Biomarkers for alzheimer s disease drug development methods in molecular biology 2785 perneczky 202
Biomarkers
s Disease Drug Development Methods in Molecular Biology 2785 Perneczky
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Biomarkers for Alzheimer s Disease Drug Development 1st Edition Robert Perneczky (Eds.)
Biomarkers for Alzheimer’s Disease Drug Development
Second Edition
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BiomarkersforAlzheimer’s DiseaseDrugDevelopment
Second Edition
Edited by Robert Perneczky
Department of Psychiatry and Psychotherapy, LMU Hospital, Ludwig-Maximilians-University Munich, Munich, Germany
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Preface
Alzheimer’s disease is a devastating neurodegenerative disorder that affects millions of people worldwide. Despite significant progress in understanding the underlying biology of the disease, developing effective treatments for Alzheimer’s disease remains a major challenge. In recent years, there have been several promising developments in the field of Alzheimer’s disease drug development, including the discovery of new drug targets and the development of novel therapeutic approaches.
One exciting development is the approval of new disease-modifying drugs targeting amyloid in several countries. These first-generation drugs represent a new era in Alzheimer’s disease drug development, with the potential for greater benefits for affected individuals and society. For example, aducanumab is a human immunoglobulin G1 monoclonal antibody that binds to the linear epitope formed by amino acids 3–7 of the Aβ peptide, with a higher affinity for fibrillar aggregates as compared with monomers. It has become the first amyloidtargeting antibody to be approved in the United States for Alzheimer’s disease patients with mild cognitive impairment or the mild dementia stage of the disease.
This second edition of our textbook provides an up-to-date and comprehensive overview of the current state of technologies helping to accelerate Alzheimer’s disease drug development. We cover the latest advances in preclinical and clinical research, including new insights into the molecular mechanisms of Alzheimer’s disease and emerging therapeutic strategies. We also discuss the challenges and opportunities in Alzheimer’s disease drug development, including the need for improved biomarkers and diagnostic tools, as well as the importance of collaboration and data sharing among researchers. Our laboratory protocols help researchers to use cutting-edge methods in their work. This book also includes emerging new approaches such as digital biomarkers and advanced neuroimaging analysis since those tools will transform how clinical trials in the Alzheimer’s disease field are performed.
Our goal with this textbook is to provide students, researchers (both from academia and industry), and clinicians with a valuable resource for understanding the complexities of Alzheimer’s disease drug development. We hope that this book will inspire and inform future efforts to develop effective treatments for this devastating disease. Our expectation is that this book will complement other excellent volumes and monographs on Alzheimer’s disease that cover the basic science or clinical aspects of the disease.
Munich, GermanyRobert Perneczky
1 Blood-Based Biomarkers for Early Alzheimer’s Disease Diagnosis in Real-World Settings 3
Robert Perneczky, Niels Hansen, Anna Hofmann, Christoph Laske, Josef Priller, Timo Grimmer, Lutz Fro¨lich, Emrah Du ¨ zel, Frank Jessen, and Jens Wiltfang
2 Alzheimer’s Disease Prevention and Treatment Based on Population-Based Approaches
Robert Perneczky
3 CSF N-Glycomics Using High-Throughput UPLC-ESI Techniques in Alzheimer’s Disease
Angela Messina, Rita Barone, Luisa Sturiale, Mario Zappia, Angelo Palmigiano, and Domenico Garozzo
4 CSF N-Glycomics Using MALDI MS Techniques
Angela Messina, Donata Agata Romeo, Rita Barone, Luisa Sturiale, Angelo Palmigiano, Mario Zappia, and Domenico Garozzo
5 Optimized Pre-analytical Handling Protocol for Blood-Based Biomarkers of Alzheimer’s Disease
Alexander Jethwa and Laura Stockl
6 Mass Spectrometry-Based Metabolomics Multi-platform for Alzheimer’s Disease Research
A ´ lvaro Gonza ´ lez-Domı ´ nguez, Ana Sayago, A ´ ngeles Ferna ´ ndez-Recamales, and Rau ´ l Gonza ´ lez-Domı ´ nguez PART III MAGNETIC
7 Analysis of Resting-State Functional Magnetic Resonance Imaging in Alzheimer’s Disease
Ersin Ersozlu ¨ and Boris-Stephan Rauchmann
8 Diffusion Tensor Imaging in Alzheimer’s Studies
Adriana L. Ruiz-Rizzo, Kathrin Finke, and Mario E. Archila-Mele´ndez
9 Magnetic Resonance Spectroscopy (MRS) in Alzheimer’s Disease
Nasim Sheikh-Bahaei
10 Neuroimaging Methods for MRI Analysis in CSF Biomarkers Studies 143
Carles Falcon, Gre´gory Operto, Jose´ Luis Molinuevo, and Juan Domingo Gispert
PART IV MOLECULAR IMAGING APPROACHES
11
Amyloid PET Imaging: Standard Procedures and Semiquantification
Francesca D’Amico, Luca Sofia, Matteo Bauckneht, and Silvia Morbelli
12 PET Imaging to Measure Neuroinflammation In Vivo
Maura Malpetti, Nicolai Franzmeier, and Matthias Brendel
13 Imaging Neuroinflammation: Quantification of Astrocytosis in a Multitracer PET Approach
Elena Rodriguez-Vieitez, Amit Kumar, Mona-Lisa Malarte, Konstantinos Ioannou, Filipa M. Rocha, and Konstantinos Chiotis
PART VNEUROPATHOLOGY
14 High-Throughput Lipidomic and Metabolomic Profiling for Brain Tissue and Biofluid Samples in Neurodegenerative Disorders .
Bonne M. Thompson and Giuseppe Astarita
15 Neuropathological Assessment as an Endpoint in Clinical Trial Design
Steve M. Gentleman and Alan King Lun Liu
16 Noninvasive Visualization of Amyloid-Beta Deposits in Alzheimer’s Amyloidosis Mice via Fluorescence Molecular Tomography Using Contrast Agent
Wuwei Ren and Ruiqing Ni
17 Brain Banking in Dementia Studies
Ahmet Turan Isik, Derya Kaya, and Murat Gokden
Simona Schafer, Janna Herrmann, Sol Tovar, Nicklas Linz, and Johannes Troger
19 cCOG Web-Based Cognitive Assessment Tool.
