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Discovery of biomarkers for amyotrophic lateral sclerosis from human cerebrospinal fluid using mass spectrometry-based proteomics

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is progressive neurodegenerative diseases characterized by the relentless loss of upper and lower motor neurons, eventually leading to death. Critical to the mission of developing effective therapies for ALS is the discovery of biomarkers that can illuminate mechanisms of neurodegeneration, as well as that, can be used for diagnostic, prognostic, or pharmacodynamic value across the disease. Here, we merged unbiased discovery-based approaches and targeted quantitative comparative analyses to identify proteins that are altered in cerebrospinal fluid (CSF) from ALS. Mass spectrometry (MS)-based proteomic approaches employing tandem mass tags (TMT) quantification methods from 40 CSF samples comprising 20 patients with ALS and 20 healthy control (HC) individuals identified 53 candidate biomarker proteins after CSF fractionation. Notably, these candidate biomarkers included both previously identified proteins validating our approach and novel ones, expanding the applicability of the biomarkers. Candidate biomarkers were subsequently examined using parallel reaction monitoring (PRM) MS methods on 61 unfractionated CSF samples comprising 30 patients with ALS and 31 HC individuals. Fifteen candidate biomarkers (APOB, APP, CAMK2A, CHI3L1, CHIT1, CLSTN3, ERAP2, FSTL4, GPNMB, JCHAIN, L1CAM, NPTX2, SERPINA1, SERPINA3 and UCHL1) showed significant differences between ALS and Control. Taken together, this study identifies multiple novel proteins that are altered in ALS, which provides the foundation for developing biomarkers for ALS.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: chanhyun na

LAB HEAD: Chan-Hyun NA

PROVIDER: PXD038846 | Pride | 2023-05-22

REPOSITORIES: Pride

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Project description:Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen postmortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72 -RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72 -RE status revealed 330 protein alterations in the C9ORF72 -RE+ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72 -RE on synaptic composition.

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Discovery of biomarkers for amyotrophic lateral sclerosis from human cerebrospinal fluid using mass spectrometry-based proteomics

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