2021 CMSC Annual Meeting

RGC-32 Contributes to Reactive Astrocytosis in Experimental Autoimmune Encephalomyelitis By Maintaining a Mature Phenotype


Astrocytes are increasingly recognized as critical contributors to the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Our research has shown that Response Gene to Complement 32 (RGC-32) knock-out (KO) mice have an attenuated EAE disease course. We also showed that RGC-32 KO astrocytes express markers of immaturity such as vimentin and display a morphology reminiscent of their radial glia precursors, suggesting that RGC-32 may contribute to astrocyte reactivity in part by promoting their differentiation.
Our objective was to use RNA sequencing to investigate whether the genes differentially regulated by RGC-32 in astrocytes are enriched in categories related to development and differentiation. In addition, we sought to investigate how the lack of RGC-32 affects tissue expression of markers of astrocyte maturity and proliferation.
We purified neonatal astrocytes from WT and RGC-32 KO mice, stimulated them with TGF-?, and then performed RNA-seq. Pathway enrichment analysis was performed using PANTHER software. The genes most differentially expressed in categories of interest were confirmed by Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE were harvested at day 0 and at the peak of disease (day 14) and subjected to immunohistochemistry for astrocytes lineage marker GFAP, radial glial marker HOPX, neural stem cell marker CD133, and proliferative marker Ki-67.
Pathway enrichment analysis revealed overrepresentation of genes in categories such as brain development, axonal guidance, cell migration, and extracellular matrix (ECM) regulation. Real-Time PCR confirmed upregulation of genes related to brain development, such as Ephrin A7, bone morphogenetic protein 1, and polycystin 1, as well as significant upregulation of genes related to ECM, such as heparan sulfate proteoglycan 2, versican, testican, fibrillin-1, fibrillin-2, and fibulin-2. Immunohistochemistry showed that RGC-32 KO mice display a higher number of HOPX+ cells at day 0 and day 14. Similarly, the number of CD133+ cells with a radial glial morphology was significantly higher in RGC-32 KO mice spinal cords at both day 0 and day 14 as compared to WT mice. In addition, we found that RGC-32 KO astrocytes have a higher proliferative index at the peak of EAE, as shown by their increased expression of Ki-67.
This data suggests that in the absence of RGC-32, astrocytes are unable to fully mature and to become reactive, retaining a radial glial phenotype with high proliferative capacities and progenitor cell potential. In addition, it suggests that RGC-32 is crucial for the ECM remodeling capacities of astrocytes, which could have important implications in both developmental and reparatory processes.