Hanneke F. M. Rhodius-Meester, Teemu Paajanen, and Jyrki Lotjonen
Ange´lique Sadlon
Contributors
MARIO E. ARCHILA-MELE ´ NDEZ • Department of Neurosurgery, Jena University Hospital, Jena, Germany
GIUSEPPE ASTARITA • Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA
RITA BARONE • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy; Pediatric Neurology Unit, Department of Pediatrics, University of Catania, Catania, Italy
MATTEO BAUCKNEHT • Nuclear Medicine Unit, Department of Health Sciences, University of Genoa, Genoa, Italy; IRCCS Ospedale Policlinico San Martino, Genoa, Italy
MATTHIAS BRENDEL • Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Department of Nuclear Medicine, LMU University Hospital, LMU Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
KONSTANTINOS CHIOTIS • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden; Department of Neurology, Karolinska University Hospital, Stockholm, Sweden
FRANCESCA D’AMICO • Nuclear Medicine Unit, Department of Health Sciences, University of Genoa, Genoa, Italy; IRCCS Ospedale Policlinico San Martino, Genoa, Italy
EMRAH DU ¨ ZEL • Institute of Cognitive Neurology and Dementia Research (IKND), Ottovon-Guericke University, Magdeburg, Germany; German Center for Neurodegenerative Diseases (DZNE) Magdeburg, Magdeburg, Germany
ERSIN ERSO ¨ ZLU ¨ • Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany; Department of Geriatric Psychiatry and Developmental Disorders, kbo-Isar-Amper-Klinikum Munich East, Academic Teaching Hospital of LMU Munich, Munich, Germany
CARLES FALCON • Barcelonaβ eta Brain Research Center, Pasqual Maragall Foundation, Barcelona, Spain; Centro de Investigacion Biome´dica en Red de Bioingenierı ´ a, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain
A ´ NGELES FERNA ´ NDEZ-RECAMALES • Department of Chemistry, Faculty of Experimental Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence CeiA3, University of Huelva, Huelva, Spain
KATHRIN FINKE • Department of Neurology, Jena University Hospital, Jena, Germany
NICOLAI FRANZMEIER • Institute for Stroke and Dementia Research, LMU University Hospital, LMU Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, Mo¨lndal and Gothenburg, Sweden
LUTZ FRO ¨ LICH • Department of Geriatric Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany
DOMENICO GAROZZO • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy
STEVE M. GENTLEMAN • Department of Brain Sciences, Imperial College London, London, UK
JUAN DOMINGO GISPERT • Barcelonaβ eta Brain Research Center, Pasqual Maragall Foundation, Barcelona, Spain; Centro de Investigacion Biome´dica en Red de Bioingenierı ´ a, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain
MURAT GOKDEN • Division of Neuropathology, Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
A ´ LVARO GONZA ´ LEZ-DOMI ´ NGUEZ • Instituto de Investigacion e Innovacion Biome´dica de Ca ´ diz (INiBICA), Hospital Universitario Puerta del Mar, Universidad de Ca ´ diz, Ca ´ diz, Spain
RAU ´ L GONZA ´ LEZ-DOMI ´ NGUEZ • Instituto de Investigacion e Innovacion Biome´dica de Ca ´ diz (INiBICA), Hospital Universitario Puerta del Mar, Universidad de Ca ´ diz, Ca ´ diz, Spain
TIMO GRIMMER • Department of Psychiatry and Psychotherapy, Klinikum rechts der Isar, Technical University Munich, Munich, Germany
NIELS HANSEN • Department of Psychiatry and Psychotherapy, University Medical Center Go¨ttingen, Go¨ttingen, Germany
ANNA HOFMANN • Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany; Department of Neurology, University Hospital Tuebingen, Tuebingen, Germany; German Center for Neurodegenerative Diseases (DZNE) Tuebingen, Tuebingen, Germany
KONSTANTINOS IOANNOU • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
AHMET TURAN ISIK • Unit for Aging Brain and Dementia, Department of Geriatric Medicine, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
FRANK JESSEN • Department of Psychiatry and Psychotherapy, University of Cologne, Medical Faculty, Cologne, Germany; German Center for Neurodegenerative Diseases (DZNE) Bonn, Bonn, Germany; Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
ALEXANDER JETHWA • Roche Diagnostics GmbH, Penzberg, Germany
DERYA KAYA • Unit for Aging Brain and Dementia, Department of Geriatric Medicine, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
AMIT KUMAR • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
CHRISTOPH LASKE • German Center for Neurodegenerative Diseases (DZNE) Tuebingen, Tuebingen, Germany; Section for Dementia Research, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany; Department of Psychiatry and Psychotherapy, University of Tuebingen, Tuebingen, Germany
ALAN KING LUN LIU • Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
JYRKI LO ¨ TJO ¨ NEN • Combinostics Oy, Tampere, Finland
MONA-LISA MALARTE • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
MAURA MALPETTI • Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
ANGELA MESSINA • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy
JOSE ´ LUIS MOLINUEVO • Barcelonaβ eta Brain Research Center, Pasqual Maragall Foundation, Barcelona, Spain; CIBER Fragilidad y Envejecimiento Saludable (CIBERFES), Madrid, Spain
SILVIA MORBELLI • Nuclear Medicine Unit, Department of Health Sciences, University of Genoa, Genoa, Italy; IRCCS Ospedale Policlinico San Martino, Genoa, Italy
RUIQING NI • Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland; Institute for Biomedical Engineering, ETH Zurich & University of Zurich, Zurich, Switzerland
GRE ´ GORY OPERTO • Barcelonaβ eta Brain Research Center, Pasqual Maragall Foundation, Barcelona, Spain
TEEMU PAAJANEN • Work ability and Working Careers, Finnish Institute of Occupational Health, Helsinki, Finland
ANGELO PALMIGIANO • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy
ROBERT PERNECZKY • Department of Psychiatry and Psychotherapy, LMU Hospital, LudwigMaximilians-University Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Ageing Epidemiology (AGE) Research Unit, School of Public Health, Imperial College London, London, UK; Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK
JOSEF PRILLER • Department of Psychiatry and Psychotherapy, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE) Berlin, Berlin, Germany; Department of Psychiatry and Psychotherapy, Charite University Medicine, Berlin, Germany; Dementia Research Institute, University of Edinburgh, Edinburgh, UK
BORIS-STEPHAN RAUCHMANN • Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany; Department of Neuroradiology, University Hospital, LMU Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, Germany; Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK
WUWEI REN • School of Information Science and Technology, ShanghaiTech University, Shanghai, China
HANNEKE F. M. RHODIUS-MEESTER • Alzheimer Center Amsterdam, Neurology, Vrije Universiteit Amsterdam, Amsterdam UMC location VUmc, Amsterdam, The Netherlands; Amsterdam Neuroscience, Neurodegeneration, Amsterdam, The Netherlands; Department of Internal medicine, Geriatric Medicine section, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, the Netherlands; Department of Geriatric Medicine, The Memory Clinic, Oslo University Hospital, Oslo, Norway
