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Methods in
Molecular Biology 1750

Robert Perneczky Editor

Biomarkers
for Alzheimer’s
Disease Drug
Development
Methods in Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Biomarkers for Alzheimer’s
Disease Drug Development

Edited by

Robert Perneczky
Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-Universität München,
Munich, Germany
German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, Germany
Neuroepidemiology and Ageing Research Unit, School of Public Health, The Imperial College of Science,
Technology and Medicine, London, UK
West London Mental Health NHS Trust, London, UK
Editor
Robert Perneczky
Department of Psychiatry and Psychotherapy
Ludwig-Maximilians-Universität München
Munich, Germany
German Center for Neurodegenerative Diseases (DZNE) Munich
Munich, Germany
Neuroepidemiology and Ageing Research Unit, School of Public Health
The Imperial College of Science, Technology and Medicine
London, UK
West London Mental Health NHS Trust
London, UK

ISSN 1064-3745     ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7703-1    ISBN 978-1-4939-7704-8 (eBook)
https://doi.org/10.1007/978-1-4939-7704-8

Library of Congress Control Number: 2018932034

© Springer Science+Business Media, LLC 2018

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Preface

The prevalence of Alzheimer’s disease and its healthcare and socioeconomic impact are
exploding worldwide, and drug development success rates need to be improved urgently;
the traditional linear model of pharmaceutical R&D has become outdated and virtually all
new drugs have failed since the cholinesterase inhibitors were introduced to the markets
two decades ago. Drug development has been more successful in other fields of medicine
such as infectious diseases and cancer, in which translational models are applied, linking
population-based cohorts and genetic data with potential drug targets and study endpoints.
This powerful translational approach is fuelled by technology platforms such as n ­ euroimaging,
-omics, and fluid biomarkers. The Alzheimer’s disease field requires a significant cultural
change to discover and develop effective disease-modifying treatment options (until 2025,
as postulated at the 2013 G8 Dementia Summit).
The main objective of this book is to bridge and converge population, -omics, and
imaging sciences (typically owned by academia) with R&D approaches for new t­ echnologies
and novel, effective drugs (the traditional remit of industry). The book will help building a
new generation of experts with a broader understanding of key topics to initiate the
­disruptive innovation required to make real progress. Given the high complexity and
multifactorial disease nature related to dementias, a precision/personalized medicine
­
approach in designing the next generation R&D strategies is now urgently required.
This publication comprises nine parts: In Part I (Chapters 1–3) we explain why Alzheimer’s
disease is one of the major challenges for the global societies and healthcare systems, and how
population-based and systems biology approaches can be leveraged to develop more effective
treatments. In Part II (Chapters 4–6) we present innovative approaches to the discovery of novel
biomarkers in cerebrospinal fluid, whereas in Part III (Chapters 7–9) innovation in blood-based
biomarkers is discussed. Part IV (Chapters 10–12) and Part V (Chapters 13–16) provide a
­comprehensive ­overview of magnetic resonance imaging and molecular imaging approaches and
their value for developing drugs for Alzheimer’s disease, respectively. In Part VI (Chapters 17 and
18) cutting-edge d ­ evelopments in neuropathology and their relevance for Alzheimer’s disease
trials are presented. Part VII (Chapters 19–21) covers novel genomic strategies for biomarker
development. Part VIII (Chapters 22 and 23) ­highlights the contribution of preclinical research
to the ­development of novel b ­ iomarkers and drugs. Finally, in Part IX (Chapters 24 and 25), we
consider r­elevant related topics including neuropsychological testing and advanced analytical
methods.
The book is targeted at individuals with an interest in the use of advanced biomarker ­strategies
to accelerate the development of effective, disease-modifying drugs for Alzheimer’s disease. This
includes researchers, clinicians, and those interested in regulatory and medical affairs, both from
academia and industry. We wish to present biomarker development approaches as a strategy for
the study of Alzheimer’s disease with the hope and expectation that the results will translate into
more effective treatments. We expect this book to ­complement other excellent volumes and
monographs on Alzheimer’s disease that cover basic science or clinical aspects of the disease.

Munich, Germany Robert Perneczky

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   xi
Part I The Need for Alzheimer’s Disease Biomarkers
1 Epidemiology of Dementia: The Burden on Society,
the Challenges for Research�����������������������������������������������������������������������������   3
Frank J. Wolters and M. Arfan Ikram
2 Population-Based Approaches to Alzheimer’s Disease Prevention���������������������   15
Robert Perneczky
3 Systems Biology Methods for Alzheimer’s Disease Research
Toward Molecular Signatures, Subtypes, and Stages
and Precision Medicine: Application in Cohort Studies and Trials���������������������   31
Juan I. Castrillo, Simone Lista, Harald Hampel, and Craig W. Ritchie

Part II Innovative Approaches to Cerebrospinal

Fluid Biomarker Discovery
4 CSF Lipidomics Analysis: High-Resolution Mass
Spectrometry Analytical Platform���������������������������������������������������������������������   69
Paul L. Wood and Randall L. Woltjer
5 CSF N-Glycomics Using MALDI MS Techniques
in Alzheimer’s Disease�������������������������������������������������������������������������������������   75
Angelo Palmigiano, Angela Messina, Rosaria Ornella Bua,
Rita Barone, Luisa Sturiale, Mario Zappia, and Domenico Garozzo
6 MicroRNA Profiling of Alzheimer’s Disease Cerebrospinal Fluid ���������������������   93
Johannes Denk and Holger Jahn

Part III Novel Blood-Based Biomarkers

7 Validation of a Chemiluminescence Immunoassay
for Measuring Amyloid-β in Human Blood Plasma������������������������������������������� 111
Jonathan Vogelgsang, Jens Wiltfang, and Hans W. Klafki
8 Mass Spectrometry-Based Metabolomic Multiplatform
for Alzheimer’s Disease Research��������������������������������������������������������������������� 125
Raúl González-Domínguez, Álvaro González-Domínguez, Ana Sayago,
and Ángeles Fernández-Recamales
9 Blood-Based Biomarker Screening with Agnostic Biological
Definitions for an Accurate Diagnosis Within the Dimensional
Spectrum of Neurodegenerative Diseases��������������������������������������������������������� 139
Filippo Baldacci, Simone Lista, Sid E. O’Bryant, Roberto Ceravolo,
Nicola Toschi, Harald Hampel, and for the Alzheimer Precision Medicine
Initiative (APMI)

vii
viii Contents

Part IV Magnetic Resonance Imaging Methods

10 Functional Magnetic Resonance Imaging in Alzheimer’
Disease Drug Development����������������������������������������������������������������������������� 159
Stefan Holiga, Ahmed Abdulkadir, Stefan Klöppel, and Juergen Dukart
11 Neuroimaging Methods for MRI Analysis in CSF Biomarkers Studies��������������� 165
Carles Falcon, Grégory Operto, José Luis Molinuevo,
and Juan Domingo Gispert
12 Hybrid PET-MRI in Alzheimer’s Disease Research������������������������������������������� 185
Ismini C. Mainta, Maria I. Vargas, Sara Trombella,
Giovanni B. Frisoni, Paul G. Unschuld, and Valentina Garibotto

