Toombs, Jamie; (2019) Amyloid beta: from pre-analytical factors to disease mechanisms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Biomarkers are powerful tools for interrogating the basic science of disease processes, in the clinical detection of disease states, and as both targets and endpoints in therapeutic strategy. Amyloid beta (Aβ) is a core biomarker for Alzheimer’s disease (AD), but measurement variation between sites and experiments limits its potential. Furthermore, although the role of brain Aβ accumulation early in AD is extremely well attested, the biological mechanisms underlying this remain poorly understood. // Methods: To contribute to the development of treatments for AD patients and those at risk, this thesis set out to identify important pre-analytical confounding factors in Aβ measurement, strategies to mitigate them, and identify disease relevant patterns of Aβ peptide production in human CSF and an induced pluripotent stem cell-derived cortical neuron model of familial AD (fAD). // Results: A series of experiments demonstrated the importance of sample surface exposure to the measurement of Aβ peptides and tau. The volume at which samples are stored and iterative contact with fresh surfaces had profound effect on Aβ, but not tau, with greater surface exposure resulting in depletion of Aβ concentration. The mechanism was demonstrated to be protein surface adsorption. Importantly, the different Aβ peptides did not absorb to polypropylene to the same extent; Aβ42 concentration decreased proportionally more with surface exposure treatment than Aβ40 and Aβ38. It was observed that the addition of a non-ionic surfactant (Tween 20) to samples significantly mitigated the effect of surface exposure treatments on Aβ peptides and tau. However, the use of this additive did not meaningfully improve variability when sample storage conditions were standardised. Furthermore, variances in clinic to laboratory temperature and time interval did not significantly affect Aβ or tau concentration. Validation of an in vitro model of fAD was conducted. Experiment identified the use of Aβ ratios as a robust method for normalising data variability between and within cell lines over extended time periods. Furthermore, comparison of paired CSF, cell media, cell lysates, and post-mortem cortical tissue from the same individual demonstrated physiologically consistent patterns of Aβ ratios across sample types. Finally, comparison of multiple fAD mutation and control cell lines demonstrated quantitative and qualitative differences in secreted Aβ. APP V717I neurons increased secretion of Aβ42 and Aβ38 relative to Aβ43 and Aβ40. PSEN1 mutations increased secretion of longer Aβ peptides relative to shorter Aβ peptides, with mutation specific differences such as greatly increased Aβ43 in PSEN1 R278I.// Conclusions: This work demonstrated several novel considerations in the use of Aβ peptides as biomarkers for AD. Data principally highlight the importance of Aβ ratios to AD biomarker research, the necessity of controlling pre-analytical sample surface exposure intended for the measurement of ‘sticky’ protein biomarkers such as Aβ peptides, and the validity of iPSC-derived neuronal models for exploring the production of Aβ in AD and health.

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