FILIPA M. ROCHA • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
ELENA RODRIGUEZ-VIEITEZ • Division of Clinical Geriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden; Division of Neurogeriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
DONATA AGATA ROMEO • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy
ADRIANA L. RUIZ-RIZZO • Department of Neurology, Jena University Hospital, Jena, Germany
ANGE ´ LIQUE SADLON • Department of Clinical Chemistry, Pharmacogenomics Research Group, Bern University Hospital & University of Bern, Bern, Switzerland
ANA SAYAGO • Department of Chemistry, Faculty of Experimental Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence CeiA3, University of Huelva, Huelva, Spain
NASIM SHEIKH-BAHAEI • Department of Radiology, Keck School of Medicine of USC, Los Angeles, CA, USA
LUCA SOFIA • Nuclear Medicine Unit, Department of Health Sciences, University of Genoa, Genoa, Italy; IRCCS Ospedale Policlinico San Martino, Genoa, Italy
LAURA STO ¨ CKL • Roche Diagnostics GmbH, Penzberg, Germany
LUISA STURIALE • CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania, Catania, Italy
BONNE M. THOMPSON • Arkuda Therapeutics, Watertown, MA, USA
SOL TOVAR • ki:elements GmbH, Saarbru¨cken, Germany
JOHANNES TRO ¨ GER • ki:elements GmbH, Saarbru¨cken, Germany
JENS WILTFANG • Department of Psychiatry and Psychotherapy, University Medical Center Go¨ttingen, Go¨ttingen, Germany; German Center for Neurodegenerative Diseases (DZNE) Goettingen, Goettingen, Germany; Neurosciences and Signaling Group, Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Aveiro, Portugal
MARIO ZAPPIA • Section of Neurosciences-Department GF Ingrassia, University of Catania, Catania, Italy
Part I
Strategies to Improve Alzheimer’s Disease Biomarkers
Chapter 1
Blood-Based Biomarkers for Early Alzheimer’s Disease Diagnosis in Real-World Settings
Robert Perneczky, Niels Hansen, Anna Hofmann, Christoph Laske, Josef Priller, Timo Grimmer, Lutz Fro ¨ lich, Emrah Du ¨ zel, Frank Jessen, and Jens Wiltfang and for
the German Network Memory Clinics – Diagnostic Tools Working Group
Abstract
As our knowledge about the biology of Alzheimer’s disease (AD) expands and we recognize the significance of early intervention for effective treatment, there is a shift in focus toward detecting the disease at an early stage. AD is characterized by the accumulation of misfolded amyloid-β (Aβ) and phosphorylated tau proteins in the brain, leading to the formation of senile plaques and neurofibrillary tangles. While a definitive diagnosis of AD can only be confirmed through autopsy by examining these pathological features, there are now reliable methods available for diagnosing the disease in living individuals. These methods involve analyzing cerebrospinal fluid and using positron emission tomography to accurately assess the presence of Aβ and tau proteins. While these diagnostic markers have shown high accuracy in memoryclinic populations, they do have limitations such as the requirement for invasive lumbar puncture or exposure to ionizing radiation. Additionally, they are not easily accessible outside of specialized healthcare settings. Blood-based biomarkers of the core pathological features of AD are being developed, showing promise for less invasive, scalable identification of AD cases in the community. The advantages for the healthcare systems of this development are obvious, but the diagnostic performance of blood-based biomarkers in broader, non-selected populations outside of retrospective analyses and research cohorts still requires further investigation, including the combination with more effective neuropsychological assessments such as digital cognitive test solutions.
Key words Early diagnosis of Alzheimer’s disease and dementia, Subjective cognitive impairment and mild cognitive impairment, Scalable diagnostic technologies, Patient and service user value, Early and targeted treatment and prevention of neurodegeneration
1 A Global Dementia Pandemic
Modern societies have achieved longer life expectancies, which is a significant accomplishment. However, the aging global population
has led to a rapid increase in the number of individuals affected by dementia. This has had a profound negative impact on the lives of affected families and national budgets. In 2018, the global cost of dementia was estimated to be US$1 trillion, and it is projected to reach US$2 trillion by 2030. Among various causes of dementia, Alzheimer’s disease (AD) is the most prevalent, currently affecting approximately 55 million individuals worldwide. This number is expected to rise to 132 million by 2050 [1].
To address this global challenge, the World Health Organization (WHO) developed a dementia plan in 2017, outlining priorities for action. These priorities include enhancing dementia diagnosis, treatment, and care [2]. Similar national strategies, such as the 2020 German National Dementia Strategy [3], have also been implemented. However, a recent study indicates that healthcare systems are not adequately equipped to effectively detect cognitive decline, accurately diagnose early-stage disease, and determine the most suitable interventions or deliver diseasemodifying treatments in the future [4].
2 A Paradigm Shift Toward Early Disease Detection
As our understanding of the biology of AD increases and we recognize the importance of early intervention for effective treatment, there is a shift in focus toward early disease detection. Pathologically, AD is characterized by the accumulation of misfolded amyloid-β (Aβ) and phosphorylated tau proteins in the brain, forming senile plaques and neurofibrillary tangles, respectively. While a definite diagnosis of AD requires confirmation of these pathological features through autopsy, there are now reliable methods available for diagnosing AD in living individuals. This is made possible through the use of cerebrospinal fluid (CSF) and positron emission tomography (PET) measures, which accurately assess the burden of Aβ and tau proteins. These diagnostic markers have shown high accuracy in memory-clinic populations, but they do have limitations such as the need for invasive lumbar puncture or exposure to ionizing radiation. Additionally, they are not readily accessible outside of specialized healthcare settings [5]. Furthermore, their diagnostic performance in broader, non-selected populations still requires further investigation.
3 Targeting the Early Diagnostic Window with Therapies
AD exhibits a prolonged presymptomatic stage that can last for many years. Recent advancements in imaging and fluid marker technologies have enabled the detection of increasing biological abnormalities during this stage. Once AD pathology surpasses a
specific threshold determined by an individual’s resilience against neurodegeneration [6], cognitive symptoms begin to manifest. The terms mild cognitive impairment (MCI) or prodromal AD are used to describe cases where cognitive performance falls below the expected norm for age and education, while basic daily activities remain mostly unaffected [7, 8].