Part V Molecular Imaging Approaches

13 Amyloid PET Imaging: Standardization and Integration
with Other Alzheimer’s Disease Biomarkers����������������������������������������������������� 203
Silvia Morbelli and Matteo Bauckneht
14 The Use of 18F-FDG PET in the Diagnostic Workup
of Alzheimer’s Dementia��������������������������������������������������������������������������������� 213
Marion M. Ortner
15 Quantification of Tau Load in Alzheimer’s Disease Clinical
Trials Using Positron Emission Tomography ��������������������������������������������������� 221
Tessa Timmers, Bart N.M. van Berckel, Adriaan A. Lammertsma,
and Rik Ossenkoppele
16 Imaging Neuroinflammation: Quantification of Astrocytosis
in a Multitracer PET Approach ����������������������������������������������������������������������� 231
Elena Rodriguez-Vieitez and Agneta Nordberg

Part VI Neuropathology
17 Unbiased Lipidomics and Metabolomics of Human Brain Samples������������������� 255
Giuseppe Astarita, Matteo Stocchero, and Giuseppe Paglia
18 Neuropathological Assessment as an Endpoint in Clinical Trial Design������������� 271
Steve Gentleman and Alan King Lun Liu

Part VII Genomic Methods

19 Analysis of Micro-RNA Expression by qPCR on a Microfluidics Platform
for Alzheimer’s Disease Biomarker Discovery��������������������������������������������������� 283
Petros Takousis
20 Telomere Length Shortening in Alzheimer’s Disease: Procedures
for a Causal Investigation Using Single Nucleotide Polymorphisms
in a Mendelian Randomization Study��������������������������������������������������������������� 293
Yiqiang Zhan and Sara Hägg
21 Quantifying miRNA Deregulation in Alzheimer’s Disease ������������������������������� 307
Ana L. Cardoso and Joana R. Guedes
 Contents ix

Part VIII Preclinical Research

22 Imaging of Microglial Activation in Alzheimer’s Disease
by [11C]PBR28 PET ��������������������������������������������������������������������������������������� 323
Cornelius K. Donat, Nazanin Mirzaei, Sac-Pharm Tang,
Paul Edison, and Magdalena Sastre
23 In Vivo Two-Photon Calcium Imaging of Hippocampal Neurons
in Alzheimer Mouse Models����������������������������������������������������������������������������� 341
Marc Aurel Busche

Part IX Related Concepts: Cognitive Testing

and Advanced Analytics

24 Cognitive Assessment in Alzheimer’s Disease Clinical Trials����������������������������� 355

Geraint Price
25 Data Mining and Machine Learning Methods for Dementia Research ������������� 363
Rui Li

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Contributors

Ahmed Abdulkadir · University Hospital of Old Age Psychiatry and Psychotherapy Bern,

Bern, Switzerland
M. Arfan Ikram · Departments of Epidemiology, Radiology, Neurology, Erasmus
University Medical Center Rotterdam, Rotterdam, The Netherlands
Giuseppe Astarita · Department of Biochemistry and Molecular and Cellular Biology,
Georgetown University, Washington, DC, USA
Filippo Baldacci · AXA Research Fund & UPMC Chair, Paris, France; Sorbonne
Université, AP-HP, GRC n° 21, Alzheimer Precision Medicine (APM), Hôpital de la
Pitié-Salpêtrière, Boulevard de l’hôpital, Paris, France; Institut du Cerveau et de la
Moelle Épinière (ICM), INSERM U 1127, CNRS UMR 7225, Boulevard de l’hôpital,
Paris, France; Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A),
Département de Neurologie, Hôpital de la Pitié-Salpêtrière, AP-HP, Boulevard de
l’hôpital, Paris, France; Department of Clinical and Experimental Medicine,
University of Pisa, Pisa, Italy
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, Polyclinic San Martino Hospital, Genova,
Italy; Department of Health Sciences, University of Genoa, Genoa, Italy
Bart N.M. van Berckel · Department of Radiology and Nuclear Medicine, Neuroscience
Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
Rosaria Ornella Bua · CNR, Istituto per i Polimeri, Compositi e i Biomateriali
Catania, Catania, Italy
Marc Aurel Busche · Department of Neurology, Massachusetts General Hospital,
Harvard Medical School, MassGeneral Institute for Neurodegenerative Disease,
Charlestown, MA, USA; Department of Psychiatry and Psychotherapy, Technical
University of Munich, Munich, Germany; Munich Cluster for Systems Neurology, Munich,
Germany
Ana L. Cardoso · Center for Neuroscience and Cell Biology, University of Coimbra,
Coimbra, Portugal
Juan I. Castrillo · Genetadi Biotech S.L. Parque Tecnológico de Bizkaia, Derio, Bizkaia,
Spain
Roberto Ceravolo · Department of Clinical and Experimental Medicine, University of
Pisa, Pisa, Italy
Johannes Denk · Department of Psychiatry and Psychotherapy, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
Cornelius K. Donat · Division of Brain Sciences, Imperial College London,
Hammersmith Hospital, London, UK
Juergen Dukart · F. Hoffmann-La Roche, Pharma Research Early Development, Roche
Innovation Centre Basel, Basel, Switzerland; Roche Pharmaceutical Research and Early
Development, Neuroscience, Ophthalmology and Rare Diseases, Discovery and
Translational Area, Roche Innovation Center Basel, Basel, Switzerland

xi
xii Contributors

Paul Edison · Division of Brain Sciences, Imperial College London, Hammersmith

Hospital, London, UK
Carles Falcon · Barcelonaβeta Brain Research Center, Pasqual Maragall Foundation,
Barcelona, Spain; Centro de Investigación Biomédica en Red de Bioingeniería,
Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain
Ángeles Fernández-Recamales · Department of Chemistry, Faculty of Experimental
Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence
CeiA3, University of Huelva, Huelva, Spain
Giovanni B. Frisoni · Faculty of Medicine, Nuclear Medicine Department, Geneva
University Medical Center, University of Geneva, Geneva, Switzerland; Department of
Internal Medicine, Geneva University Hospitals, Geneva, Switzerland
Valentina Garibotto · Division of Nuclear Medicine and Molecular Imaging, Geneva
University Hospitals, Geneva, Switzerland; Faculty of Medicine, Nuclear Medicine
Department, Geneva University Medical Center, University of Geneva, Geneva,
Switzerland
Domenico Garozzo · CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania,
Catania, Italy
Steve Gentleman · Division of Brain Sciences, Department of Medicine, Imperial College
London, London, UK
Juan Domingo Gispert · Barcelonaβeta Brain Research Center, Pasqual Maragall
Foundation, Barcelona, Spain; Centro de Investigación Biomédica en Red de
Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, Spain
Raúl González-Domínguez · Department of Chemistry, Faculty of Experimental
Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence
CeiA3, University of Huelva, Huelva, Spain
Álvaro González-Domínguez · Department of Chemistry, Faculty of Experimental
Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence
CeiA3, University of Huelva, Huelva, Spain
Joana R. Guedes · Center for Neuroscience and Cell Biology, University of Coimbra,
Coimbra, Portugal
Sara Hägg · Department of Medical Epidemiology and Biostatistics, Karolinska Institutet,
Stockholm, Sweden
Harald Hampel · AXA Research Fund & UPMC Chair, Paris, France; Sorbonne
Université, AP-HP, GRC n° 21, Alzheimer Precision Medicine (APM), Hôpital de la
Pitié-Salpêtrière, Boulevard de l’hôpital, Paris, France; Institut du Cerveau et de la
Moelle Épinière (ICM), INSERM U 1127, CNRS UMR 7225, Boulevard de l’hôpital,
Paris, France; Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A),
Département de Neurologie, Hôpital de la Pitié-Salpêtrière, AP-HP, Boulevard de
l’hôpital, Paris, France
Stefan Holiga · F. Hoffmann-La Roche, pharma Research Early Development, Roche
Innovation Centre Basel, Basel, Switzerland
Holger Jahn · Department of Psychiatry and Psychotherapy, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
Hans W. Klafki · Department of Psychiatry and Psychotherapy, University Medical Center
Goettingen, Georg-August-University, Goettingen, Germany
 Contributors xiii

