| Summary: | <p>Amyotrophic lateral sclerosis (ALS), the commonest form of motor neuron disease (MND), is a fatal, typically rapidly-progressive neurodegenerative syndrome without highlyeffective disease-modifying treatment. A diverse range of upstream cellular perturbations converge on a common clinical pathway. A major impediment to the development of efficacious treatment is the lack of sensitive, objective measures of the disease process to enable early diagnosis, stratification and monitoring necessary for therapeutic trials. The experiments described in this thesis aim to develop novel biomarkers using mass spectrometrybased proteomic analysis of cerebrospinal fluid (CSF) and, to provide improved access to the intracellular compartment of central nervous system (CNS) cells, CSF-derived extracellular vesicles (EVs).</p>
<p>A high-throughput proteomic analysis of individual whole CSF samples from patients with ALS and healthy controls, patients with ALS-mimicking conditions and the upper motor neuron only variant primary lateral sclerosis (PLS) was performed. This analysis demonstrated elevation of three chitinase proteins, Chitotriosidase-1 (CHIT1), Chitinase- 3-like protein 1 (CHI3L1) and Chitinase-3-like protein 2 (CHI3L2) in the CSF of ALS patients. Chitinase proteins correlated with the rate of progression of the disease and the marker of axonal degeneraton phosphorylated neurofilament heavy chain (pNFH) and changed little over time in longitudinal analysis.</p>
<p>The elevation of chitinase proteins was validated using a commercially-available enzymelinked immunosorbent assay (ELISA). Additional analysis of chitinase proteins and clinical parameters identified correlations between the burden of upper motor neuron (UMN) signs and degree of cognitive dysfunction with CHI3L1, whilst CHI3L2 and CHIT1 correlated with the rate of symptom progression and axonal degeneration as measured by pNFH. This provides evidence that the different chitinase proteins reflect different dimensions of the glial response in ALS and supports their use as a measure of target engagement in trials of therapies directed at non-cell-autonomous mechanisms.</p>
<p>In order to enhance access to the intracellular proteome, a method of extraction of EVs from CSF based on size-exclusion chromatography was employed. Comparison of this method with current standard methods showed improvement in EV recovery and proteomic depth. Enrichment analysis comparing whole CSF and CSF EV-enriched proteomes suggested that the EV proteome reflects intracellular processes, contrasting with the secreted and plasma-derived proteome of whole CSF. Analysis of open access datasets suggests a predominantly white matter and choroid plexus origin of the EV proteome and additional overlap with the proteome of cortical and hippocampal neurons. This optimised EV extraction method was applied to individual CSF samples from a cohort of ALS patients and controls. This identified alterations in the levels of proteins involved in proteostasis pathways in ALS CSF, in particular the hexameric proteasome-like protease bleomycin hydrolase.</p>
<p>In conclusion, the work described in this thesis suggest that the whole CSF proteome in ALS predominantly reflects a secretory glial response and provides promising biomarkers of glial activity in ALS, whilst the CSF EV proteome is more reflective of core intracellular events implicated in ALS.</p>
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