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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 regulates p75NTR expression after injury. (A) Transverse sections throughout rat lesion sites show cells double labeled with p75NTR-specific antibody (red) and with TUNEL (green). Bar, 50 μm. (B) The number of p75NTR-labeled cells (left) and p75NTR cells positive for TUNEL (right) in transverse sections of rat spinal cord after injury. (C) The p75NTR protein levels increase after SCI but not after treatment with C3–05. Detection of p75NTR by Western blot after SCI and treatment with C3–05. The same tissue homogenates used to show active Rho, shown in bottom panel, were probed with a p75NTR-specific polyclonal antibody (top) and an anti-C3 antibody (panel 2). RhoA in whole tissue homogenate from the same animals is also shown (panel 3). Last panel shows GTP-bound active Rho.
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fig8: Rho regulates p75NTR expression after injury. (A) Transverse sections throughout rat lesion sites show cells double labeled with p75NTR-specific antibody (red) and with TUNEL (green). Bar, 50 μm. (B) The number of p75NTR-labeled cells (left) and p75NTR cells positive for TUNEL (right) in transverse sections of rat spinal cord after injury. (C) The p75NTR protein levels increase after SCI but not after treatment with C3–05. Detection of p75NTR by Western blot after SCI and treatment with C3–05. The same tissue homogenates used to show active Rho, shown in bottom panel, were probed with a p75NTR-specific polyclonal antibody (top) and an anti-C3 antibody (panel 2). RhoA in whole tissue homogenate from the same animals is also shown (panel 3). Last panel shows GTP-bound active Rho.

Mentions: The p75NTR has been implicated in the regulation of apoptosis after injury in the nervous system (Cheema et al., 1996; Frade and Barde, 1999; Dechant and Barde, 2002). In addition to counting TUNEL cells after SCI (Fig. 6 B), we counted p75NTR-labeled cells and double labeled cells. Many cells express p75NTR after SCI (Fig. 8 B), and 74% of these cells were also TUNEL positive. Treatment with C3–05 resulted in a decrease in p75NTR alone and in p75NTR and TUNEL (Fig. 8, A and B). These results suggest a correlation between Rho activation, p75NTR expression, and cell death. To further investigate the involvement of p75NTR in Rho activation after SCI, we probed the homogenates of transected spinal cord with p75NTR-specific antibodies using the same homogenates as shown in the bottom panel of Fig. 8 C. There was very low p75NTR detected in Western blots in the adult spinal cord (Fig. 7 B and Fig. 8 C, controls). Levels of p75NTR increased as early as 24 h after SCI, and high levels were detected 3 and 7 d postinjury, a finding consistent with previous reports of p75NTR up-regulation after SCI (Casha et al., 2001; Widenfalk et al., 2001; Beattie et al., 2002). Treatment with C3–05 not only blocked the increase in p75NTR protein levels after SCI, as detected by Western blot (Fig. 8 C), but also reduced the number of p75NTR-TUNEL–labeled cells (Fig. 8 B). These results suggest, that early on, Rho activation after SCI is mediated, at least in part, by p75NTR. These results also indicate that Rho activation is important for p75NTR up-regulation after SCI.


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 regulates p75NTR expression after injury. (A) Transverse sections throughout rat lesion sites show cells double labeled with p75NTR-specific antibody (red) and with TUNEL (green). Bar, 50 μm. (B) The number of p75NTR-labeled cells (left) and p75NTR cells positive for TUNEL (right) in transverse sections of rat spinal cord after injury. (C) The p75NTR protein levels increase after SCI but not after treatment with C3–05. Detection of p75NTR by Western blot after SCI and treatment with C3–05. The same tissue homogenates used to show active Rho, shown in bottom panel, were probed with a p75NTR-specific polyclonal antibody (top) and an anti-C3 antibody (panel 2). RhoA in whole tissue homogenate from the same animals is also shown (panel 3). Last panel shows GTP-bound active Rho.
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Related In: Results  -  Collection

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fig8: Rho regulates p75NTR expression after injury. (A) Transverse sections throughout rat lesion sites show cells double labeled with p75NTR-specific antibody (red) and with TUNEL (green). Bar, 50 μm. (B) The number of p75NTR-labeled cells (left) and p75NTR cells positive for TUNEL (right) in transverse sections of rat spinal cord after injury. (C) The p75NTR protein levels increase after SCI but not after treatment with C3–05. Detection of p75NTR by Western blot after SCI and treatment with C3–05. The same tissue homogenates used to show active Rho, shown in bottom panel, were probed with a p75NTR-specific polyclonal antibody (top) and an anti-C3 antibody (panel 2). RhoA in whole tissue homogenate from the same animals is also shown (panel 3). Last panel shows GTP-bound active Rho.
Mentions: The p75NTR has been implicated in the regulation of apoptosis after injury in the nervous system (Cheema et al., 1996; Frade and Barde, 1999; Dechant and Barde, 2002). In addition to counting TUNEL cells after SCI (Fig. 6 B), we counted p75NTR-labeled cells and double labeled cells. Many cells express p75NTR after SCI (Fig. 8 B), and 74% of these cells were also TUNEL positive. Treatment with C3–05 resulted in a decrease in p75NTR alone and in p75NTR and TUNEL (Fig. 8, A and B). These results suggest a correlation between Rho activation, p75NTR expression, and cell death. To further investigate the involvement of p75NTR in Rho activation after SCI, we probed the homogenates of transected spinal cord with p75NTR-specific antibodies using the same homogenates as shown in the bottom panel of Fig. 8 C. There was very low p75NTR detected in Western blots in the adult spinal cord (Fig. 7 B and Fig. 8 C, controls). Levels of p75NTR increased as early as 24 h after SCI, and high levels were detected 3 and 7 d postinjury, a finding consistent with previous reports of p75NTR up-regulation after SCI (Casha et al., 2001; Widenfalk et al., 2001; Beattie et al., 2002). Treatment with C3–05 not only blocked the increase in p75NTR protein levels after SCI, as detected by Western blot (Fig. 8 C), but also reduced the number of p75NTR-TUNEL–labeled cells (Fig. 8 B). These results suggest, that early on, Rho activation after SCI is mediated, at least in part, by p75NTR. These results also indicate that Rho activation is important for p75NTR up-regulation after SCI.

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