Stefan Klöppel · University Hospital of Old Age Psychiatry and Psychotherapy Bern, Bern,
Switzerland
Adriaan A. Lammertsma · Department of Radiology and Nuclear Medicine, Neuroscience
Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
Rui Li · Alibaba, Hangzhou, Zhejiang, China
Simone Lista · AXA Research Fund & UPMC Chair, Paris, France; Sorbonne Université,
AP-HP, GRC n° 21, Alzheimer Precision Medicine (APM), Hôpital de la Pitié-
Salpêtrière, Boulevard de l’hôpital, Paris, France; Institut du Cerveau et de la Moelle
Épinière (ICM), INSERM U 1127, CNRS UMR 7225, Boulevard de l’hôpital, Paris,
France; Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A), Département de
Neurologie, Hôpital de la Pitié-Salpêtrière, AP-HP, Boulevard de l’hôpital, Paris, France
Alan King Lun Liu · Division of Brain Sciences, Department of Medicine, Imperial
College London, London, UK
Ismini C. Mainta · Division of Nuclear Medicine and Molecular Imaging, Geneva
University Hospitals, Geneva, Switzerland; Faculty of Medicine, Nuclear Medicine
Department, Geneva University Medical Center, University of Geneva, Geneva,
Switzerland
Angela Messina · CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania,
Catania, Italy
Nazanin Mirzaei · Division of Brain Sciences, Imperial College London, Hammersmith
Hospital, London, UK
José Luis Molinuevo · Barcelonaβeta Brain Research Center, Pasqual Maragall
Foundation, Barcelona, Spain; CIBER Fragilidad y Envejecimiento Saludable
(CIBERFES), Madrid, Spain
Silvia Morbelli · Nuclear Medicine Unit, Polyclinic San Martino Hospital, Genova,
Italy; Department of Health Sciences, University of Genoa, Genoa, Italy
Agneta Nordberg · Division of Translational Alzheimer Neurobiology, Center for
Alzheimer Research, Department of Neurobiology, Care Sciences and Society, Karolinska
Institutet, Stockholm, Sweden; Department of Geriatric Medicine, Karolinska University
Hospital Huddinge, Stockholm, Sweden
Sid E. O’Bryant · Institute for Healthy Aging, University of North Texas Health Science
Center, Fort Worth, TX, USA
Grégory Operto · Barcelonaβeta Brain Research Center, Pasqual Maragall Foundation,
Barcelona, Spain
Marion M. Ortner · Department of Psychiatry and Psychotherapy, Klinikum rechts der
Isar, Technische Universität München, Munich, Germany
Rik Ossenkoppele · Department of Neurology and Alzheimer Center, Neuroscience
Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands;
Department of Radiology and Nuclear Medicine, Neuroscience Campus Amsterdam,
VU University Medical Center, Amsterdam, The Netherlands
Giuseppe Paglia · EURAC Institute for Biomedicine, Institute for Biomedicine, European
Academy of Bolzano/Bozen, Bolzano, Italy
Angelo Palmigiano · CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania,
Catania, Italy
xiv Contributors

Robert Perneczky · Department of Psychiatry and Psychotherapy,

Ludwig-Maximilians-­Universität München, Munich, Germany; German Center for
Neurodegenerative Diseases (DZNE) Munich, Munich, Germany; Neuroepidemiology
and Ageing Research Unit, School of Public Health, The Imperial College of Science,
Technology and Medicine, London, UK; West London Mental Health NHS Trust,
London, UK
Geraint Price · Faculty of Medicine, Neuroepidemiology and Ageing Research Unit,
School of Public Health, The Imperial College of Science Technology and Medicine,
London, UK
Craig W. Ritchie · Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
Elena Rodriguez-Vieitez · Division of Translational Alzheimer Neurobiology, Center
for Alzheimer Research, Department of Neurobiology, Care Sciences and Society,
Karolinska Institutet, Stockholm, Sweden
Magdalena Sastre · Division of Brain Sciences, Imperial College London, Hammersmith
Hospital, London, UK
Ana Sayago · Department of Chemistry, Faculty of Experimental Sciences, University of
Huelva, Huelva, Spain; International Campus of Excellence CeiA3, University of
Huelva, Huelva, Spain
Matteo Stocchero · Department of Women’s and Children’s Health, University of
Padova, Padova, Italy
Luisa Sturiale · CNR, Istituto per i Polimeri, Compositi e i Biomateriali Catania,
Catania, Italy
Petros Takousis · Neuroepidemiology and Ageing Research Unit, School of Public Health,
Imperial College, London, UK
Sac-Pharm Tang · Imanova Limited, London, UK
Tessa Timmers · Department of Neurology and Alzheimer Center, Neuroscience Campus
Amsterdam, VU University Medical Center, Amsterdam, The Netherlands; Department
of Radiology and Nuclear Medicine, Neuroscience Campus Amsterdam, VU University
Medical Center, Amsterdam, The Netherlands
Nicola Toschi · Department of Biomedicine and Prevention, University of Rome
“Tor Vergata”, Rome, Italy; Department of Radiology “Athinoula A. Martinos”, Center
for Biomedical Imaging, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
Sara Trombella · Faculty of Medicine, Nuclear Medicine Department, Geneva University
Medical Center, University of Geneva, Geneva, Switzerland
Paul G. Unschuld · Institute for Regenerative Medicine and Hospital for Psychogeriatric
Medicine, University of Zurich, Zurich, Switzerland
Maria I. Vargas · Faculty of Medicine, Nuclear Medicine Department, Geneva University
Medical Center, University of Geneva, Geneva, Switzerland; Division of Neuroradiology,
Geneva University Hospitals, Geneva, Switzerland
Jonathan Vogelgsang · Department of Psychiatry and Psychotherapy, University Medical
Center Goettingen, Georg-August-University, Goettingen, Germany
Jens Wiltfang · Department of Psychiatry and Psychotherapy, University Medical Center
Goettingen, Georg-August-University, Goettingen, Germany; German Center for
Neurodegenerative Diseases (DZNE), Goettingen, Germany; Medical Sciences
Department, iBiMED, University of Aveiro, Aveiro, Portugal
 Contributors xv

Frank J. Wolters · Department of Epidemiology, Erasmus Medical Centre, Rotterdam,