Recent evidence suggests that concerns regarding cognitive decline could be an indication of early AD, even in the absence of objective below-average results on psychometric tests required for an MCI diagnosis [9]. When AD-related biomarker changes are present during this stage of subjective cognitive decline (SCD), the risk of progressing to MCI and dementia significantly increases [10]. The accurate identification of individuals with AD in the SCD stage is crucial as it offers a window of opportunity for early intervention and disease prevention. It is important to identify individuals in the community who have cognitive complaints and subtle deficits before their condition progresses to more severe stages. However, in most countries worldwide, there is currently a lack of infrastructure to accurately identify individuals with very early AD outside of specialized clinics, leading to low diagnostic rates [11]. Despite evidence showing that structured screening programs can significantly improve the detection of dementia cases [12], such programs are not widely implemented. The implementation of financial incentives aimed at increasing specialist referrals by primary care physicians has not yielded improved diagnostic rates for early cognitive decline in the UK [13]. Likewise, in Germany a significant number of dementia cases still go undiagnosed [14], and primary care physicians’ motivation to conduct cognitive screening is limited due to the absence of effective disease-modifying treatments [15]. Similar challenges are observed in other high-income countries. On the contrary, previous research suggests that community-based screening programs targeting memory concerns and minor cognitive deficits are both feasible and efficient in various settings [16]. A recent systematic review conducted for the US Preventive Services Task Force indicated that screening instruments can adequately detect cognitive impairment, but it remains uncertain whether systematic screening for cognitive impairment improves outcomes for patients and caregivers [17]. Therefore, further research is urgently needed to investigate the acceptance, effectiveness, and costs of community-based cognitive evaluations. Additionally, exploring the attitudes of seniors and primary care physicians toward opportunistic cognitive screening is crucial for developing an acceptable approach that can drive changes in current healthcare practices.
4 The Benefits of Digital Cognitive Testing
Standardized questionnaires like the SCD-Q [18] are available for assessing subjective complaints, but their validity and usefulness outside of specialized memory clinics still need to be determined. Objective psychometric tests such as the Mini-Mental-State Examination (MMSE) [19] or the Montreal Cognitive Assessment (MoCA) [20] can enhance diagnostic accuracy. Digital cognitive tests may offer significant advantages over traditional paper-andpencil assessments, particularly in community settings and primary care. They can be self-administered without requiring extensive involvement from healthcare professionals, potentially reducing rater bias and improving the reliability of longitudinal assessments. This could make early intervention more feasible [21]. However, it is yet to be determined whether self-administered tests provide enough additional diagnostic value to justify their integration into routine clinical care. Only if the test results offer actionable information for healthcare professionals will digital testing have a tangible impact on patient care.
5 Complementary Digital and Fluid Biomarkers
To enhance the accuracy of population-based cognitive screening, one potential approach is to integrate digital cognitive tests with pathophysiological biomarkers. Recent advancements in the identification of blood biomarkers provide a unique opportunity for noninvasive and scalable targeted diagnostics, which can inform decisions regarding specialist referral in primary care medicine. By combining precise blood protein assays with digital cognitive tests, it could be possible to create a robust screening tool that reduces errors in identifying preclinical or prodromal AD. However, the real-world effectiveness, cost-efficiency, and acceptance of this innovative approach still need to be fully explored.
The development of a blood-based biomarker algorithm holds critical importance due to its ease of measurement and costeffectiveness compared to the current gold standard assessments using CSF and PET. Recent studies have shown promising results in clinical and population-based cohorts, utilizing new ultrasensitive assay technology to measure blood concentrations of various AD biomarkers. These biomarkers include p-tau 181 and p-tau 217 (but not total tau) and neurofilament light chain (NfL) [22], although there have been conflicting results for Aβ42/40 [23].
An analysis of the US healthcare system indicated that integrating cognitive screening tests with blood-based biomarkers could eliminate wait lists for specialist assessments within the first 3 years and potentially reduce annual costs by US$400–700 million.
Additionally, it could lead to an increase of approximately 120,000 correctly identified cases per year [24]. In cases of SCD or MCI, the most effective prediction model for AD appears to be a combination of plasma pTau-181/217, APOE genotype, and a brief cognitive assessment of memory and executive function [25]. However, it is important to conduct a real-world evaluation of triage strategies, as genetic analyses have their own limitations, including increased requirements for patient consent and data protection.
6 Defining Appropriate Reference Ranges for Blood Biomarkers
Previous studies have demonstrated that concentrations of biomarkers in CSF [26] and blood [27, 28] are influenced not only by neurodegeneration but also by age. As a result, it is necessary to establish age-specific normal ranges for prodromal AD to ensure accurate diagnostic assessments. A recent study aimed to define reference ranges for plasma neurofilament light chain (NfL) in healthy individuals ranging from 5 to 90 years of age [29], and similar efforts will be required for other blood biomarkers and early symptomatic stages of AD. Adjusted reference ranges for NfL have also been proposed, taking into account factors such as renal function and body mass index [30], and there is some evidence suggesting higher NfL levels in men compared to women, although the clinical significance of this difference remains to be determined [31]. Plasma markers such as Aβ42, Aβ40, t-tau, and p-tau can be influenced by comorbidities such as chronic kidney disease, hypertension, hyperlipidemia, diabetes, myocardial infarction, and stroke [32]. Ethnicity [33] and food intake (postprandial vs fasting) [34] may also impact blood protein levels, and if these factors are not appropriately addressed, the proportion of misdiagnosed individuals may increase, especially in non-specialized settings and early stages of the disease.
Another impor tant consideration is determining the optimal blood compartment for analysis, as concentrations in plasma and serum may provide similar diagnostic information but may differ in certain situations. For instance, studies have shown comparable diagnostic performance and strong correlations between serum and plasma pairs, suggesting that p-tau analysis can be extended to healthcare settings with a preference for serum over other blood matrices. However, it should be noted that absolute biomarker concentrations may not be interchangeable, indicating that plasma and serum samples should be used independently [35]. Standardized values may prove helpful when combining results from different blood matrices, as demonstrated for NfL [36].
7 Lessons from Familial Alzheimer’s Disease
Familial AD exhibits distinct characteristics from the sporadic form, including different associated genes and comorbidities. However, genetic AD cohorts still provide a valuable model system for studying biomarkers relevant to individuals with late symptom onset. Longitudinal biomarker analyses spanning up to 15 years are available for participants in the Dominantly Inherited AD Network (DIAN), and published data demonstrates a strong correlation and elevated levels of NfL in both CSF and blood at the presymptomatic stages of familial AD. Longitudinal analysis of NfL dynamics in serum revealed the ability to differentiate mutation carriers from non-carriers almost a decade earlier than cross-sectional absolute NfL levels [37]. Thus, serum NfL dynamics can predict disease progression and brain neurodegeneration in the early presymptomatic stages of familial AD, indicating its potential utility as a clinically useful biomarker, even in early sporadic disease.
In the University College London Longitudinal Study of Familial AD, plasma p-tau181 concentrations were found to be elevated in both symptomatic and presymptomatic mutation carriers compared to non-carriers [38]. Despite significant intraindividual variability, individual values of plasma p-tau181 effectively discriminated symptomatic and presymptomatic carriers from non-carriers of the same age and sex. P-tau181 concentrations were higher in mutation carriers compared to non-carriers as early as 16 years before the estimated symptom onset, supporting its value as an early marker of AD.
