Abstract
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.
Highlights
Response Gene to Complement (RGC)-32 was first identified in rat oligodendrocytes as a novel gene induced by complement activation [1]
We found the most differentially expressed to be insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF), with the expression of these proteins being downregulated in Response Gene to Complement 32 (RGC-32)-depleted astrocytes
We found that astrocytes displayed a higher density of processes in WT spinal cords in areas rich in inflammatory infiltrate (Figures 1A, D) when compared to their RGC-32 KO counterparts (Figures 1G, J), which showed a drastic reduction in inflammatory infiltrate accumulation
Summary
Response Gene to Complement (RGC)-32 was first identified in rat oligodendrocytes as a novel gene induced by complement activation [1]. Astrocytes were initially viewed as passive bystanders in CNS diseases, but their ability to undergo complex morphological and molecular changes in response to CNS damage has been increasingly recognized [12]. This complex set of changes is collectively termed reactive astrogliosis [13, 14]. Together with neurons and other glial cells, reactive astrocytes sustain neuroinflammation and dictate the outcome, either toward recovery and resolution of the injury or toward permanent damage and disease progression [15]. This relatively delicate balance depends mostly on the heterogeneous nature of the reactive astrocytes and is time-, region-, and context-dependent
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