CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis

Roman Fischer,Imre Mäger,Mathew Wood,Benedikt M. Kessler,Elizabeth Gray,Alexander G. Thompson,Kevin Talbot,Philip D. Charles,Marie-Laëtitia Thézénas,Martin R. Turner

doi:10.1186/s12014-020-09294-7

Roman Fischer, Imre Mäger + Show 8 more

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https://doi.org/10.1186/s12014-020-09294-7

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Journal: Clinical Proteomics	Publication Date: Aug 17, 2020
Citations: 29	License type: open-access

Affiliation: University of Oxford

Abstract

BackgroundExtracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers.MethodsCSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed.ResultsCSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases.ConclusionsProteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.

Highlights

The pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) implicates an expanding range of cellular pathways [1], for which biomarkersThompson et al Clin Proteom (2020) 17:31cerebrospinal fluid (CSF) Extracellular vesicles (EVs) have unique potential to provide insight into intracellular processes beyond that of studying the whole CSF proteome, which contains largely secreted proteins [5]
Inhibition of exosome biogenesis leads to increases in insoluble TDP-43 in a TDP-43-mutant mouse model, and reduced EV secretion is observed in fibroblasts and iPSC-derived motor neurons from patients carrying the C9orf72 hexanucleotide repeat expansion [10, 11]
Gene Ontology (GO) overrepresentation analysis, comparing proteins identified in proteomic analysis with a large dataset of the whole CSF proteome [19] indicated significant enrichment of component terms “extracellular exosome”, “extracellular vesicle” (557/988, OR 1.64, p < 0.001) and “blood microparticle” (73/85, OR 2.49, p < 0.001; Fig. 2d; top 200 GO component GO terms can be found in Additional file 1: Table S1)

Summary

Methods

CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatographytandem mass spectrometry proteomic analysis performed

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