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Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system.

Dubreuil CI, Winton MJ, McKerracher L - J. Cell Biol. (2003)

Bottom Line: After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia.Treatment with C3-05 blocked the increase in p75NTR expression.Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.

ABSTRACT
Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR- mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

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Rho is activated after SCI by a p75NTR-dependent mechanism. (A) p75NTR-labeled cells colocalize with active GTP-Rho in injured spinal cord. Cells labeled with p75NTR (red) and GST-RBD (green) in gray matter (top) and in white matter (bottom). Bars, 100 μM. (B) Rho activation after SCI in normal and p75NTR−/− mice. Active GTP-RhoA was isolated by pull-down assay and detected by immunoblotting with anti-RhoA antibody (top). In p75NTR−/− mice, 24 h after injury only basal levels of active Rho are detected compared with normal mice. Paired samples were run on the same gel, and blots were developed under the same conditions. Total Rho in the tissue homogenates from the same animals was detected by immunoblotting with anti-RhoA antibody. MAG was detected in the same homogenates by Western blot (apparent MW 100 kD). The p75NTR levels (apparent MW 75 kD) are shown in bottom panel. In control uninjured animals low levels of p75NTR are detected, with p75NTR only being up-regulated after injury. (C) Active Rho is detected in p75NTR−/− mice 3 d after SCI.
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fig7: Rho is activated after SCI by a p75NTR-dependent mechanism. (A) p75NTR-labeled cells colocalize with active GTP-Rho in injured spinal cord. Cells labeled with p75NTR (red) and GST-RBD (green) in gray matter (top) and in white matter (bottom). Bars, 100 μM. (B) Rho activation after SCI in normal and p75NTR−/− mice. Active GTP-RhoA was isolated by pull-down assay and detected by immunoblotting with anti-RhoA antibody (top). In p75NTR−/− mice, 24 h after injury only basal levels of active Rho are detected compared with normal mice. Paired samples were run on the same gel, and blots were developed under the same conditions. Total Rho in the tissue homogenates from the same animals was detected by immunoblotting with anti-RhoA antibody. MAG was detected in the same homogenates by Western blot (apparent MW 100 kD). The p75NTR levels (apparent MW 75 kD) are shown in bottom panel. In control uninjured animals low levels of p75NTR are detected, with p75NTR only being up-regulated after injury. (C) Active Rho is detected in p75NTR−/− mice 3 d after SCI.

Mentions: It has been shown recently that MAG activates Rho in the presence of p75NTR (Yamashita et al., 2002) and that MAG interacts with neuronal lipid rafts containing NgR, GT1b, p75NTR, and Rho (Vinson et al., 2003). We have shown that after SCI, MAG is present at the lesion sites (Fig. 2 D). To determine the mechanism by which Rho is activated after injury, we examined if Rho activation was p75NTR dependent. We first examined if p75NTR was present in cells containing active Rho. We found that p75NTR colocalizes with active Rho in both gray (Fig. 7 A, top) and white matter (Fig. 7 A, bottom) 24 h after SCI. We then examined levels of active Rho in mice lacking the p75NTR gene (p75NTR−/−). No change in Rho activation was detected 24 h after SCI (Fig. 7 B). Rho activation was, however, detected in these animals 3 d after injury (Fig. 7 C). These results indicate that Rho is activated through a p75NTR-dependent mechanism early after SCI, but at later time points p75NTR-independent activation occurs.


Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system.

Dubreuil CI, Winton MJ, McKerracher L - J. Cell Biol. (2003)

Rho is activated after SCI by a p75NTR-dependent mechanism. (A) p75NTR-labeled cells colocalize with active GTP-Rho in injured spinal cord. Cells labeled with p75NTR (red) and GST-RBD (green) in gray matter (top) and in white matter (bottom). Bars, 100 μM. (B) Rho activation after SCI in normal and p75NTR−/− mice. Active GTP-RhoA was isolated by pull-down assay and detected by immunoblotting with anti-RhoA antibody (top). In p75NTR−/− mice, 24 h after injury only basal levels of active Rho are detected compared with normal mice. Paired samples were run on the same gel, and blots were developed under the same conditions. Total Rho in the tissue homogenates from the same animals was detected by immunoblotting with anti-RhoA antibody. MAG was detected in the same homogenates by Western blot (apparent MW 100 kD). The p75NTR levels (apparent MW 75 kD) are shown in bottom panel. In control uninjured animals low levels of p75NTR are detected, with p75NTR only being up-regulated after injury. (C) Active Rho is detected in p75NTR−/− mice 3 d after SCI.
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fig7: Rho is activated after SCI by a p75NTR-dependent mechanism. (A) p75NTR-labeled cells colocalize with active GTP-Rho in injured spinal cord. Cells labeled with p75NTR (red) and GST-RBD (green) in gray matter (top) and in white matter (bottom). Bars, 100 μM. (B) Rho activation after SCI in normal and p75NTR−/− mice. Active GTP-RhoA was isolated by pull-down assay and detected by immunoblotting with anti-RhoA antibody (top). In p75NTR−/− mice, 24 h after injury only basal levels of active Rho are detected compared with normal mice. Paired samples were run on the same gel, and blots were developed under the same conditions. Total Rho in the tissue homogenates from the same animals was detected by immunoblotting with anti-RhoA antibody. MAG was detected in the same homogenates by Western blot (apparent MW 100 kD). The p75NTR levels (apparent MW 75 kD) are shown in bottom panel. In control uninjured animals low levels of p75NTR are detected, with p75NTR only being up-regulated after injury. (C) Active Rho is detected in p75NTR−/− mice 3 d after SCI.
Mentions: It has been shown recently that MAG activates Rho in the presence of p75NTR (Yamashita et al., 2002) and that MAG interacts with neuronal lipid rafts containing NgR, GT1b, p75NTR, and Rho (Vinson et al., 2003). We have shown that after SCI, MAG is present at the lesion sites (Fig. 2 D). To determine the mechanism by which Rho is activated after injury, we examined if Rho activation was p75NTR dependent. We first examined if p75NTR was present in cells containing active Rho. We found that p75NTR colocalizes with active Rho in both gray (Fig. 7 A, top) and white matter (Fig. 7 A, bottom) 24 h after SCI. We then examined levels of active Rho in mice lacking the p75NTR gene (p75NTR−/−). No change in Rho activation was detected 24 h after SCI (Fig. 7 B). Rho activation was, however, detected in these animals 3 d after injury (Fig. 7 C). These results indicate that Rho is activated through a p75NTR-dependent mechanism early after SCI, but at later time points p75NTR-independent activation occurs.

Bottom Line: After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia.Treatment with C3-05 blocked the increase in p75NTR expression.Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, QC H3T 1J4, Canada.

ABSTRACT
Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR- mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

Show MeSH
Related in: MedlinePlus