RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis

Abstract

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.

Role of RGC-32 in multiple sclerosis and neuroinflammation – few answers and many questions

“…In vitro experiments have demonstrated that RGC-32 KO brain astrocytes stimulated with TGF-β produce lower levels of extracellular matrix (ECM) components such as pro-collagen I, IV, and V; fibronectin; fibrillin; and fibulin, as well as proteoglycan core proteins such as heparan sulfate proteoglycan 2 (HSPG2), versican (VCAN), and testican (SPOCK3) ( 17 , 19 ). Mechanistically, RGC-32 physically associates with the transcription factor SMAD3 and is translocated to the nucleus through a process that requires SMAD3 phosphorylation and RhoA-Rho kinase activation ( 17 ).…”

“…In a quest to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we have performed next-generation RNA sequencing on brain astrocytes purified from WT and RGC-32 KO mice, under basal conditions and after TGF-β stimulation. We found that a lack of RGC-32 has a significant impact on the transcriptomic programs normally associated with brain development, neurogenesis, cell motility, and cell projection ( 19 ). Of special note is the fact that the differential regulation of pathways ontologically related to cell motility suggests that RGC-32 may be involved in astrocyte migration, as we have already described for other cell types such as endothelial cells and vascular smooth muscle cells ( 49 , 50 ).…”

“…Functional enrichment analysis has shown that many pathways impaired by lack of RGC-32 are associated with processes such as neurogenesis and nervous system development ( 19 ). Connectivity analysis has further revealed a particular network of interconnected molecules involved in axonal guidance that is differentially regulated only in WT astrocytes ( 19 ). These axonal guidance molecules (AGM) play vital roles during brain development, providing axons with cues for normal wiring ( 51 ).…”

“…In adult brains, reactive astrocytes are a major pool for AGM synthesis and secretion and, thanks to their ability to inhibit axonal regeneration and to regulate the immune system, AGM are thought to play an important role in MS pathogenesis ( 52 – 54 ). RGC-32 KO astrocytes have lower mRNA levels of AGM family members such as ephrin receptor A type 7 (EPHA7), plexin A1 (PLXNA1), and Slit guidance ligand 2 (SLIT2) ( 19 ). On a similar note, we have found a lower number of EPHA7/GFAP-double positive cells in the spinal cords of RGC-32 KO mice during peak EAE ( 19 ).…”

“…RGC-32 modulates a number of cellular processes, including cell cycle regulation, cell migration, cellular differentiation, and fibrosis, and influences pathological processes such as carcinogenesis, metabolic disorders, atherosclerosis, and autoimmunity ( 13 – 15 ). Our work has demonstrated that RGC-32 plays an important role in the pathogenesis of EAE by regulating the differentiation of Th17 cells ( 16 ) as well as the ability of astrocytes to undergo reactive changes ( 17 – 19 ).…”