The same University College London longitudinal study also revealed increased levels of glial fibrillary acidic protein (GFAP) in plasma of both presymptomatic and symptomatic mutation carriers compared to non-carriers, as well as in symptomatic carriers compared to presymptomatic carriers [39]. These differences were observed up to 16 years prior to symptom onset, consistent with findings of elevated plasma GFAP concentrations in Aβ-positive compared to Aβ-negative cognitively normal older individuals [40]. Overall, these findings support plasma GFAP as a potential biomarker for early AD and highlight the involvement of activated astrocytes in the early stages of the disease.
8 Performance of Blood Biomarkers Across the Alzheimer’s Continuum
Molecular blood biomarkers show promise in optimizing diagnostic approaches and potentially enabling early diagnoses of AD. These markers can also assist in stratifying patients by predicting the progression from early stages, such as SCD or MCI, to manifest stages of AD dementia. Several blood biomarker
candidates are currently being investigated for diagnosing AD and assessing prognosis. Markers related to Aβ pathology, which are part of current CSF and Aβ-PET biomarkers, are of significant interest for blood-based diagnostics in AD.
A recent study compared plasma biomarkers across two independent cohorts, BioFINDER and the AD Neuroimaging Initiative. The study demonstrated that combining immunoprecipitation with mass spectrometry analysis platform was superior to standard immunoassays for detecting Aβ, particularly by relying on the Aβ-42/Aβ-40 ratio within the brain [41]. Immunoprecipitation plasma assays with mass spectrometry exhibited moderate to good diagnostic accuracy in detecting brain amyloidosis in large AD cohorts, both in cognitively unimpaired and impaired individuals [42]. The diagnostic accuracy of this analysis platform improved further when considering the APOE status [42]. Automated analysis of the Aβ-42/Aβ-40 ratio [43] (and p-tau181 [44]) using the Lumipulse platform was also shown to provide high accuracy in AD diagnosis. However, other measurement methods such as SIMOA technology [45] or using carbon nanotubes for detecting Aβ-42/ Aβ-40 in plasma samples did not yield convincing results in detecting brain amyloidosis [46].
In addition to measuring Aβ peptides, p-tau forms such as p-tau181, p-tau217, and p-tau231 are currently under intense investigation as promising biomarkers for early diagnosis and disease progression in AD. These markers have shown utility in diagnosing AD early, differentiating AD from other neurodegenerative diseases, and predicting cognitive decline and neurodegenerative processes in AD [41, 47–53]. A comprehensive retrospective study presented evidence that p-tau217 outperforms p-tau181 in distinguishing AD from other neurodegenerative conditions such as frontotemporal dementia [49] (Thijssen, La Joie et al., 2021), and p-tau217 has been highlighted as a potential marker for preclinical and prodromal stages of AD [54].
In the preclinical stage, another blood biomarker of interest is GFAP, a marker of astroglial inflammation. GFAP has shown potential for detecting Aβ pathology in preclinical stages and predicting the progression from MCI to dementia [50, 55]. Furthermore, a cytoskeleton marker of axonal neurodegeneration in blood has demonstrated a close correlation with CSF concentration [56]. Plasma NfL has been explored to predict the disease course and cognitive outcome in AD [50, 57]. Currently available tests have shortcomings in access, throughput, and scalability that limit widespread implementation. A high-throughput and fully automated Lumipulse plasma p-tau181 assay for the detection of AD has been evaluated and showed robust performance in differentiating AD from control participants [44]. However, the Lumipulse technique needs to be validated in multicenter cohorts and for other AD biomarkers.
In larger multicenter cohorts, there is a need to explore ultrasensitive assays capable of covering the full biomarker-based classification of AD using blood biomarkers, offering a less invasive approach than CSF. A recent study [58] demonstrated a strong correlation between CSF and blood markers, suggesting the feasibility of implementing biomarker-based classification in blood. However, a limitation of Aβ-42/Aβ-40 is that its levels in blood are considerably lower than in CSF [59]. Developing composite marker panels that provide higher diagnostic accuracy than individual biomarkers may prove beneficial. In the future, the range of blood biomarkers for AD is expected to expand. For example, serum β-synuclein has emerged as a novel candidate for indicating synaptic degeneration in the temporal lobe in AD [60]. These studies and considerations underscore the increasing importance of blood biomarkers in the future early and differential diagnosis of AD.
9 Outlook and Next Steps Required
Ensuring the long-term effectiveness of any new diagnostic algorithm is crucial to create tangible benefits to the older population. It is essential to develop tailored approaches that align with the specific requirements of local healthcare systems, thus ensuring their sustained commitment. The availability and coverage of various interventions vary significantly across different regions worldwide. In this regard, employing low-cost assessments like digital and blood biomarkers may offer advantages over more expensive alternatives such as PET scans. Currently, one of the primary challenges in identifying early-stage cognitive decline cases is the limited preparedness of healthcare systems to offer proactive screening visits and specialist assessments following initial check-ups in primary care settings. As soon as AD-modifying drug will be available more broadly, the demand for accurate early diagnostics will dramatically increase, necessitating the readiness of healthcare systems to address this need. Objective digital tests can provide valuable information to healthcare professionals for making better care decisions, while enabling primary care doctors to make informed decisions about specialist referrals at an earlier stage can alleviate the burden on the general practitioner sector. To support coverage decisions, it is important to gather more quantitative data on the diagnostic yield and cost per identified patient associated with combined digital and blood biomarkers, as this information will be vital for payers. The envisioned diagnostic and care pathways will include close collaboration across different healthcare sectors, and specialized specialist memory clinics will play an increasingly important role in verifying early AD diagnoses, making treatment decisions and monitoring dug efficacy and safety [61] (Fig. 1).
Fig. 1 Potential pathway for case identification, diagnostic ascertainment, and treatment initiation/monitoring in early Alzheimer’s disease (reprinted with permission from [61]). A proposed model has been suggested for a care pathway aimed at identifying older individuals who exhibit minor cognitive complaints or deficits in primary care. This would be followed by a secondary care step where subjects with a very low likelihood of Alzheimer’s disease pathology would be excluded, possibly through the use of blood-based biomarkers. Diagnostic confirmation would then be conducted by a dementia specialist, potentially employing positron emission tomography and cerebrospinal fluid markers. Following a careful evaluation of potential risks and benefits, including the monitoring of side effects, disease-modifying treatment would be initiated. It is important to note that individuals may also move backward in this model if they fail to meet the criteria for progressing to the next stage, such as having a negative blood biomarker for Alzheimer’s disease
Acknowledgments
R.P. is supported by the German Center for Neurodegenerative Disorders (Deutsches Zentrum fu¨r Neurodegenerative Erkrankungen, DZNE), the Deutsche Forschungsgemeinschaft (DFG, 1007 German Research Foundation) under Germany’s Excellence Strategy within the framework of 1008 the Munich Cluster for Systems Neurology (EXC 2145 SyNergy – ID 390857198), the Davos Alzheimer’s Collaborative, the VERUM Foundation, the RobertVogel-Foundation, the National Institute for Health and Care Research (NIHR) Sheffield Biomedical Research Centre (NIHR203321), the University of Cambridge – LudwigMaximilians-University Munich Strategic Partnership within the
framework of the German Excellence Initiative and Excellence Strategy, and the European Commission under the Innovative Health Initiative program (project 101132356). 12RobertPerneczkyetal.
