Synaptic and complement markers in extracellular vesicles in multiple sclerosis

Abstract

Background: Synaptic loss is a feature of multiple sclerosis pathology that can be seen even in normal-appearing gray matter. Opsonization of synapses with complement components may underlie pathologic synapse loss.

Objective: We sought to determine whether circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (NEVs and AEVs) provide biomarkers reflecting complement-mediated synaptic loss in multiple sclerosis.

Methods: From plasma of 61 people with multiple sclerosis (46 relapsing-remitting multiple sclerosis (RRMS) and 15 progressive MS) and 31 healthy controls, we immunocaptured L1CAM + NEVs and GLAST + AEVs. We measured pre- and post-synaptic proteins synaptopodin and synaptophysin in NEVs and complement components (C1q, C3, C3b/iC3b, C4, C5, C5a, C9, Factor B, and Factor H) in AEVs, total circulating EVs, and neat plasma.

Results: We found lower levels of NEV synaptopodin and synaptophysin in MS compared to controls (p < 0.0001 for both). In AEVs, we found higher levels of multiple complement cascade components in people with MS compared to controls; these differences were not noted in total EVs or neat plasma. Strikingly, there were strong inverse correlations between NEV synaptic proteins and multiple AEV complement components in MS, but not in controls.

Conclusion: Circulating EVs could identify synaptic loss in MS and suggest a link between astrocytic complement production and synaptic loss.

Keywords: Extracellular vesicles; astrocytes; biomarkers; complement; multiple sclerosis; synaptic dysfunction.

Figure 1.. Synaptic proteins in NEVs are reduced in MS

(A) Schematic representation of the study involving 60 MS patients (45 with RRMS, 15 with PMS) and 31 HC. (B) Concentration of NEVs in MS patient and HC plasma. (C) Average diameter (size) of NEVs in MS and HC groups. (D) NEV Synaptopodin and (E) Synaptophysin concentrations in MS and HC groups. (F) Tight correlation of nEV synaptopodin and synaptophysin concentrations in MS and HC groups. (G) NEV synaptopodin and (H) synaptophysin concentrations in HC, RRMS and PMS groups. (I) Scatter plot of NEV synaptopodin concentration by age in the HC group. (J) Scatter plot of nEV synaptopodin concentration by age in the MS group. p values for B, C, D & E are derived from Student’s t tests. p values for G & H are derived from one-way ANOVA with Dunnett’s multiple comparisons test. rho and p values for F, I & J are derived from Spearman’s correlation.

Figure 2.. Complement components in AEVs are increased in MS

Complement component levels in AEVs from HC, RRMS and PMS groups – (A) C1q, (B) C3, (C) C3b/iC3b, (D) C4, (E) C5, (F) C5a, (G) C9, (H) Factor B and (I) Factor H. p values for A-I are derived from one-way ANOVA with Dunnett’s multiple comparisons test.

Figure 3.. Complement levels in total circulating EVs and neat plasma do not differ between MS and HC groups

C5, C5a and C9 protein levels in total circulating EVs (A) and neat plasma (B) from 10 MS patients and 10 HC. Subjects were selected from the larger cohort so that their complement levels in AEVs were around the group average to serve as group representatives. All C9 signals from neat plasma samples were bellow the LOD and hence not shown. p values for A and B are derived from two-tailed unpaired Student’s t tests.

Figure 4.. Complement component levels in AEVs are strongly correlated with NEV synaptic protein levels

(A) Heat map of correlations between NEV synaptic proteins and AEV complement components in MS and HC groups demonstrates strong correlations in MS but not in HC. (B) Scatter plots depicting correlation between selected AEV complement components (C1q, C5, Factor H) and NEV synaptopodin (left) or synaptophysin (right). Rho and p values for J & K are derived from Spearman’s correlations.