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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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			Action	DRS
	121322_ALS_PRM_first.sky	Other
	121322_ALS_PRM_first.sky.view	Other
	121322_ALS_PRM_first.skyd	Other
	121322_ALS_PRM_first.skyl	Other
	121322_ALS_rescue.sky	Other

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Project description:Unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of Amyotrophic Lateral Sclerosis (ALS). However, high abundance proteins mask the presence of lower abundance proteins that may hold valuable diagnostic and prognostic use. Developments in mass spectrometry proteomic data acquisition methods therefore hold promise for improved protein depth and more sensitive biomarker discovery. In this study, mass spectrometry with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of 40 people with ALS, 15 healthy controls and 8 people with mimicking conditions. Qthen quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins significantly upregulated in ALS vs healthy controls, and 9 with altered abundance in ALS vs mimicking conditions (FDR <0.1). Elevated Chitotriosidase 1 (CHIT1) was common to both comparisons and showed strong correlation with disease progression rate (Pearson r =0.41, FDR-adjusted p = 0.035), but no association with survival (Pearson correlation and Kaplan-Meier survival analysis). Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS vs healthy controls) showed a stronger correlation with disease progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and significant association with survival in Kaplan Meier analysis (log rank p = 0.013) but no independent effect in proportional hazards models. Weighted correlation network analysis was then used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was strongly associated with age at disease onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, negatively correlated with disease progression rate (r = -0.42, FDR-adjusted p =0.109). This study confirms that UCHL1 is strongly associated with disease progression and survival, and highlights potential of inflammatory and endoplasmic reticulum proteins to provide prognostic biomarker potential.

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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