Conflicts of Interest R.P. has received honoraria for advisory boards and speaker engagements from Roche, EISAI, Eli Lilly, Biogen, Janssen-Cilag, Astra Zeneca, Schwabe, Grifols, Novo Nordisk, and Tabuk. J. W. has been an honorary speaker for Actelion, Amgen, Beeijing Yibai Science and Technology Ltd., Gloryren, Janssen Cilag, Med Update GmbH, Pfizer, and Roche Pharma and has been a member of the advisory boards of Abbott, Biogen, Boehringer Ingelheim, Lilly, MSD Sharp & Dohme, and Roche Pharma and receives fees as a consultant for Immungenetics, Noselab, and Roboscreen and holds the following patents: PCT/EP 2011 001724 and PCT/EP 2015 052945.
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Chapter 2
Alzheimer’s Disease Prevention and Treatment Based on Population-Based Approaches
Robert Perneczky
Abstract
The development of effective prevention and treatment strategies for Alzheimer’s disease (AD) and dementia is hindered by limited knowledge of the underlying biological and environmental causes. While certain genetic factors have been associated with AD, and various lifestyle and environmental factors have been linked to dementia risk, the interactions between genes and the environment are not yet fully understood. To identify new avenues for dementia prevention, coordinated global efforts are needed to utilize existing cohorts and resources effectively and efficiently. This chapter provides an overview of current research on risk and protective factors for AD and dementia and discusses the opportunities and challenges associated with population-based approaches.
Key words Alzheimer’s disease, Dementia, Drug development, Population-based, Cohort studies, Lifestyle, Environment, Genetics
1 Introduction
As the prevalence of Alzheimer’s disease (AD) and its impact on healthcare and socioeconomic systems continue to grow worldwide, there is an urgent need to improve drug development success rates, and the approval of the first anti-amyloid-β (Aβ) targeting antibody drugs is initiating an exciting new era of AD treatment and prevention [1]. Successful drug discovery and development in other fields, such as cancer and infectious diseases, have demonstrated the potential of translational models that link epidemiological cohort and genetic data to potential drug targets and study endpoints [2]. Technology platforms, including neuroimaging, -omics, and biomarkers, have proven to be powerful tools in this process. To discover and develop novel disease-modifying treatments beyond the first generation of Aβ-targeting compounds, as called for since the 2013 G8 Dementia Summit, a significant cultural shift must be initiated [3].
Increased life expectancy is a remarkable achievement of modern society, but it also brings with it the challenge of a growing prevalence of age-related disorders, placing a significant burden on global healthcare systems. Over the next 30–40 years, the number of people affected by late-onset neurodegenerative disorders such as AD and Parkinson’s disease is expected to increase three-to fourfold worldwide [4], with national and international dementia plans calling for immediate action, such as the 2017 WHO Action Plan [5]. The individual risk for these disorders varies greatly, and the development of effective prevention and treatment strategies is hindered by our incomplete understanding of the factors that influence vulnerability to neurodegeneration [6]. There is growing consensus among researchers that an individual’s susceptibility to AD is determined by a combination of biological and environmental factors, with important implications for the development of effective interventions. While AD has a significant hereditary component, monogenic familial forms caused by clearly pathogenic mutations in specific genes are extremely rare. Most AD cases are likely the result of complex interactions between common genetic variants and other factors. The biological mechanisms by which genes influence an individual’s vulnerability to AD are not yet fully understood, but recent discoveries of risk genes suggest the involvement of certain biological pathways, such as Aβ and tau processing, immune response, and lipid processing [7].
An individual’s vulnerability to AD is influenced by a complex interplay of genetic and non-genetic factors, including environmental and epigenetic mechanisms. While genetic susceptibility is innate, the risk associated with certain environmental factors may be modifiable, offering opportunities for prevention or treatment through changes in lifestyle. Many known environmental risk factors are related to conditions such as obesity, diabetes, and vascular disease, which can potentially be modified. Protective environmental factors, such as a Mediterranean diet and cognitive and physical activity, can also be actively strengthened. An individual’s personality and inherent beliefs can influence their behavior and lifestyle choices, affecting their dementia risk. Additionally, factors outside an individual’s control, such as the healthcare system, air pollution, and the wider economy, can also impact their chronic disease risk [8] (Fig. 1).
2 Epidemiological Approaches to Alzheimer’s Disease Research
While the association between AD risk and certain environmental and lifestyle factors is well established, the underlying biological mechanisms are not yet fully understood. The use of highthroughput “omics” technologies in prospective cohort studies
2.1 The Value of Established Global Infrastructures
with stored biospecimens offers a unique opportunity to characterize individuals at the molecular level. Advanced technologies, such as epigenomics and metabolomics, can be applied to pre-diagnostic biosamples to identify early disease markers. By combining epidemiological and molecular data, with a focus on gene-environment interactions, etiological research can pave the way for the identification of etiopathogenetic pathways through systems biology approaches.
In recent decades, several large-scale prospective cohort studies on aging and neurodegenerative disease have been established worldwide. These studies have collected detailed dietary, lifestyle, and clinical information, as well as biological samples and neuroimaging data, from many participants at various points in their lives. The continued follow-up of these existing infrastructures and the collection of additional detailed information from these aging populations is a highly efficient strategy for addressing existing knowledge gaps. However, data analysis has mostly been restricted to individual cohorts due to differences in assessment procedures and methods between countries. As a result, there is an urgent need for
Fig. 1 A multicausal model of Alzheimer’s disease
concerted action to facilitate the comprehensive analysis of large datasets and to increase knowledge transfer between experts in different countries. Standardized data analysis across global aging and neurodegeneration cohorts and effective networking activities between researchers in the field are crucial. The main goals of such networking activities are to develop harmonized measures for key outcomes used in different cohort studies (e.g., genome-wide data, environmental exposures, neuroimaging data, blood and cerebrospinal fluid proteins, etc.) and to facilitate knowledge transfer and expert exchanges between countries and institutions. This includes providing the best possible training and career opportunities for early-stage researchers. Examples of large-scale initiatives that effectively integrate information from diverse cohorts include the Dementias Platform UK (http://www.dementiasplatform.uk/) and the EU Joint Program for Neurodegenerative Disease Research (JPND) longitudinal cohort studies action group [9].
