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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 active in neurons and glial cells after SCI, detected by in situ pull-down assay. (A) Control uninjured animals (control + GST-RBD) and C3–05 treatment of animals after SCI (C3–05 + GST-RBD) probed with GST-RBD show no active Rho. Sections of animals after SCI incubated with lysate from empty pGEX vector expressing only GST protein (lesion + GST-pGEX) show only background levels of GST in the spinal cord and no active Rho. Bar, 50 μm. (B) Double labeling of spinal cord with cell type–specific markers (red) and GST antibody to detect GST-RBD (green). At all time points tested (24 h, 3 d and 7d), GTP-Rho was detected in neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) after SCI. Bars, 50 μm.
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fig5: Rho is active in neurons and glial cells after SCI, detected by in situ pull-down assay. (A) Control uninjured animals (control + GST-RBD) and C3–05 treatment of animals after SCI (C3–05 + GST-RBD) probed with GST-RBD show no active Rho. Sections of animals after SCI incubated with lysate from empty pGEX vector expressing only GST protein (lesion + GST-pGEX) show only background levels of GST in the spinal cord and no active Rho. Bar, 50 μm. (B) Double labeling of spinal cord with cell type–specific markers (red) and GST antibody to detect GST-RBD (green). At all time points tested (24 h, 3 d and 7d), GTP-Rho was detected in neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) after SCI. Bars, 50 μm.

Mentions: Although the location of C3–05 can indicate the potential to suppress Rho activation, it does not permit us to determine which cells have increased Rho activation after SCI. To further examine increased GTP-Rho after SCI, we used a modified in situ pull-down method, omitting cell transfection with recombinant Rho (Li et al., 2002) to detect endogenous Rho activation levels. We incubated sections with GST-RBD, and cells that bound high levels of RBD were detected with an anti-GST antibody. Active Rho was detected in many cells in both the gray (Fig. 4, panels 1 and 2) and white matter (Fig. 4, panel 3) of injured spinal cord. We also found that Rho was activated both rostral (Fig. 4, panel 1) and caudal (Fig. 4, panel 2) to the lesion site. At further distances from the lesion site, staining for active Rho was very faint or absent (Fig. 4, panel 4). Rho-GTP was not detected in uninjured spinal cord (Fig. 5 A, left) or after C3–05 treatment was used to reverse the increase in Rho activation after SCI (Fig. 5 A, middle). To assess the specificity of the technique, we incubated sections with GST without RBD, and no positive cellular active Rho staining is visible (Fig. 5 A, right).


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 active in neurons and glial cells after SCI, detected by in situ pull-down assay. (A) Control uninjured animals (control + GST-RBD) and C3–05 treatment of animals after SCI (C3–05 + GST-RBD) probed with GST-RBD show no active Rho. Sections of animals after SCI incubated with lysate from empty pGEX vector expressing only GST protein (lesion + GST-pGEX) show only background levels of GST in the spinal cord and no active Rho. Bar, 50 μm. (B) Double labeling of spinal cord with cell type–specific markers (red) and GST antibody to detect GST-RBD (green). At all time points tested (24 h, 3 d and 7d), GTP-Rho was detected in neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) after SCI. Bars, 50 μm.
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fig5: Rho is active in neurons and glial cells after SCI, detected by in situ pull-down assay. (A) Control uninjured animals (control + GST-RBD) and C3–05 treatment of animals after SCI (C3–05 + GST-RBD) probed with GST-RBD show no active Rho. Sections of animals after SCI incubated with lysate from empty pGEX vector expressing only GST protein (lesion + GST-pGEX) show only background levels of GST in the spinal cord and no active Rho. Bar, 50 μm. (B) Double labeling of spinal cord with cell type–specific markers (red) and GST antibody to detect GST-RBD (green). At all time points tested (24 h, 3 d and 7d), GTP-Rho was detected in neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) after SCI. Bars, 50 μm.
Mentions: Although the location of C3–05 can indicate the potential to suppress Rho activation, it does not permit us to determine which cells have increased Rho activation after SCI. To further examine increased GTP-Rho after SCI, we used a modified in situ pull-down method, omitting cell transfection with recombinant Rho (Li et al., 2002) to detect endogenous Rho activation levels. We incubated sections with GST-RBD, and cells that bound high levels of RBD were detected with an anti-GST antibody. Active Rho was detected in many cells in both the gray (Fig. 4, panels 1 and 2) and white matter (Fig. 4, panel 3) of injured spinal cord. We also found that Rho was activated both rostral (Fig. 4, panel 1) and caudal (Fig. 4, panel 2) to the lesion site. At further distances from the lesion site, staining for active Rho was very faint or absent (Fig. 4, panel 4). Rho-GTP was not detected in uninjured spinal cord (Fig. 5 A, left) or after C3–05 treatment was used to reverse the increase in Rho activation after SCI (Fig. 5 A, middle). To assess the specificity of the technique, we incubated sections with GST without RBD, and no positive cellular active Rho staining is visible (Fig. 5 A, right).

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