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

Abstract

Background: Extracellular 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.

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 chromatography-tandem mass spectrometry proteomic analysis performed.

Results: CSF 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.

Conclusions: Proteomic 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.

Keywords: Amyotrophic lateral sclerosis; Biomarker; CSF; Exosome; Extracellular vesicle.

Fig. 1

Experimental workflow. CSF samples obtained by lumbar puncture from patients with ALS and healthy controls were stored at – 80 °C until use. EVs were extracted from patient CSF samples using ultrafiltration liquid chromatography, with resulting EV samples subjected to liquid chromatography-tandem mass spectrometry proteomic analysis with label-free quantification, with subsequent multivariate, univariate and pathway analysis. ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; EV: extracellular vesicle

Fig. 2

EV characterisation. a nanoparticle tracking analysis of extracted CSF EVs (grey lines indicate individual sample particle counts, solid green line indicates mean count for each particle size). b Transmission electron micrograph of extracted EVs (obtained from a pooled control sample). c EV marker proteins and contaminants (denoted by gene symbol) identified within the proteomic dataset in healthy control (yellow) and ALS first-visit (red) samples. d Gene ontology cellular component overrepresentation analysis, using identifications from the CSF proteome resource as the background list, indicates significant enrichment of exosome, microvesicle and extracellular vesicle terms. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; FDR: false discovery rate; CSF: cerebrospinal fluid; CD9—UniProt P21926; SDCBP—O00560; CD81—P60033; PDCD6IP—Q8WUM4; HSPA8—P11142; CD82—P27701; CD63—P08962; TSG101—Q99816; FLOT1—O75955; FLOT2—Q14254; HSP90AB1—P08238; LAMP1—P11279; LAMP2—P13473; ALB—P02768; APOA1—P02647; APOA2—P02652; APOB—P04114

Fig. 3

a Mean size distribution of CSF EVs in patients with ALS (n = 20) and healthy controls (n = 9) overlaid. Mean ± SD for groups shown in neighbouring plots. b Total number of EVs per mL CSF and C modal size of extracted EVs. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; CSF: cerebrospinal fluid; HC: healthy control

Fig. 4

a Mean size distribution of CSF EVs in patients with C9orf72-associated (n = 4) and non-C9orf72-associated ALS (n = 16) overlaid. Mean ± SD for groups shown in neighbouring plots b total number of EVs per mL CSF and c modal size of extracted EVs. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; CSF: cerebrospinal fluid; C9: C9orf72 hexanucleotide repeat expansion

Fig. 5

a Heatmap and hierarchical clustering of protein abundance in the CSF EV proteome. Column colour bar indicates sample group. b PCA biplot of CSF EV samples. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; CSF: cerebrospinal fluid; HC: healthy control; PC: principal component; C9: C9orf72 hexanucleotide repeat expansion

Fig. 6

The CSF EV proteome in ALS. a Volcano plot of the CSF EV proteome in ALS (n = 12, first visit samples only) compared with healthy control subjects (n = 5). Highlighted points indicate FDR-adjusted p < 0.1. Negative log fold change indicates proteins downregulated in ALS compared with controls. Dotted lines indicate p < 0.01 and log₂ fold change ± 0.5. b Volcano plot of Gene Ontology (GO) terms by gene set enrichment analysis, ordered by the product of -log₁₀ p-value and fold change. GO terms with FDR-adjusted p < 0.1 labelled. c Volcano plot of GO overrepresentation analysis of proteins upregulated or downregulated in ALS compared with healthy controls, p < 0.05 and log fold change > 0.5 or < − 0.5 compared with healthy controls. Selected GO terms labelled. d Volcano plot of proteomic analysis data, proteins annotated to “proteasome core complex” labelled and highlighted. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; CSF: cerebrospinal fluid; HC: healthy control; BLMH: Bleomycin hydrolase (UniProt Q13867); PSMA: Proteasome alpha subunit (PSMA4—P25789; PSMA5—P28066; PSMA6—P60900; PSMA7—O14818); PSMB: Proteasome beta subunit (PSMB1—P20618; PSMB2—P49721; PSMB3—P49720; PSMB6—P28072); GO: Gene ontology

Fig. 7

a Volcano plot of the CSF EV proteome in C9orf72 ALS (n = 3) compared with non-C9orf72 ALS subjects (n = 9, first-visit samples only). Red points indicate FDR-adjusted p < 0.1, higher in non-C9orf72 ALS, blue points FDR-adjusted p < 0.1 higher in C9orf72 ALS. Negative log fold change indicates proteins downregulated in non-C9orf72 ALS compared with C9orf72 ALS. Dotted lines indicate p < 0.01 and log₂ fold change ± 0.5. b Gene ontology (GO) gene set enrichment analysis of the CSF EV proteome in C9orf72 ALS compared with non-C9orf72 ALS. Representative up- and down-regulated terms labelled. ALS: amyotrophic lateral sclerosis; EV: extracellular vesicle; CSF: cerebrospinal fluid; GO: gene ontology; C9: C9orf72 hexanucleotide repeat expansion-associated ALS; CFL1—UniProt P23528; CYBB—P04839; UBA1—P22314; ANXA11—P50955; GPNMB—Q14956; TAGLN—Q01995; TGM2—P21980; KRT13—P13646