Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.
Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.
Sublytic levels of C5b-9 increase the survival of oligodendrocytes (OLGs) and induce the cell cycle. We have previously observed that SIRT1 co-localizes with surviving OLGs in multiple sclerosis (MS) plaques, but it is not yet known whether SIRT1 is involved in OLGs survival after exposure to sublytic C5b-9. We have now investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 and phosphorylated-SIRT1 (Ser27) expression. We also examined the downstream effects of SIRT1 by measuring histone H3 lysine 9 trimethylation (H3K9me3) and the expression of cyclin D1 as a marker of cell cycle activation. OLG progenitor cells (OPCs) purified from the brain of rat pups were differentiated in vitro and treated with sublytic C5b-9 or C5b6. To investigate the signaling pathway activated by C5b-9 and required for SIRT1 expression, we pretreated OLGs with a c-jun antisense oligonucleotide, a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and a protein kinase C (PKC) inhibitor (H7). Our data show a significant reduction in phospho-SIRT1 and SIRT1 expression during OPCs differentiation, associated with a decrease in H3K9me3 and a peak of cyclin D1 expression in the first 24 h. Stimulation of OLGs with sublytic C5b-9 resulted in an increase in the expression of SIRT1 and phospho-SIRT1, H3K9me3, cyclin D1 and decreased expression of myelin-specific genes. C5b-9-stimulated SIRT1 expression was significantly reduced after pretreatment with c-jun antisense oligonucleotide, H7 or LY294002. Inhibition of SIRT1 with sirtinol also abolished C5b-9-induced DNA synthesis. Taken together, these data show that induction of SIRT1 expression by C5b-9 is required for cell cycle activation and is mediated through multiple signaling pathways.
We previously showed that lack of Response Gene to Complement 32 (RGC-32) decreases the severity of clinical scores of mice with EAE and renders spinal cord astrocytes a progenitor phenotype reminiscent of radial glia. In this study, we used RNA-sequencing to investigate whether RGC-32 deferentially regulates biological processes related to astrocyte maturation and activation. We purified neonatal astrocytes from WT and RGC-32 knock-out (KO) mice, stimulated them with TGF-β and then performed RNA-seq (Genewiz, S. Plainfield, NJ). 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, as well as radial glial marker HOPX, neural stem cell marker CD133, and proliferation marker Ki-67. Pathway enrichment analysis revealed that the genes deferentially expressed in WT but not RGC-32 KO astrocytes belonged to GO categories related to brain development, axonal guidance, cell migration and extracellular matrix (ECM) regulation. Real-Time PCR confirmed that Ephrin A7, bone morphogenetic protein 1 and polycystin 1, other genes related to brain development and ECM were significantly up-regulated in WT astrocytes after TGF-β stimulation. We found that spinal cords of RGC-32 KO mice displayed a higher number of HOPX+ cells at day 14. Similarly, the number of CD133+ cells with a radial glial morphology was higher in RGC-32 KO mice. In addition, we found that RGC-32 KO astrocytes had a higher proliferative index at the peak of EAE, as shown by their increased expression of Ki-67. These data suggest that RGC-32 is an important regulator of astrocyte maturation and reactive astrocyte formation during EAE
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