The Netherlands
Randall L. Woltjer · Department of Neurology, Oregon Health Science University and
Portland VA Medical Center, Portland, OR, USA
Paul L. Wood · Metabolomics Unit, College of Veterinary Medicine, Lincoln Memorial
University, Harrogate, TN, USA
Mario Zappia · Section of Neurosciences, Department of GF Ingrassia, University of
Catania, Catania, Italy
Yiqiang Zhan · Department of Medical Epidemiology and Biostatistics, Karolinska
Institutet, Stockholm, Sweden
 Part I

The Need for Alzheimer’s Disease Biomarkers

 Chapter 1

Epidemiology of Dementia: The Burden on Society,

the Challenges for Research
Frank J. Wolters and M. Arfan Ikram

Abstract
Dementia is among the leading causes of death and disability. Due to the ageing population, its prevalence
is expected to nearly triple worldwide by 2050, urging the development of preventive and curative inter-
ventions. Various modifiable risk factors have been identified in community-based cohort studies, but
insight into the underlying pathophysiological mechanisms is lacking. Clinical trials have thus far failed in
the development of disease-modifying therapy in patients with dementia, thereby triggering a shift of focus
toward the presymptomatic phase of disease. The extensive preclinical disease course of Alzheimer’s disease
warrants reliable, easily obtainable biomarkers to aid in timely application of preventive strategies, selecting
participants for neuroprotective trials, and disease monitoring in trials and clinical practice. Biomarker and
drug discovery may yield the fruits from technology-driven developments in the field of genomics, epi-
genetics, metabolomics, and brain imaging. In that context, bridging the gap between translational and
population research may well prove a giant leap toward development of successful preventive and curative
interventions against dementia.

Key words Dementia, Alzheimer’s disease, Epidemiology, Population-based, Omics, Prevention,

Genetics, Imaging

1 Introduction: The Burden of Disease

At present, 48 million people worldwide are suffering from demen-

tia, and this number is projected to increase to 131 million by 2050
[1]. The enormous burden this poses on patients, their caregivers,
and society as a whole is only in part reflected by the estimated
$818 billion per annum needed for dementia care worldwide. Costs
for dementia already exceed those for any other disease [2, 3], and
as the number of patients with dementia grows the annual health
expenditure is projected to reach one trillion dollars as soon as 2018
[1]. Every year, nearly ten million people develop dementia, with a
median age at diagnosis of around 80 years (Fig. 1). Similar to other
disorders with steeply increasing prevalence at higher ages, the bur-
den of dementia arises predominantly from disability rather than
mortality. In industrialized countries, 80% of those in care homes

Robert Perneczky (ed.), Biomarkers for Alzheimer’s Disease Drug Development, Methods in Molecular Biology, vol. 1750,
https://doi.org/10.1007/978-1-4939-7704-8_1, © Springer Science+Business Media, LLC 2018

3
4 Frank J. Wolters and M. Arfan Ikram

Fig. 1 Age-specific incidence of dementia per geographic area. Data source:

World Alzheimer Report, 2015

have dementia or significant cognitive problems, and despite good

care less than half experience a good quality of life [4]. Prior to
institutionalization, caregivers alleviate much of the burden of dis-
ease, but often at the cost of their own well-being [5].
Meanwhile, health expenditure continues to fall short in antic-
ipating long-term care costs. A 2012 study in the UK found that
every £10 of health and social care costs attributable to dementia
are counterbalanced by only £0.08 for dementia research [6]. This
is particularly striking, as investments in long-term prevention are
projected to result in cost-effective gains in quality-adjusted life
years in the population [7]. Driven by the projections of the bur-
den on public health, governments have only recently designated
dementia as a major public health threat, and started to prioritize
research in their federal budgets accordingly. This will undoubt-
edly spark advances in research and clinical care, but time is not on
our side. Subclinical alterations in brain health are thought to
occur decades before diagnosis of dementia [8], and the social and
economic burden of dementia will thus increase enormously, unless
curative and preventive measures can be rapidly established.
As the burden of dementia grows across the globe, the largest
increase will occur in low- and middle-income countries, such that
by 2050 roughly two-thirds of people with dementia will live in
these regions (Fig. 2). Yet, only a minority of patients receive
(extensive) investigation or a formal diagnosis, particularly in set-
tings poor in resources [9, 10]. Together with differences in patient
care, the low rate of diagnoses accounts for a stark contrast in
global healthcare expenditure, where over 90% of current demen-
tia care costs are incurred in high-income countries [11]. It shows
above all that wide availability and accessibility of diagnostics, pre-
ventive measures, and feature disease-modifying agents will be vital
to control the global dementia epidemic.
 Epidemiology of Dementia: The Burden on Society, the Challenges for Research 5

Fig. 2 The numbers of people living with dementia in millions (black box) per geographic area in 2015 (light
grey), with projections for 2030 (dark blue) and 2050 (dark grey). Corresponding percentage increase com-
pared to 2015 is depicted in red labels. Data source: World Alzheimer Report, 2015

In this chapter, we shall provide a brief overview of the current

state of affairs in preventive and curative medicine, and highlight
the main challenges and opportunities encountered in etiological
research and drug development.

2 Preventive and Curative Efforts: Small Successes in the Light of Failure

The urgency for new drugs is reflected in a steep increase in the num-
ber of trials registered for drugs against Alzheimer’s disease. Over the
past 20 years, hundreds of registered trials set out to determine the
efficacy of potential drugs for dementia, but this has resulted in only
four drugs offering limited symptom relief (Table 1), while thus far no
disease-modifying therapies are available. The cost of these trials cul-
minates to billions of euros. Of 244 compounds that were assessed in
413 trials in the past decade, none have made it to the market since
the approval of memantine for symptom relief in 2003 [12]. Of every
100 compounds entering phase 1 trials for Alzheimer’s disease, 28%
make it to phase 2, in which 8% succeed [12]. For comparison, success
rates of phase 1 and phase 2 trials for infectious disease are 70% and
43%, respectively, while for cardiovascular disease these percentages
are 59% and 24% [13]. As of 2016, another large phase 3 Alzheimer
trial, assessing the efficacy of amyloid antibody solanezumab, has
joined the long list of unsuccessful intervention for (mild) Alzheimer’s
disease, along with the 5-HT6 antagonist idalopirdine that was aimed
6 Frank J. Wolters and M. Arfan Ikram

Table 1
Overview of currently approved drugs for Alzheimer’s disease

Generic drug (brand name) Approval for disease stage Year of approval Indication
Donepezil (Aricept) All stages 1996 Symptom relief
Rivastigmine (Exelon) All stages 2000 Symptom relief
Galantamine (Razadyne) Mild-moderate 2001 Symptom relief
Memantine (Namenda) Moderate-severe 2003 Symptom relief

