Discovery of Biomarkers for Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration From Human Cerebrospinal Fluid Using Mass Spectrometry-Based Proteomics

Abstract

BackgroundAmyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are progressive neurodegenerative diseases that share clinical and neuropathologic features. Critical to the mission of developing effective therapies for ALS and FTLD is the discovery of biomarkers that can illuminate shared mechanisms of neurodegeneration, which can then be evaluated for diagnostic, prognostic or pharmacodynamic value across the disease spectrums. MethodsHere, we merged unbiased discovery-based approaches and targeted quantitative comparative analyses between ALS and FTLD cerebrospinal fluid (CSF) to identify proteins that are altered in ALS and FTLD. ResultsDiscovery mass spectrometry (MS)-based proteomic approaches combined with tandem mass tags (TMT) quantification methods from 40 CSF samples comprising 20 patients with ALS and 20 healthy control (HC) individuals identified 19 differentially expressed candidate biomarker proteins after CSF fractionation. Notably, these candidate biomarkers included novel and previously identified proteins, thus validating our approach. Candidate biomarkers were subsequently examined using parallel reaction monitoring (PRM) MS methods on 80 unfractionated CSF samples comprising 30 patients with ALS, 19 patients with FTLD, and 31 HC individuals. Two candidate biomarkers (CNTNAP2 and CLSTN1) were downregulated in both ALS and FTLD compared to healthy controls, and 11 further candidate proteins were significantly downregulated in FTLD compared to HC. ConclusionsTaken together, this study identifies multiple novel proteins that are altered in ALS and FTLD, which provides the foundation for their evaluation and development as biomarkers for these diseases.

Data‐independent acquisition proteomics of cerebrospinal fluid implicates endoplasmic reticulum and inflammatory mechanisms in amyotrophic lateral sclerosis

“…Using a simplified pre‐analytical preparation protocol combined with label‐free LC–MS/MS in DIA mode, we were able to quantify a total of 1832 protein groups from only 50 μL of CSF and identified alterations in the levels of individual CSF proteins and network‐level changes in people with ALS. This depth is similar to that achieved with DIA in recent studies for non‐fractionated CSF (1493–1732 proteins, Cho et al., 2021; Karayel et al., 2022), although somewhat lower than previous studies in ALS that have made use of labelling methods (1936–2303 proteins, Barschke et al., 2020; Oeckl et al., 2020; Oh et al., 2023). Importantly, however, the DIA approach resulted in a much lower rate of missing values than typically achieved with DDA, with between‐group comparisons carried out on 1593 proteins after a stringent (50% per group) filtering threshold, thus allowing for highly robust comparisons.…”

“…Furthermore, in the lysosomal storage disorder Gaucher disease, for which secreted CHIT1 is a key diagnostic marker (Hollak et al., 1994), the characteristically lipid‐laden macrophages show increases in the abundance of chemokine CCL18 and Glycoprotein nonmetastatic melanoma protein B (GPNMB) that are typically more indicative of alternatively activated microglia (Hüttenrauch et al., 2018; Kramer et al., 2016; Zigdon et al., 2015). Whilst significantly increased abundance was not found in this study, GPNMB has also been identified as increased in CSF of ALS patients in other studies (Oeckl et al., 2020; Oh et al., 2023). Given that in transgenic SOD1 models of ALS there is evidence for a shift from alternatively activated to classically activated microglia over the disease course (Gravel et al., 2016; Liao et al., 2012), defining the specific activation state of the cellular source of all the chitinase proteins in CSF will further contribute to understanding of the contribution of neuroinflammation to the clinical heterogeneity of the disease.…”

“…Univariate analysis identified seven protein groups as significantly different in ALS versus healthy controls and nine in ALS versus disease controls at an FDR threshold of 0.1 (Figure 1a,b). All differentially abundant proteins in ALS versus healthy controls were proteins that were increased in ALS and included CHIT1 and CHI3L1, along with UCHL1 and Alpha 1‐antichymotrypsin (SERPINA3) which have recently been independently identified as significantly higher in ALS patients (Oeckl et al., 2020; Oh et al., 2023). The remaining three protein groups were low‐affinity immunoglobulin gamma Fc region receptor III‐A (FCGR3A), myoglobin (MB) and complement factor D (CFD).…”

“…As an unbiased approach, shotgun proteomics using data‐dependent acquisition (DDA) liquid chromatography–tandem mass spectrometry (LC–MS/MS) has proved itself a powerful tool for biomarker discovery in ALS, identifying an increase in a set of glial proteins—the chitinase proteins chitotriosidase‐1 (CHIT1), chitinase‐3‐like protein‐1 (CHI3L1 or YKL‐40) and chitinase‐3‐like protein‐2 (CHI3L2 or YKL‐39) (Thompson et al., 2018; Varghese et al., 2013). Additional proteins such as glycoprotein nonmetastatic melanoma protein B (GPNMB), macrophage‐capping protein (CAPG), alpha 1‐antichymotrypsin (SERPINA3) identified in mass spectrometry, and chemokines or cytokines including monocyte chemotactic protein 1 (MCP‐1), macrophage inflammatory protein‐1α and β (MIP‐1α and MIP‐1β), interleukin‐8, and granulocyte colony‐stimulating factor (GM‐CSF) point towards a strong inflammatory involvement in the disease (Baron et al., 2005; Huang et al., 2020; Kuhle et al., 2009; Mitchell et al., 2009; Oeckl et al., 2020; Oh et al., 2023; Yang et al., 2016). However, the temporal profile of inflammatory involvement in ALS is poorly understood, with mixed data on the association of these proteins with survival and disability progression.…”

“…In CSF, the abundant plasma proteins, such as albumin and immunoglobulins, mask the detection of CNS‐derived proteins, which have much lower absolute abundance (Feneberg et al., 2014). Improving proteomic depth by immuno‐depletion of abundant proteins and strategies combining isobaric labelling with pre‐fractionation have been undertaken, yielding several promising candidate biomarkers including Ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) and Neuronal pentraxin‐2 (NPTX2; Collins et al., 2015; Oeckl et al., 2020; Oh et al., 2023) in CSF in ALS. These methods are vulnerable to higher preanalytical variability and a greater proportion of missing values than a typical label‐free workflow, with a subsequent impact on statistical power (Carlyle et al., 2018).…”