Cerebrospinal fluid biomarkers link toxic astrogliosis and microglial activation to multiple sclerosis severity

Abstract

Background: Once multiple sclerosis (MS) reaches the progressive stage, immunomodulatory treatments have limited efficacy. This suggests that processes other than activation of innate immunity may at least partially underlie disability progression during late stages of MS. Pathology identified these alternative processes as aberrant activation of astrocytes and microglia, and subsequent degeneration of oligodendrocytes and neurons. However, we mostly lack biomarkers that could measure central nervous system (CNS) cell-specific intrathecal processes in living subjects. This prevents differentiating pathogenic processes from an epiphenomenon. Therefore, we sought to develop biomarkers of CNS cell-specific processes and link them to disability progression in MS.

Methods: In a blinded manner, we measured over 1000 proteins in the cerebrospinal fluid (CSF) of 431 patients with neuroimmunological diseases and healthy volunteers using modified DNA-aptamers (SOMAscan®). We defined CNS cell type-enriched clusters using variable cluster analysis, combined with in vitro modeling. Differences between diagnostic categories were identified in the training cohort (n = 217) and their correlation to disability measures were assessed; results were validated in an independent validation cohort (n = 214).

Results: Astrocyte cluster 8 (MMP7, SERPINA3, GZMA and CLIC1) and microglial cluster 2 (DSG2 and TNFRSF25) were reproducibly elevated in MS and had a significant and reproducible correlation with MS severity suggesting their pathogenic role. In vitro studies demonstrated that proteins of astrocyte cluster 8 are noticeably released upon stimulation with proinflammatory stimuli and overlap with the phenotype of recently described neuro-toxic (A1) astrocytes.

Conclusion: Microglial activation and toxic astrogliosis are associated with MS disease process and may partake in CNS tissue destruction. This hypothesis should be tested in new clinical trials.

Keywords: CNS tissue destruction; CSF biomarkers; MS severity; Microglial activation; Multiple sclerosis; Toxic astrogliosis.

Published by Elsevier B.V.

Figures

Figure 1:

Cluster scores adjusted based on age were compared among diagnosis subgroups. Only clusters which have reproducible statistically significant differences between disease diagnosis subgroups are shown. The y-axis as selected to have better visual assessment of majority of patients’ cluster scores. Thus, few of individual patients’ points may be missing in these graphs. Data were analyzed using ANOVA, with multiple comparisons using Tukey’s method; *p

Figure 2:

Scatter plots for Astrocyte Cluster 8 and Microglia Cluster 2; cluster score (adjusted for age) versus MS disability (CombiWISE and EDSS), CNS tissue destruction (COMRIS-CTD) and disease severity (MS-DSS) measures. Both clusters exhibited reproducible statistically significant (p value

Figure 3:

CNS cells were cultured either without any treatment (native) or treated with pro-inflammatory stimuli (stimulated PBMCs supernatant, 50% volume/volume) or mitochondrial stressor (Rotenone, 100 nM). Secretion of individual biomarker under these culture conditions was measured by computing standardized RFU differences between start and end of culture conditions (24h – 0h), and then the mean of each cluster was computed. Only data for clinically important clusters is depicted.