at symptom relief. Nevertheless, over 20 phase 3 trials are currently

ongoing, of which the majority is directed against amyloid, either by
antibodies, vaccines, or inhibiting beta-secretase 1 (BACE).
With regard to prevention, various modifiable risk factors have
been identified in community-based cohort studies over the past
20–30 years. Midlife hypertension, obesity, smoking, diabetes mel-
litus, low educational attainment, and physical activity are consis-
tently associated with an increased risk of disease, and account for
about 25% of all cases of dementia [14, 15]. As these risk factors are
largely shared with other (cardiovascular) diseases, improvements in
cardiovascular risk management may have had positive effects on
dementia risk over the past decades also. In fact, multiple reports
now suggest a decline in the incidence of dementia in industrialized
countries by about 20% per decade since the 1970s [16–18].
However, the precise causes of this trend have not been accounted
for, data from low- and middle-income countries are not available,
and even if the current trend continues it will not suffice to offset
the effects of a rapidly ageing population on the number of patients
with dementia. In addition, trials assessing the efficacy of specific
preventive interventions for lowering the risk of dementia have thus
far been largely unsuccessful [19]. Following the failures of these
dementia prevention trials in the late 1990s and early 2000s, subse-
quent trials have determined the effect of interventions on cogni-
tion as a more sensitive outcome measure than dementia, with more
positive results in particular for trials assessing the efficacy of physi-
cal activity and multi-domain interventions [19]. For example, the
Scandinavian FINGER trial (a multi-domain intervention involving
diet, exercise, vascular risk monitoring, and cognitive training) has
shown benefit on cognitive function [20], and participants con-
tinue to be surveyed for the incidence of dementia. Despite gener-
ally modest effect sizes at the individual level, these preventive
efforts could greatly reduce the burden of disease at the population
level. If preventive efforts succeed in delaying the onset of dementia
by merely 5 years, this could reduce the incidence of dementia by as
much as 50% over the next decades [3]. Yet again, in an unselected
population of elderly Dutch people (the preDIVA trial), multi-
 Epidemiology of Dementia: The Burden on Society, the Challenges for Research 7

domain vascular care intervention did not significantly lower

dementia incidence, and the French Multi-domain Alzheimer
Prevention Trial (MAPT—testing similar interventions plus
omega-3 supplements) found no significant benefit on cognitive
decline over a 3-year period [21, 22]. These inconsistencies across
trials employing closely aligned interventions emphasize that much
work remains to be done to understand the specific pathways under-
lying successes and failures of these preventive trials.
Summarizing the small successes and many disappointing past
attempts to halt dementia at any stage of disease, the question rises
why so many efforts have thus far failed. What are the difficulties
medical research faces in developing treatments against dementia
and specifically Alzheimer’s disease, while successful battles have
been fought against many other similarly common and age-related
diseases? The next section will therefore focus on the challenges
and opportunities in striving to improve the process and outcomes
of drug discovery.

3 Challenges in Clinical Trials

Part of the lagging behind of dementia treatments, compared to

improvements in treatment of cardiovascular disease and cancer,
could be linked to lack of research funding [6]. Every £10 of health
and social care costs attributable to dementia are counterbalanced by
£0.08 for dementia research in the UK, compared with £1.08 for
cancer and £0.65 for heart disease [6]. This 8–14-fold difference in
research investment may reflect the differences in disease perception
and heterogeneous nature of cancer as a disease entity, as well as less
direct effects of preventive measures for dementia, compared to
acute vascular events. Today’s intervention might prevent tomor-
row’s myocardial infarction, whereas the long preclinical phase of
dementia precludes return of investment within several years from
initiation of preventive measures. Nevertheless, a Swedish-Finnish
study that modeled cost and utilities of such investments suggests
that long-term prevention can in fact result in a cost-effective gain in
quality-adjusted life years in the population [7], emphasizing the
necessity of prioritizing dementia research funding.
Other challenges in dementia research pertain to the disease
course and impairment of dementia itself, which make it different
from other diseases that medicine has successfully engaged. These
include high attrition rates in trials, insensitive or inaccurate out-
come assessments [23], and—perhaps foremost—a long and vari-
able presymptomatic disease phase that has repeatedly been put
forward as an explanation for why so many clinical trials have failed.
By the time of diagnosis, the brain of patients with dementia shows
high degrees of degeneration that vary greatly across patients and
are unlikely to be ameliorated by treatment. Moreover, markers for
8 Frank J. Wolters and M. Arfan Ikram

risk of neurodegeneration may arise years before diagnosis of

dementia [8], and intervention at the time of disease may conse-
quently be too late. Trials have therefore moved from including
patients in any stage of disease to those with mild dementia only,
and, driven by yet again disappointing results, ongoing trials now
focus on presymptomatic individuals. As such, the Alzheimer
Prevention Initiative’s A4 trial currently enrols cognitively healthy
individuals in the USA, Canada, and Australia for 39 months of
treatment with intravenous solanezumab or placebo (ClinicalTrials.
gov Identifier NCT02008357). However, when targeting earlier,
presymptomatic stages of the disease, enrichment of people at
highest risk of developing the disease is vital for the trial to be suf-
ficiently powered. Strategies for enrichment include targeting indi-
viduals who are at increased genetic risk, and several studies now
focus exclusively on genetically high-risk individuals, such as cren-
ezumab for preclinical PSEN1 mutation carriers of Colombian kin-
dred (ClinicalTrials.gov Identifier NCT01998841), and the
Generation Study for individuals aged 60–75 years who carry two
copies of the apolipoprotein E epsilon 4 allele (ClinicalTrials.gov
Identifier NCT02565511). Although this might in part circum-
vent the problem of eleventh-hour interventions, it could render
results less applicable to the large population at risk due to the
strong selection on relatively rare genetic variants. In addition,
time to event in these preventive trials remains prolonged, making
them costly and unable to provide rapid answers to urgent ques-
tions. The feasibility of future trials will therefore depend on the
ability to either recruit (representative) individuals at risk of devel-
oping disease during the trial period or develop reliable biomarkers
that can serve as surrogate endpoints for preventive trials.

4 Biomarkers and Treatment Targets: The Search Continues

Risk stratification of individuals for a certain disease generally starts

with easily obtainable demographics and details about personal history.
This approach has been highly effective in predicting for instance long-
term risk of cardiovascular disease in the community [24], short-term
risk of stroke in patients with transient ischemic attack [25], or odds of
having pulmonary embolism [26]. However, for dementia, such sim-
ple models using demographics and personal history have thus far
proven of limited predictive value in the general population [27]. Of
potential predictors, educational attainment, a positive family history,
and subjective memory complaints appear most informative, but their
incremental value over merely age and gender is limited. In the setting
of memory clinics, additional, more advanced tools are generally avail-
able for diagnosis. Biomarker levels of (phosphorylated) tau and
β-amyloid in cerebrospinal fluid are consistently associated with a diag-
nosis of dementia or mild cognitive impairment due to AD [28], but
Epidemiology of Dementia: The Burden on Society, the Challenges for Research 9

