Contributions
Abstract: EP1452
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Introduction: Extracellular matrix (ECM) deposition in multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. RGC-32 plays an important role in the mediation of TGF-β effects, but its role in astrogliosis has not been investigated.
Objectives: We are proposing to investigate RGC-32 involvement in the expression of TGF-β-induced ECM components collagen type I, type IV, type V, fibronectin and reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin, together with the molecular mechanisms underlying RGC-32 expression and function. We also investigate the effect of lack of RGC-32 on astrocyte phenotype during experimental autoimmune encephalomyelitis (EAE).
Aims: Our aim is to evaluate the role of RGC-32 in astrogliosis.
Methods: We purified neonatal astrocytes from wild type (WT) and RGC-32 knock-out (KO) mice. The expression of ECM components and reactive astrocytic markers was assessed by Real time PCR and Western blotting. We also investigated the physical interaction between RGC-32 and Smad proteins, as well as the effect of Smad3 on RGC-32 nuclear localization. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed on spinal cords from WT and RGC-32 KO mice with EAE.
Results: Collagens I, IV and V, fibronectin, α-SMA and nestin were induced by TGF-β stimulation and siRNA-mediated RGC-32 silencing resulted in a significant reduction in their expression. We also found that the expression of TGF-β-induced collagens I, IV and V, fibronectin and α-SMA was significantly reduced in RGC-32 KO mice astrocytes when compared with WT mice. Co-immunoprecipitation assays showed that Smad3 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of Smad3 phosphorylation significantly reduced RGC-32 nuclear translocation and TGF-β-induced collagen I expression. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the Smad3 nuclear translocation was unaffected. In addition, during EAE, RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype.
Conclusions: Our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis during EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in chronic MS.
Disclosure: Alexandru Tatomir: nothing to disclose.
Cosmin Tegla: nothing to disclose.
Dallas Boodhoo: nothing to disclose.
Vinh Nguyen: nothing to disclose.
Amurgam Mekala: nothing to disclose.
Freidrich Anselmo: nothing to disclose.
Cornelia Cudrici: nothing to disclose.
Tudor Badea: nothing to disclose.
Violeta Rus: nothing to disclose.
Horea Rus: nothing to disclose.
This work was supported in part by Veterans Administration Merit Award I01BX001458 (to H.R.).
Abstract: EP1452
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Introduction: Extracellular matrix (ECM) deposition in multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. RGC-32 plays an important role in the mediation of TGF-β effects, but its role in astrogliosis has not been investigated.
Objectives: We are proposing to investigate RGC-32 involvement in the expression of TGF-β-induced ECM components collagen type I, type IV, type V, fibronectin and reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin, together with the molecular mechanisms underlying RGC-32 expression and function. We also investigate the effect of lack of RGC-32 on astrocyte phenotype during experimental autoimmune encephalomyelitis (EAE).
Aims: Our aim is to evaluate the role of RGC-32 in astrogliosis.
Methods: We purified neonatal astrocytes from wild type (WT) and RGC-32 knock-out (KO) mice. The expression of ECM components and reactive astrocytic markers was assessed by Real time PCR and Western blotting. We also investigated the physical interaction between RGC-32 and Smad proteins, as well as the effect of Smad3 on RGC-32 nuclear localization. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed on spinal cords from WT and RGC-32 KO mice with EAE.
Results: Collagens I, IV and V, fibronectin, α-SMA and nestin were induced by TGF-β stimulation and siRNA-mediated RGC-32 silencing resulted in a significant reduction in their expression. We also found that the expression of TGF-β-induced collagens I, IV and V, fibronectin and α-SMA was significantly reduced in RGC-32 KO mice astrocytes when compared with WT mice. Co-immunoprecipitation assays showed that Smad3 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of Smad3 phosphorylation significantly reduced RGC-32 nuclear translocation and TGF-β-induced collagen I expression. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the Smad3 nuclear translocation was unaffected. In addition, during EAE, RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype.
Conclusions: Our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis during EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in chronic MS.
Disclosure: Alexandru Tatomir: nothing to disclose.
Cosmin Tegla: nothing to disclose.
Dallas Boodhoo: nothing to disclose.
Vinh Nguyen: nothing to disclose.
Amurgam Mekala: nothing to disclose.
Freidrich Anselmo: nothing to disclose.
Cornelia Cudrici: nothing to disclose.
Tudor Badea: nothing to disclose.
Violeta Rus: nothing to disclose.
Horea Rus: nothing to disclose.
This work was supported in part by Veterans Administration Merit Award I01BX001458 (to H.R.).