;111(2):829-32. Re DB, Le Verche V, Yu C, et al. Necroptosis Drives Motor Neuron Death in Models of Both Sporadic and Familial ALS. Neuron. Feb 4, 2014.

Cell-to-cell Spread of Misfolded SOD1 Seen in Cell Model Mutant SOD1 is responsible for 13 percent of familial ALS. Researchers showed that both misfolded mutant SOD1 protein and misfolded normal protein could be released by one cell and picked up by another cell, and that the uptake of misfolded protein could propagate the misfolding process from cell to cell. Cell-to-cell transmission could be reduced by antibodies against the SOD1 protein. “These results are intriguing, and potentially important in understanding the ALS disease process,” said Dr. Bruijn. “More work will need to be done to determine whether misfolded SOD1 does in fact move from cell to cell in humans, and whether this process contributes to the pattern of disease progression we see. Answering these important questions takes on new urgency with the publication of this study.” Grad LI, Yerbury JJ, Turner BJ, et al. Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and-independent mechanisms. Proc Natl Acad Sci USA. Mar 4, 2014;111(9):3620-5.

High-Caloric Intake is Safe in ALS, Setting Stage to Test for Effect on Survival

Previous studies in ALS mice have shown that high-caloric intake is associated with increased survival, and ALS patients who are slightly obese tend to have longer survival. Now, researchers have shown that in people with ALS with a feeding tube in place, a high-calorie diet was safe over a fivemonth trial. “These encouraging results provide support for proceeding Unprecedented Details Reported of C9ORF72 Mutation’s Effects with a larger trial,” said Dr. Bruijn. “That trial will tell us whether this simple Mutations in the C9ORF72 gene account for up to 40 percent of familial ALS intervention can improve survival, an effect predicted from the animal and six percent of sporadic ALS. The mutation is an expansion of a six-nucle- model.” otide GGGGCC section of the gene, from as few as two units in the normal Wills AM, Hubbard J, Macklin EA, et al. Hypercaloric enteral nutrition in patients with gene to hundreds to thousands. Much recent work has begun to elucidate amyotrophic lateral sclerosis: a randomized, double-blind, placebo-controlled phase 2 Acknowledgement: Richard Robinson, Science Writer