this does not automatically translate into improved diagnostic accuracy

above and beyond clinical observations [29]. Similarly, prediction of
conversion from mild cognitive impairment to dementia might be
improved by advanced (PET) imaging, but currently available data are
deemed insufficient to recommend these expensive investigations for
routine use in clinical setting [30]. Regardless, the stage at which
patients present at memory clinics may be too late in the disease pro-
cess to initiate a prevention trial, because those patients are symptom-
atic per definition. Standardization of any marker will be vital to prevent
the large inter-site variability in measurements that currently hampers
definition of prediction rules across clinical sites [31]. Moreover, diag-
nostic value cannot simply be inferred from association studies, and
diagnostic accuracy strongly depends on the screening strategy and
setting [32]. For example a biomarker with high sensitivity and low
specificity can be very useful as a first screening phase in primary care,
while it will likely not be very helpful in clinical setting. Similarly, a
biomarker that is more reflective of late-state disease can be very useful
in the monitoring disease course, whereas it will be of limited value in
risk stratification in asymptomatic individuals. Screening strategy and
setting will also largely affect cost-effectiveness, which should be taken
into account in the development of any biomarker. While investiga-
tions in memory clinics generally include state-of-the-art brain imaging
and cerebrospinal fluid samples, screening strategies in the general
population, by contrast, should rely on more readily attainable mea-
sures from for instance blood, urine, or saliva. In any case, high intra-
individual reliability is essential for disease monitoring, and for these
tests to be applied in clinical trials. Given the multifactorial nature of
dementia, incremental value of biomarkers for the prediction of demen-
tia, or even Alzheimer’s disease, is likely to be found only by the com-
bination of multiple entities, rather than individual markers. Lastly, any
candidate (set of) biomarker(s) will require validation in independent
samples to confirm and establish their predictive value.
Similar to biomarker development, identification and validation
of potential treatment targets is an important combined task for
population, clinical, and translational medicine. As highlighted
above, many modifiable risk factors for dementia have been identi-
fied, but lack of understanding of their biological underpinning has
long hampered drug development. Amyloid has been the major tar-
get for drug interventions, but the string of negative trial results has
led to fierce criticism in the field that amyloid might be a down-
stream result of changes leading to Alzheimer’s disease [33]. The
challenge for alternative theories is to explain why amyloid accumu-
lates in the brain if it is not causing neurodegeneration while propos-
ing reasonable alternative mechanisms for neurodegeneration in
Alzheimer’s disease. Alternatives that have been put forward include
(but are not limited to) mechanisms related to microvascular func-
tion and neurovascular coupling, inflammation, angiogenesis, and
lipid or iron metabolism. While these ideas frequently originate from
10 Frank J. Wolters and M. Arfan Ikram

observations in clinical or population studies, identification of the

biological underpinning of these mechanisms relies on translational
medicine. Yet, also at the preclinical stage, models in the past decades
have been mostly aimed at overexpression of amyloid or tau, thereby
overlooking a range of important determinants of dementia in pop-
ulation studies. Animal models are generally built on rare familial
types of Alzheimer’s disease, and may therefore not reflect the
pathology of the common late onset, in which for example vascular
disease and diabetes are prominent features. Although therapeutic
targets may produce desired effects in transgenic mouse models,
current animal studies have not been able to aid discovery of
Alzheimer therapy, and have consequently faced criticism about
questionable validity to reflect human disease [34]. At the same
time, limitations to in vivo imaging of amyloid and tau have long
precluded etiological research of amyloid and tau in humans, and
still the required tracers at a price of around €1500 per acquired
brain scan are generally beyond the scope of (large) population stud-
ies. Aside efforts to incorporate these types of research on a popula-
tion level, the matching of findings from preclinical models to
observations in clinical and population studies is vital to unravel
pathophysiological mechanisms. It is this translation of group differ-
ences to the individual variation that is the biological underpinning
of the disease. Understanding the interindividual differences in
pathology and clinical presentation is the step forward to developing
and electing proper treatments for very individual (future) patient.

5 Big Data and Precision Medicine

“Doctors have always recognized that every patient is unique, and

doctors have always tried to tailor their treatments as best they can
to individuals. You can match a blood transfusion to a blood type –
that was an important discovery. What if matching a cancer cure to
our genetic code was just as easy, just as standard? What if figuring
out the right dose of medicine was as simple as taking our tempera-
ture?” With these words US President Barack Obama kicked of the
Precision Medicine Initiative in early 2015. Aside short-term goals
for personalizing cancer care, the long-term ambitions of the pro-
gram focus on all areas of health, including neurological disease.
And given the multifactorial and complex pathophysiological nature
of dementia, a personalized approach in the development of inter-
ventions may benefit dementia as much as cancer therapy.
Over the past decades, the quest for unraveling the etiology of
common diseases has led to instigation of multiple large studies in
the general population. These studies’ sizes range from 10,000 to
15,000 participants for community-based cohorts like the
Framingham Heart Study and the Rotterdam Study [35, 36] to an
intended 500,000 subjects examined for the UK Biobank and
Epidemiology of Dementia: The Burden on Society, the Challenges for Research 11

1,000,000 for the US Precision Medicine Initiative. Already, these

types of studies have shown to be valuable in identifying genetic risk
alleles and imaging markers predisposing for dementia. Since the
first genome-wide association studies in the early 2000s, increasingly
large quantities of genetic data have revolutionized the way afore-
mentioned research questions can be addressed. Together with epi-
genetics and proteomics, genomics and neuroimaging have yielded
previously unimagined opportunities to obtain rich information
about the influence of an individual’s biological sketch and the envi-
ronment on virtually any phenotype. But big data does not just
allude to large quantity. More than sheer numbers, it is the opportu-
nities presented by the combination of aforementioned modalities
that allows hypothesis-free research of the -omics data. Also, the
linkage of these cohort data to potential drug targets holds promise
for drug development, as is increasingly acknowledged among
researchers, funding agencies (e.g., the U.S. National Institutes of
Health BD2K project), pharmaceutical industry, and consultant
companies alike. By no means does such an approach substitute
detailed study about disease pathways and singular treatment tar-
gets. On the contrary, sometimes this may lead directly to develop-
ment of new drugs, such as for PCSK9 inhibitors [37–39], while
arguably more often (genetic) data could be applied to increase suc-
cess rate of drug development by selecting genetically supported
drug targets from potential candidates [40, 41]. Also for Alzheimer’s
disease, genomics approaches have recently shown interest in identi-
fying disease-relevant tissues, variants, and regulators [42].
At the same time, the diverse and often complex methods pose
new challenges of their own. Data harmonization, storage, access
and sharing, and analysis are some of the main hurdles to large-
scale analyses and collaborations that can be taken only by interna-
tional standardization, improvements in infrastructure for
collaboration, and advanced technology for data acquisition, stor-
age, and analyses. In addition, unlike genetic studies, application of
proteomics and epigenetics is vulnerable to selection bias, con-
founding, and other biases that traditionally hamper research [43].
For example, while the UK Biobank achieves its intended 10%
response rate [44], this is far lower than the response rates of about
60–80% in population-based cohort studies. Sufficient response
rates are important to avoid selection bias and guarantee generaliz-
ability of findings to a larger population. Similarly, other funda-
mentals of epidemiology need to be upheld, such as avoidance of
attrition, accuracy of outcome assessment, and accounting for
potential biases. These methodological challenges cannot be coun-
terbalanced by quantity, and warrant careful study design and
choice of appropriate methods for data analysis. If, and only if,
these hurdles can be overcome, the combination of powerful com-
putational methods and large-scale -omics data with biological
information about the disease from translational models has the
12 Frank J. Wolters and M. Arfan Ikram

potential to boost drug discovery and biomarker development. It

may well be the bridge between observations in large-scale popula-
tion data and (molecular) biology of a disease that can transform
the -omics era into that of precision medicine, with Alzheimer’s
disease as one of its primary targets.

