RGC-32 facilitates reactive astrocytosis by modulating the expression of axonal guidance molecules

Abstract Response Gene to Complement (RGC)-32 modulates TGF-β-induced extracellular matrix secretion and the ability of astrocytes to undergo reactive changes in vivo during experimental autoimmune encephalomyelitis (EAE). However, the molecular pathways underlying these effects are still not well understood. In this study, we investigated how lack of RGC-32 affects the transcriptomic profile and the expression of axonal guidance molecules (AGM) in astrocytes during EAE. We performed next-generation RNA sequencing on brain neonatal astrocytes isolated from wild type (WT) and RGC-32 knock-out (KO) mice, either unstimulated or stimulated with TGF-β. Results were then validated by using Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE (at days 0 and 14) were stained by immunohistochemistry for the astrocyte marker GFAP, AGM, and for nuclear factor IA (NFIA), a gliogenic factor and transcriptional regulator of AGM. Lack of RGC-32 had a significant impact on the transcriptomic programs normally associated with brain development whose re-expression is usually seen in reactive astrocytes. Connectivity analysis revealed that genes coding for AGM were particularly affected. We found lower transcript levels of ephrin receptor A type 7 (Epha7), plexin A1 and Slit guidance ligand 2 in RGC-32 KO astrocytes. Moreover, our results showed that NFIA and EPHA7 are expressed by astrocytes during EAE. We found a lower number of astrocytes expressing EPHA7 and NFIA in RGC-32 KO mice with acute EAE when compared with WT mice. These results suggest that RGC-32 might facilitate reactive astrogliosis during acute EAE through regulating the expression of AGM and NFIA. Veterans Administration Merit Award I01BX001458 (to HR)