3 Gene-Environment Interaction Analyses
There is a growing consensus among researchers that AD and other forms of dementia result from a combination of genetic and environmental factors and their interactions [10, 11]. Subtle differences in DNA sequence composition, such as different allele status at polymorphic sites, can cause individuals to respond differently to the same environmental exposure. This partly explains why some individuals have a lower risk of developing the disease due to an environmental insult, while others are more vulnerable. For instance, not all carriers of the apolipoprotein (APOE) ε4 risk allele, the most significant genetic risk factor for AD, develop the disease or dementia even at an advanced age [12]. The presence of unknown gene-environment patterns may also contribute to the difficulties in identifying other major genetic effects besides APOE Population-based studies have consistently shown that 40–50% of individuals who reach the age of 90 are free of dementia, indicating that dementia is not an inevitable consequence of aging [13]. However, several important questions remain unanswered, including why some individuals escape dementia and what genetic and/or environmental factors distinguish them from those who develop the disease. Epidemiological studies have provided some insights into these questions, but the picture is still incomplete, particularly regarding the interactions among different factors at different stages of life.
Previous research has suggested interactions between genes and various environmental factors. For example, gene-diet interactions have been found between the APOE gene and nutrients such as n-3 polyunsaturated fatty acids, vitamin E, total fat, and energy intake in relation to the risk of AD and dementia [14]. Emerging
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the Divine Teacher and the listener who was drinking in such soul-absorbing truths!
Percival. Surely it was not in blind ignorance of the meaning of what she did, that Mary brought her precious ointment to pour on the feet of the Master! He who could read her inmost thoughts said, "She did it for My burial." Were not Mary's thoughts, then, something like this—
"He hath said it—alas! alas!—and all that my Lord says must be true. The holy Jesus will be delivered into the hands of the Gentiles. He will be mocked, scourged, and slain. Yes, He who called my brother from the grave must Himself die! And they who murder Him will not, perhaps, suffer due honour to be paid to the holy corpse; I may not be able to approach the sacred form! I will be beforehand with Christ's cruel foes; what I may not be allowed to do after His death, I will do ere the awful moment come when the Lamb of God must be sacrificed for our sins. I will anoint Him for His burial!"
Seyton. And when the sacrifice had been offered, you believe that Mary of Bethany, unlike any of the apostles, had faith to look beyond death to Christ's Resurrection?
Percival. There is nothing in Scripture that I know of to lead us to doubt it. The piety of Mary of Bethany seems to have been of a higher, a more spiritual type, than that of her sister. It was not Mary who exclaimed against the removal of the stone from the sepulchre's mouth. Mary perhaps saw in the resurrection of Lazarus a type and pledge of that of her Lord. If so, her joy must have been yet more intense than that of Martha, even as her gratitude took a more palpable form.
Seyton. It is interesting and refreshing to the spirit thus to meditate over Scripture characters. What to some are merely like ancient statues, when we gaze on them thus, become human beings instinct with life.
Percival. And such meditation makes us realize the tie which binds Christians of to-day to saints of the olden time; at least, it has that effect with me. I feel almost as if those whose forms I have attempted to depict on my canvas had become my familiar friends. I look forward to meeting those three Maries hereafter: perhaps that time may not be far off.
Seyton. There are two male figures in the background of your picture, represented as just about to enter the room. One, the elder, appears to be struggling to retire: he is unwilling to intrude on the sacredness of grief.
Percival. Can you not read sorrow and shame on his halfaverted face?
Seyton. His younger companion is using loving persuasion to draw him forward: his arm is thrown around the elder, and his face expresses compassion and love. The two figures must represent John and Peter.
Percival. Such was the idea in my mind. Where would poor broken-hearted Peter hide himself when, pierced by that look of his Lord, he went forth and bitterly wept? Would he not seek the solemn shade of the olives in the Garden of Gethsemane, and prostrate himself on the spot where the Master had knelt in agonized prayer? Would not Peter lay his throbbing brow on the sod where he could trace red signs of the bloody sweat, and try to efface them with his hot tears?
Percival went on: How terrible to Peter must have been the darkness which for three hours covered the earth, a sign that the fearful deed was being done, on which the sun could not look! He who had thrice denied his Lord dared not go near His cross; but the disciple's anguished soul would vividly picture its horrors. Peter must have trembled at the shock of the earthquake which told that all was over. How could he rise from the earth? How endure ever to look again on the face of a fellow-apostle?
Seyton. And you have imagined John, with tender sympathy, seeking out his erring brother in the place where he would be most likely to find him.
Percival. And entreating Peter not to remain apart from all his brethren; not to give way to despair: but to join those who, like himself, were mourning their crucified Lord.
Seyton. No marvel that Peter should shrink from entering the presence of the bereaved mother of Christ!
Percival. Mary would not turn from him; she would utter no word of reproach: she would raise her tearful eyes, and give the penitent Peter a look which would remind him of that which he had last seen on the sacred face of her Son.
CHAPTER X.
The Legend of the Roman Soldier.
I HAD fastened up several of Percival's pictures on the wall of the room which he now occupied; and in which he received frequent visits from my aunt. On one occasion the following conversation was held between them.
Lady Mar. Percival, I cannot take my eyes from that picture of yours hung in the corner: it is so dramatic in composition, so vigorous in execution. Yet I find it so difficult to trace any connexion between it and any narrative contained in the Bible. I understood from my nephew that you only illustrate passages from Scripture.
Percival. Not exactly so, dear Lady Mar. The connection of my poor fancies with the Scriptures is like that of the mistletoe with the oak. The mistletoe is a weak little plant; of a nature different to, and far less noble than, the tree on which it rests: yet from that tree, it derives both nourishment and support.
Lady Mar. And the mistletoe bears delicate white berries, which serve to make winter brighter. But this picture before me has red berries rather than white ones. Despondency and attempted suicide appear to form its subject. A powerfully-made soldier, evidently a Roman, is about to fall on his own sword, his face expressing the despair which is driving him on to self-destruction. Another man, a Jew, has caught hold of his arm, evidently to prevent the warrior from accomplishing his desperate purpose.
Lady Mar continued: "Please tell us on what branch of the oak your parasite grows. You cannot refuse us anything this evening, as my nephew leaves us for college to-morrow; so to one of us, as you see, this will be the last night of meeting for some months to come."
I thought sadly, "Possibly indeed the last meeting. Shall I find Percival here on my return?"