6 Concluding Remarks

The increasingly heavy burden of dementia on society poses one of

the greatest challenges for modern medicine. Despite our best
efforts, to date no disease-modifying treatments have evolved from
identification of various risk factors in (clinical) populations. It is
therefore not simply an option, but a necessity to combine insights
and approaches in laboratory, clinics, and population setting
toward the development of biomarkers and treatments for demen-
tia. Science presents us with an increasing number of research tools
to this aim. In the coming years, we are to use these tools inven-
tively and in compliance with fundamental epidemiological princi-
ples, if we are to fulfil the potential for the development of
preventive and curative interventions that these approaches hold.

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 Chapter 2

Population-Based Approaches to Alzheimer’s

Disease Prevention
Robert Perneczky

Abstract
Progress in prevention and treatment of Alzheimer’s disease (AD) and dementia is hampered by the
restricted understanding of the biological and environmental causes underlying pathophysiology. It is
widely accepted that certain genetic factors are associated with AD and a number of lifestyle and other
environmental characteristics have also been linked to dementia risk. However, interactions between genes
and the environment are not yet well understood, and coordinated global action is required to utilize exist-
ing cohorts and other resources effectively and efficiently to identify new avenues for dementia prevention.
This chapter provides a brief summary of current research on risk and protective factors and opportunities
and challenges in relation to population-based approaches are discussed.

Key words Alzheimer’s disease, Dementia, Drug development, Population-based, Cohort studies,
Lifestyle, Environment, Genetics

1 Introduction

The traditional linear model of pharmaceutical research and devel-

opment has become outdated and attrition rates have almost been
100% over the last two decades in the field of Alzheimer’s disease
(AD). At the same time the prevalence of AD and its healthcare and
socioeconomic impact are exploding worldwide and drug develop-
ment success rates need to be improved urgently. Successful drug
discovery and development in other fields of medicine, such as can-
cer and infectious diseases, have shown that translational models
linking epidemiological cohort and genetic data to potential drug
targets and study end points can lead to success [1]. Technology
platforms such as neuroimaging, -omics, and biomarkers have been
shown to be powerful tools in this translational process. A signifi-
cant cultural change has to be initiated to discover and develop
novel disease-modifying treatments in the dementia field until
2025, as postulated at the 2013 G8 Dementia Summit [2].

Robert Perneczky (ed.), Biomarkers for Alzheimer’s Disease Drug Development, Methods in Molecular Biology, vol. 1750,
https://doi.org/10.1007/978-1-4939-7704-8_2, © Springer Science+Business Media, LLC 2018

15
16 Robert Perneczky

Increased life expectancy is a major achievement of modern

society, which however comes at the price of a growing prevalence
of age-associated disorders with severe consequences for the global
healthcare systems. Within the next 30–40 years, the number of
people affected by late-onset neurodegenerative disorders such as
AD and Parkinson’s disease will globally be three to four times
higher than today [3]. The individual risk for these disorders is
highly variable and the development of effective preventive and
therapeutic strategies is hindered by the still incomplete knowl-
edge about factors influencing the vulnerability to neurodegenera-
tion. There is emerging academic consensus that an individual’s
susceptibility to AD is determined by both biological and environ-
mental factors, which has important implications for the develop-
ment of effective treatment options [4]. Even though AD has a
significant heritability, monogenic familial forms caused by clearly
pathogenic mutations in certain genes are extremely rare; the vast
majority of AD cases are likely to be caused by an interplay between
common genetic variants and other factors. The biological mecha-
nisms by which genes influence the individual vulnerability to AD
are still largely unknown, but recently identified risk genes point to
the involvement of certain biological pathways such as immune
response and lipid processing [5].

1.1 A Multicausal As with most complex diseases, an individual’s vulnerability to AD is

Model of Alzheimer’s not exclusively defined by genetic factors, but also by nongenetic
Disease determinants such as the environment and epigenetic mechanisms.
Genetic susceptibility is innate but risk associated with certain envi-
ronmental factors may be modifiable and changes in lifestyle may
offer a chance to prevent or treat AD. Many of the known environ-
mental risk factors are related to obesity, diabetes mellitus, and vas-
cular disease, all of which can potentially be modified; the impact of
protective environmental factors such as Mediterranean diet and
cognitive and physical activity can also be actively strengthened. An
individual’s personality and inherent beliefs affect their behavior and
therefore have an important impact on their lifestyle choices and the
associated dementia risk [6]. Furthermore, factors outside an indi-
vidual’s control also influence their chronic disease risk, such as the
current healthcare system, air pollution, and wider economy (Fig. 1).

2 Epidemiological Approaches to Alzheimer’s Disease Research

While the link between AD risk and certain environmental and

lifestyle factors seems well established, the biological mechanisms
underlying these associations are not yet well understood. The
application of high-throughput “omics” technologies to prospec-
tive cohort studies with stored biospecimens offers a unique oppor-
tunity to phenotype individuals at the molecular level. Cutting-edge
 Population-Based Approaches to Alzheimer’s Disease Prevention 17

Fig. 1 A multicausal model of Alzheimer’s disease

technologies such as epigenomics and metabolomics can be applied

to pre-diagnostic biosamples for the detection of early disease
markers. Etiological research combining epidemiologic and molec-
ular data, with a focus on gene-environment interactions, can pave
the way to the identification of etiopathogenetic pathways via sys-
tems biology approaches.

2.1 The Value Worldwide, several large-scale prospective cohort studies on ageing
of Established Global and neurodegenerative disease have been established in the past
Infrastructures decades (for a comprehensive listing of European cohorts see [7]);
the studies have collected detailed dietary, lifestyle, and clinical
information, and in some cases biological samples and neuroimag-
ing data, from very large numbers of participants at various points
in life. The continued follow-up of these existing infrastructure and
a collection of repeated and additional detailed information from
these ageing populations is a highly efficient strategy to address the
existing knowledge gaps. However, data analysis has so far mostly
been restricted to individual cohorts due to differences in assess-
ment procedures and applied methods between countries.
Concerted action is therefore urgently required to facilitate the
comprehensive analysis of large datasets and to increase knowledge
transfer between experts in different countries. It is crucial to
achieve standardized data analysis across global ageing and neuro-
degeneration cohorts and effective networking activities between
researchers in the field. The main aims of such networking activities
Another random document with
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 — A voi velikulta, tämä elämä on heittänyt minua kahtapuolta niin
huimasti, että nyt jo käteni melkein ristiän, kun lopetti. Silloin pieksi
köyhyydellä, kun teitä ruplilla mairitteli, sitten taas löi minulle
syötävät ja rahat eteen, kun teille jo irvisti paljasta ientä, ja sitten
taas pudottaa pumautti minut nälkäsuohon, kun teillä oli jo koura
kuokan varressa ja jyvässä pitkä itu. Kiroan, kiroan minäkin jo
menneen ajan. Leikkimään vain opetti, ja sen opinkin. Mattotuolissa
makautti kesäpäivän äähkeessä ja laiskaksi sulatti. Nyt on taas
työnteko kuin haarahongan seisomista. Hyvä oli Rasutovi, ylen hyvä,
häntä en moiti, hän minulle näytti sen, minkä muut näyttivät
aikoinaan teille: kuinka rupla on soma ja rakas hypitellä kämmenellä
ja kuinka toiselta maistuu toisen vehnänenkin. En moiti, en,
Rasutovia, mies oli itse soria, mutta elämä ei ollut sijallista, liian oli
kepeätä ja leikinmukaista. Sillä minutkin rangottiin: köyhyydestä
nostettiin Rasutovin renkitupaan, kiusattiin hetkinen kaikella hyvällä
ja sitten taas laitettiin kylänlaitaan sienensyöntiin. Mutta hyvää teki
kuin tekikin! En voivottele enää, vaikka yhteen otteeseen jo
tuskailinkin. Ei ole rikkaudestakaan, ikäväksi tekee lopulta elämän,
kaikkea on mitä tahdot, tekeminen loppuu. Rikkaana olet kuin
nukkuva: milloin poskea painoi, vähän kohensit päänalaista, ja taas
oli hyvä. Tapella, tapella pitää leipä pöydälle, silloin vasta on
makeata. Sen nyt tiedän. Ja näytettiinhän se meille kaikille
järvituittulaisille: ensiksi syötiin puotivehnästä ja kehuttiin, hyvää on,
huokeata on, mitäs tätä leipää itse palstaakaan. A nyt: nyt syödään
kauransekalikkoa leipää ja sanotaan: omaa on, nyt tämä vasta
makeata onkin!

