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 spinal cords during experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation.
In this study, we investigated whether lack of RGC-32 alters the expression of glial fibrillary acidic protein (GFAP) and astrocyte progenitor markers, as well as the proliferation of astrocytes during EAE.
Spinal cords from WT and RGC-32 knock-out (KO) mice with EAE were harvested at day 0 and at the peak of disease (day 14) and subjected to immunohistochemical analysis for GFAP, vimentin, fatty acidic binding protein 7 (FABP7), Ki-67, and connective tissue growth factor (CTGF). Neonatal astrocytes were purified from WT and RGC-32 KO mice, and the expression of GFAP, vimentin, and CTGF was assessed through Real-Time PCR and Western Blot.
Immunohistochemical analysis showed that during acute EAE, WT astrocytes had a reactive morphology, whereas RGC-32 KO astrocytes had a morphology similar to radial glia and immature progenitors. Astrocyte density and GFAP expression was significantly higher in WT spinal cords (p=0.02). In addition, the elongated, radial glia-like astrocytes from RGC-32 KO mice expressed higher levels of vimentin and FABP7 compared to WT mice, confirming their immature phenotype. At day 0, GFAP expression was also higher in WT mice, whereas the number of FABP7-positive radial glia was higher in RGC-32 KO mice. We found that RGC-32 KO astrocytes had a higher proliferative index at the peak of EAE, as measured by their increased expression of Ki-67. Double staining for GFAP and CTGF, a growth factor known to be involved in astrocyte differentiation, showed a lower number of CTGF-positive astrocytes in spinal cords of RGC-32 KO mice compared to WT mice during acute EAE (p=0.002). Significantly lower levels of GFAP and CTGF and higher levels of vimentin were found in cultured RGC-32 KO astrocytes compared to WT astrocytes.
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 to become reactive astrocytes, although they retain a higher proliferative index, as part of adopting a progenitor phenotype.