"I happen to have written out my little Legend," was Percival's reply. "If Seyton cares to read it, and you to listen, it is quite at your service."
"Whilst my nephew reads, I will keep my eye on the picture," said Lady Mar. "I feel a sympathy with that stalwart Roman, who seems in such a desperate plight."
The Legend of the Roman Soldier.
A soldier sought the silence and solitude of a forest; for the presence of his fellow-creatures had become hateful to his soul. The moonbeams, piercing like silver lances between the branches, glimmered on the steel breastplate and arms which had been borne in many a fight.
Marcus was a tried warrior, who had distinguished himself from his comrades by feats of strength and deeds of daring. But now all his spirit was gone: he would not have cared to raise his powerful arm to ward off a blow; nay, he would have welcomed the sharp steel which should cut him off from the earth, which had become to him worse than a prison.
"Now let me end my misery!" exclaimed Marcus. "I am a guilty wretch not fit to live! There is only one good deed which I can perform—use this accursed hand to avenge the innocent blood which it shed."
Clenching his teeth with fierce resolution, Marcus fixed the hilt of his sword firmly between the gnarled roots of a tree; hastily unfastened his breastplate, and flung it clanging on the earth; then nerved himself for the desperate act of throwing himself on the point of his sharp weapon.
But at that moment, the muscular arm of the strong soldier was seized by a Jew, who, unseen in the shade, had watched his movements.
"Madman! In Christ's name forbear!" exclaimed the Jew.
Marcus was startled at the word. "What! Are you one of the followers of Him who died on Calvary?" cried the soldier, drawing back, and surveying almost with fear one whom by
a slight exertion of his giant strength, he could have dashed to the ground. "If you be a disciple of Christ, far from staying the execution of justice, you will slay me yourself, and trample my blood under your feet! Take yon sword, and strike home!"
"What hast thou done," asked the Christian, "that thou shouldst bid me slay thee?"
"Hear, if thou wilt; for I can no longer endure to bear my burden in silence. Hear and then strike; for I have wellmerited death from the hand of a disciple."
So saying, Marcus flung himself down on the gnarled roots, which afforded a rude kind of seat, and signed to the Jew to take his place on a large stone near.
Asahel, such was his name, obeyed the sign, and prepared himself to listen.
But for some minutes only deep groans were heard from the unhappy Roman, who seemed to shrink from beginning his terrible confession. At last, averting his eyes, he thus began:
"Is it not enough to say that I was one of the Prætorian band on that day—not many moons have waxed and waned since then—when there was darkness and an earthquake; and the Temple curtain was rent in twain."
"What! Thou wast one of those Roman soldiers! Thou didst mock the Blessed One, and crown Him with thorns!"
"I did not!" cried Marcus fiercely. "I was not base enough for that. When I looked at that calm majestic Sufferer, I thought Him more kingly in His robe of mockery, than Pontius Pilate in all his state!"
"When I heard the yelling of the savage mob, thirsty for blood, I said to myself, 'Were I in the place of our Governor, those slaves might shout as they pleased, I would never give Him up, innocent as He is, to fanatic priest or frantic people! I would not so play the coward!'"
Asahel winced, as if some acute pain had suddenly struck him.
"But it was my duty, my detestable office, to execute the sentence which I thought unjust as well as cruel. I was accustomed as a soldier to obey orders without question, and without remorse. Hardened as I am by familiarity with executions, without mercy or scruple I crucified one of the wretched thieves. His yell of agony as I did my work did not even awake a feeling of pity in my heart."
"But it was very different with me when I laid my hand— would that lightning had blasted it!—on the hammer, and did what I would now give my life to undo! He uttered no groan—no curse; He submitted like a lamb in the slaughterer's grasp. He but said—I cannot repeat what He said."
The soldier's head sank on his broad breast, and the strong man wept.
"Christ said, 'Father, forgive them; for they know not what they do!'" said Asahel, softly.
"I was present through all," continued the soldier, when he had recovered his self-command, "I saw the sudden darkness: it fell over me like a shroud! Every hour of that fearful time convinced me the more that I was helping to torture—to murder—One who was more than man. At last, when I heard the faint words, 'I thirst!' I ran; and putting a
sponge filled with vinegar on a reed, I moistened the white, parched lips of the Dying."
"Oh that I had done that!" cried the Christian Jew, bursting into tears. "Blessed man! Thou wert the only one, then, to relieve the Saviour's dying anguish!"
The Roman gazed in astonishment at his companion. "I thought, O follower of Christ!" said he. "That thou wouldst abhor me, even as I abhor mine own self."
"I am a thousandfold more guilty than thou art!" cried Asahel. "I was one of the savage mob. It was as if under the direct influence of Satan that I shouted even as they did. I saw Him suffer—and I did not pity! Thou didst act under compulsion. I—I struck Him; and yet I live!"
Marcus started to his feet with something like an imprecation. "Wretch! Thou art beyond pardon!" he exclaimed.
"I have found pardon!" cried the believer. "And where I found it, so may'st thou."
Then, in a voice trembling with emotion, Asahel recounted the wonders of the Day of Pentecost; and repeated, almost word for word, that address of Peter, on hearing which, three thousand sinners were pricked to the heart and repented.
"I was one of that three thousand," said the converted Jew. "I believed, and I was forgiven. The blood which flowed on the cross was a full, sufficient, atonement even for guilt such as mine."
"And will it avail even for me?" exclaimed Marcus, the first ray of hope glimmering on the midnight of his despair.
"Did Christ not pray for thee, O brother? And art thou not already forgiven?"
CHAPTER XI.
Asleep.
I SHALL ever remember that evening: what followed impressed it so deeply on my mind.
Percival seemed unconscious of any one's presence; his lips softly repeated the last word forgiven, and I thought that he smiled.
A brief prayer closed our meeting that night: I now doubt whether Percival heard it.
My aunt, seeing that the invalid was unusually drowsy, hastened the preparation for his nightly rest.
In the morning. I went to Percival's room early, to bid him good-bye ere I started for college. I knocked at his door: there was no reply. I knocked again: still silence within. I opened the door softly; and entering, approached his bed.
My first glance at the countenance, so white—so still—so beautiful, told me that the spirit had fled.
"For death had come in the land of sleep; And his lifeless body lay Like a worn-out fetter, which the soul Had broken and cast away!"
We had anticipated for Percival a long, slow, painful descent to the river of death: but some chord had given way within; he was free, and had cleared the river at a bound. I could not have laid a detaining hand on the freed and rejoicing spirit!
Nothing is now left, in this world, of Henry Percival—but a modest tomb, a fragrant memory, and his little gallery of pictures.
LONDON: MORGAN & SCOTT, 12, PATERNOSTER BUILDINGS.
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