Sitten moksahti taas jokunen hirsi lumeen, ja ukot tuumailivat

puheitten aukomia asioita.
 Nummisto alkoi avaroitua, seinien esteet mataloituivat, kuorma
kuormalta siirtyivät entisen elämän jätteet nummien taakse, ja sitä
mukaa kaikkosi kylmä autiuden tuntu maisemasta. Nummien
kaarteet alkoivat jälleen päästä elähdyttämään sen luonnollista
viivakkoa.

Ja talvipäivä himmeni huomaamatta iltahämäriköksi. Ukot

peittelivät työkalunsa, keräytyivät ryhmään ja katosivat mustana
juonena nummien takaiseen iltaan.

Ristian ja Juones kulkivat viimeisinä ja seisahtivat porteilla.

Harmaan hämärän läpi näkyi vielä ruskeanpunainen juova
kaukaisella ilmankartanolla. Mutta nummista ja järviaukeama sulivat
yhdeksi värittömäksi iltatanhuaksi, jossa eli vain luonnon
salamyhkäisyys. Ihmisen osuutta ei enää havainnut.

Äänettöminä kiihdyttivät Ristian ja Juones kulkuansa ja yhtyivät

taas ukkojen jonoon metsätiellä. Hiljainen jyleikkö uuvutti kaikki
sanattomiksi, ja jokainen kulki omien ajatustensa varassa.

Juoneksen silmissä kuvasteli vielä äskeinen näky, joka oli

syöpynyt porteilla seisahdettaessa silmään, ja se nostatti rinnallensa
pian toisen, jota hän oli aikoinaan ihmetellen ja oudossa riemussa
katsellut. Juones muisti elävästi ne tuokiot sairasvuodepäiviltänsä,
jolloin hän sisäisen ahdistuksensa kourailuissa jäi tuijottamaan
vastapäisen akkunan lasikierukkaa ja sen näkyä: maa oli ja taivas
oli, mutta ihmisen osuus oli vain varjonkäännähdys niiden välissä. Ja
porttinummen laella seistessään Juoneksesta tuntui, niinkuin hän
olisi siinä silmännyt huikean suurta elämänmenon kuvaista. Mutta
siinäkin näkyi vain hämärän madaltama taivas ja kylmänvalkea maa.
Ihmisen käväisy siinä oli jo kokenut varjon kohtalon. Päivän luisu
illoillensa oli laajentanut varjot yhdeksi maahämäräksi, joka yhtyi illan
hämäräkäden pyyhkäisystä taivaalta laskeutuviin yökatteisiin —
kadoksiin. Kuitenkin veresti kaukana taivaanliepeellä hetkisen
ruskea juova tuoreen päivän lupauksena ennen vanhan päivän
ratkaisevaa sammumista.

Ja yhä likemmäksi tuli taivas maata. Jonossa kulkevat ukot näkivät

toisistansa enää pakenevien varjojen tapaisia hahmoja.

*****

Aamun ristivarjoista vapautunut maa heloitti kirkkaana ja

kosteana. Oli sunnuntaipäivä kukinnan valtakuussa. Järvituitun
harmaat kylätalot olivat hukkuneet pihlajoiden kukkavaahtoon.
Huviloiden laimeiksi vaalenneet seinät pilkoittivat arasti paksujen
lehtimuurien raoista. Kujasilla käveli nuoria ihmisiä pihlajanoksat
käsissä. Päivä oli korkea ja tuuli maantuoksusta raskas.

Kyläkankaalla, lumiauran siivillä, istui tupakoivia ukkoja kepeätä

sunnuntaikäräjää käyden.

Kaukana avartui alaniitusto vihreänä metenä metsäseinämien

kaarrokseen. Pohdittiin täyteläisen kesäpäivän nostattamia
aprikkeita ja katseltiin kaukaisille niittysaarekkeille. Ukot tunsivat
hiljaista onnea, joka uutuudellaan viehätti ja riemastutti.

Kujasilta alkoi kuulua tuttua ääntä: lähenevien kärrien jyrinää. Ukot

naurahtivat kuorossa ja käänsivät silmänsä alaniittyjen katsastelusta
lähikujasten mutkan tuijotukseen. Pian työntyi sieltä ajovaljastettu
hevonen ja perässä rattaille patsastunut ukko.

Tuutija-Mikko ajoi entisekseen ruohottuneelle asema-aukealle

puomivartioonsa.
 Mutta kuorossa naurahtaneet ukot hätkähtivät pian vakaviksi, sillä
heidän ajatuksissansa kertautui nyt rinnakkain menneen ja nykyisen
päivän sisällys. Nykyinen päivä oli vihreä ja tyyni. Mutta menneessä
päivässä soi korvantäyteisenä matkariemu ja katsetta viihdytti
tienpielien huoleton vilinä.

Tupakoitiin äänettöminä. Tuutija-Mikko oli ehtinyt jo kadota

valtatien kaarteisiin. Yksi kerrallansa lähtivät ukot sydänpurussa
lumiauran istukkeelta tuvillensa.

Päivä oli korkea ja kesäntäyteinen. Tuutija-Mikko ajoi sen

keskessä pienen pieneltä näyttävässä pölypilvessä pois kylän
elämästä.

Mutta hänen jäljessänsä ajoi Järvituitun ukkojen muistojen jono.

Pellot välkkyivät satoisanvihreinä. Maantie luikerteli kukkivien

piellensä välissä riemastuttavan päättymättömänä. — — —
Talokujasilla kulki mietteliäs ukko, joka riipaisi huomaamattansa
kouransa täyteen pihlajankukkia.

Kun hän seisahtui veräjälle, avasi hän kouransa ja varisti

kukkavalkean maahan.

— Kyllä pitää olla!

Sitten hän alkoi terhakasti repiä aitaa vastaan seisovan äkeen

haarukoista mättäänriekaleita. Tuutija-Mikko jäi unohduksiin.

Kesäinen tuulilaine puhkoi omia esteitänsä ja oli täynnänsä

kukinnan kosketusta.
 Järvituittu oli hiljainen kylä peltojen äärellä, pienillä mäennenillä,
tuulille alttiina.

*****

Kirjoitettu NVL:n hopeamerkkinäytteeksi.

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