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Cerebrospinal Fluid Biomarkers in Alzheimer's Disease

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Cerebrospinal Fluid Biomarkers in Alzheimer's Disease University College London Institute of Neurology Cerebrospinal fluid biomarkers in Alzheimer’s Disease: from bedside to bench and back A thesis submitted for the Degree of Doctor of Philosophy Ross William Paterson August 2016 1 In memory of Margaret Mackinnon 2 Abstract Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in cognitive impairment and death. The pathological hallmarks are extracellular cortical amyloid plaques and intraneuronal tangles composed of hyperphosphorylated tau. Although environmental and genetic factors contribute to the development of AD, the sequence of pathophysiological events that lead to Alzheimer’s dementia is not yet completely clear. The clinical diagnosis of AD during life can be challenging and factors that explain clinical phenotypic heterogeneity and variability in rates of disease progression are not well understood. Biomarkers, objective measures of biological function, can be employed to support a clinical diagnosis of AD and may be abnormal before the onset of clinical symptoms. Imaging and cerebrospinal fluid biomarkers (CSF) are now incorporated into clinical and research diagnostic criteria. CSF, which is in direct contact with the brain, is a promising source of biomarkers and has the potential to differentiate AD from other neurodegenerative dementias, explain clinical heterogeneity within AD and elucidate the role of other pathobiological pathways. Ultimately CSF biomarkers might facilitate diagnosis of AD in its pre-clinical phase and allow for treatment responses to be measured. In this thesis CSF samples from clinical cohorts of individuals with AD, other neurodegenerative diseases and healthy controls are analysed using an extended panel of enzyme-linked immunosorbent assays (ELISA) and a novel mass spectrometry based assay. For the established CSF biomarkers, the practical issues related to collection, transportation and storage of CSF are investigated. Amyloid positron emission tomography (PET) imaging is investigated as a means of validating clinical cutpoints. An extended panel of established and emerging ELISAs is used to determine the diagnostic utility of biomarkers for differentiating AD from other neurodegenerative dementias and for explaining phenotypic heterogeneity within AD. The role of CSF biomarkers as predictors of disease progression is investigated employing robust measures of brain atrophy as surrogate measures of rates of neurodegeneration. Finally CSF samples are probed for new AD biomarkers using a novel mass spectrometry based assay. A number of practical conclusions are drawn from this work: aliquot storage volume is identified as an important confounder in measured CSF b-Amyloid concentration. CSF laboratory transportation methods are shown not to have a significant impact 3 on measured biomarker concentration. Amyloid PET is a valuable means of validating clinical diagnostic cutpoints of core CSF biomarkers. Tau/Ab1-42 ratio, Ab40/42 ratio, P-tau and NFL emerge as having diagnostic utility for differentiating AD from other neurodegenerative diseases, and have high sensitivity and specificity for distinguishing AD from bvFTD, SD and healthy controls. Important differences in T-tau, P-tau and neurofilament light distinguish different AD atypical phenotypes and may help to elucidate underlying biological differences between these syndromes: individuals with the visual variant of AD (posterior cortical atrophy) have the lowest levels of CSF Tau and lowest rates of cognitive decline while the frontal executive cases have highest levels of NFL and highest rates of cognitive decline indicating more rapid neurodegeneration. Several novel biomarkers including trefoil factor 3 and several markers involved in vascular remodeling, amyloid processing and neuroinflammation are identified as predictors of increased atrophy rates in amyloid positive individuals suggesting possible independent mechanisms driving differing rates of neurodegeneration between individuals. Other novel AD biomarkers including malate dehydrogenase are identified as distinguishing AD from controls using a novel mass spectrometry based assay. Moreover, this assay demonstrates how mass spectrometry might be used for biomarker discovery and rapid development of a high throughput multiplexed clinical CSF assay. Taken together these results address some of the unanswered questions about how CSF should be collected, handled and stored to optimize analytical standardization, and how clinical results might be validated using amyloid PET. This work establishes the clinical utility of established biomarkers for differentiating AD from other neurodegenerative diseases and identifies established and novel biomarkers that might explain clinical heterogeneity and rates of progression between individuals. Finally a method for rapidly developing new biomarkers is tested and validated. 4 Acknowledgements I am greatly indebted to my supervisors Jonathan Schott, Henrik Zetterberg and Nick Fox for their immense enthusiasm, unwavering support and for generously sharing their knowledge of the field. I have been privileged to share my time at the Dementia Research Centre with Catherine Slattery and I am grateful for her support, ideas and friendship. I am grateful to have collaborated with Jamie Toombs, Martha Foiani, Wendy Heywood and Kevin Mills. I am particularly indebted to Amanda Heslegrave for her time, patience and dedication in the laboratory. Finally, I am thankful to Michael and to my parents and family for their love and support. 5 Contents Contents ......................................................................................................................... 6 Abbreviations............................................................................................................... 13 List of Tables ............................................................................................................... 15 List of Figures ............................................................................................................. 17 Chapter 1. Introduction ............................................................................................. 20 1.1 Alzheimer’s Disease ......................................................................................... 20 The Problem ........................................................................................................ 20 1.1.2 History and Epidemiology........................................................................ 22 1.1.3 Clinical features......................................................................................... 23 1.1.4 Differential Diagnosis of AD .................................................................... 25 1.1.5 Neuropathology of AD ............................................................................. 28 1.1.6 AD pathophysiology: The Amyloid Cascade Hypothesis ........................ 36 1.1.7 Genetic determinants and risk factors ..................................................... 38 1.1.8 Alzheimer’s disease progression ............................................................... 40 1.1.9 Current hypothetical models of biomarker changes ................................ 43 1.1.10 Diagnostic Criteria ................................................................................. 45 1.2 Biomarkers for Alzheimer’s disease................................................................. 46 1.2.1 Currently Available Biomarkers............................................................... 48 1.3 Cerebrospinal Fluid ......................................................................................... 55 1.3.1 Normal physiology and function of CSF .................................................. 55 1.3.2 Why is CSF of interest in Alzheimer’s disease?....................................... 56 1.3.3 Sampling CSF ........................................................................................... 57 1.3.4 CSF Analysis............................................................................................. 59 1.3.5 Confounders in CSF biomarker measurement ......................................... 61 1.3.6 Currently available CSF biomarkers ........................................................ 63 1.3.7 Practical Application of CSF biomarkers ................................................. 73 1.3.8 Biomarkers in revised diagnostic criteria................................................. 79 1.3.9 Limitations of currently available biomarkers ......................................... 81 1.3.10 Developing new biomarkers ................................................................... 81 1.4 Aims and objectives ......................................................................................... 83 1.4.1 Aims .......................................................................................................... 83 1.4.2 Objectives .................................................................................................. 83 1.5 Publications arising from this chapter ............................................................ 84 6 Chapter 2. General Methods ..................................................................................... 85 2.1 Cohorts............................................................................................................. 85 2.1.1 Retrospective cohort ................................................................................. 88 2.1.2 Prospective Cohort ..................................................................................
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