Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of Rho activation with C3–05 protects cells from apoptosis. (A) Sections of injured spinal cords from mouse (top) and rat (bottom) were double labeled with specific cell markers NeuN, GFAP, or MAB328 (red) and by TUNEL (green) to detect apoptotic cells. Bar, 50 μm. (B) Treatment of injured spinal cord with C3–05 significantly decreased the number of TUNEL-positive cells counted in both mice (right) and rats (left). TUNEL-positive cells were counted in 40–50 sections per animal taken from a 3- or 4-mm segment of the lesion site in mice and rats, respectively, with three animals examined per group. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Sections from rat spinal cord showing that most C3 immunostained cells were not TUNEL positive. Bar, 50 μm. (D) C3-labeled cells are less likely to be TUNEL positive. C3 and TUNEL cells were counted and compared with the number of TUNEL-labeled cells in C3–05–treated animals.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172802&req=5

fig6: Inhibition of Rho activation with C3–05 protects cells from apoptosis. (A) Sections of injured spinal cords from mouse (top) and rat (bottom) were double labeled with specific cell markers NeuN, GFAP, or MAB328 (red) and by TUNEL (green) to detect apoptotic cells. Bar, 50 μm. (B) Treatment of injured spinal cord with C3–05 significantly decreased the number of TUNEL-positive cells counted in both mice (right) and rats (left). TUNEL-positive cells were counted in 40–50 sections per animal taken from a 3- or 4-mm segment of the lesion site in mice and rats, respectively, with three animals examined per group. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Sections from rat spinal cord showing that most C3 immunostained cells were not TUNEL positive. Bar, 50 μm. (D) C3-labeled cells are less likely to be TUNEL positive. C3 and TUNEL cells were counted and compared with the number of TUNEL-labeled cells in C3–05–treated animals.

Mentions: Both neurons and glia undergo apoptosis after SCI in rat, which leads to the formation of a large lesion cavity (Liu et al., 1997; Shuman et al., 1997; Grossman et al., 2001). Even though mice do not develop cavitation at the site of SCI, we detected apoptotic neurons, astrocytes, and oligodendrocytes by double staining with cell-specific markers and TUNEL (Fig. 6 A, top), similar to that observed after rat SCI (Fig. 6 A, bottom). Importantly, in both mice and rats treated with C3–05, the number of TUNEL-labeled cells was significantly reduced by ∼50% after SCI (Fig. 6 B). Not only was C3–05 present in neurons, astrocytes, and oligodendrocytes (Fig. 3 C), but most cells containing C3–05 were not TUNEL positive (Fig. 6, C and D). The small number of cells double labeled with C3 and TUNEL (16%) suggests that C3–05 penetrated into some cells that had progressed too far into the apoptotic cascade to be rescued from death. Together our results indicate that inactivation of Rho after SCI protects cells from apoptosis. These findings have clinical relevance because neuroprotective treatments after SCI lead to improved functional recovery (Liu et al., 1997).


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)

Inhibition of Rho activation with C3–05 protects cells from apoptosis. (A) Sections of injured spinal cords from mouse (top) and rat (bottom) were double labeled with specific cell markers NeuN, GFAP, or MAB328 (red) and by TUNEL (green) to detect apoptotic cells. Bar, 50 μm. (B) Treatment of injured spinal cord with C3–05 significantly decreased the number of TUNEL-positive cells counted in both mice (right) and rats (left). TUNEL-positive cells were counted in 40–50 sections per animal taken from a 3- or 4-mm segment of the lesion site in mice and rats, respectively, with three animals examined per group. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Sections from rat spinal cord showing that most C3 immunostained cells were not TUNEL positive. Bar, 50 μm. (D) C3-labeled cells are less likely to be TUNEL positive. C3 and TUNEL cells were counted and compared with the number of TUNEL-labeled cells in C3–05–treated animals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172802&req=5

fig6: Inhibition of Rho activation with C3–05 protects cells from apoptosis. (A) Sections of injured spinal cords from mouse (top) and rat (bottom) were double labeled with specific cell markers NeuN, GFAP, or MAB328 (red) and by TUNEL (green) to detect apoptotic cells. Bar, 50 μm. (B) Treatment of injured spinal cord with C3–05 significantly decreased the number of TUNEL-positive cells counted in both mice (right) and rats (left). TUNEL-positive cells were counted in 40–50 sections per animal taken from a 3- or 4-mm segment of the lesion site in mice and rats, respectively, with three animals examined per group. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Sections from rat spinal cord showing that most C3 immunostained cells were not TUNEL positive. Bar, 50 μm. (D) C3-labeled cells are less likely to be TUNEL positive. C3 and TUNEL cells were counted and compared with the number of TUNEL-labeled cells in C3–05–treated animals.
Mentions: Both neurons and glia undergo apoptosis after SCI in rat, which leads to the formation of a large lesion cavity (Liu et al., 1997; Shuman et al., 1997; Grossman et al., 2001). Even though mice do not develop cavitation at the site of SCI, we detected apoptotic neurons, astrocytes, and oligodendrocytes by double staining with cell-specific markers and TUNEL (Fig. 6 A, top), similar to that observed after rat SCI (Fig. 6 A, bottom). Importantly, in both mice and rats treated with C3–05, the number of TUNEL-labeled cells was significantly reduced by ∼50% after SCI (Fig. 6 B). Not only was C3–05 present in neurons, astrocytes, and oligodendrocytes (Fig. 3 C), but most cells containing C3–05 were not TUNEL positive (Fig. 6, C and D). The small number of cells double labeled with C3 and TUNEL (16%) suggests that C3–05 penetrated into some cells that had progressed too far into the apoptotic cascade to be rescued from death. Together our results indicate that inactivation of Rho after SCI protects cells from apoptosis. These findings have clinical relevance because neuroprotective treatments after SCI lead to improved functional recovery (Liu et al., 1997).

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