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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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RhoA is activated when cells are plated on growth inhibitory substrates. (A) Rho activation levels were examined in vitro in PC-12 cells plated on either poly-l-lysine, myelin (8 μg), or MAG (8 μg). Active GTP-bound RhoA was isolated by pull-down assay 24 h after the cells were plated on substrates and detected by immunoblotting with anti-RhoA antibody. Total Rho levels were determined from whole cell lysates as shown in the bottom panel. (B) Reversal of Rho activation by treatment of cells with C3–05. PC-12 cells plated on myelin were treated with C3–05 (1 μg/ml), and Rho-GTP levels were detected by pull-down assay. The middle panel shows total Rho levels, and the bottom panel shows whole cell lysates probed with an anti-C3 antibody. Samples for pull-down assays and total Rho and C3 blots were from the same homogenates. (C) Pull-down assay with GST-RBD without lysate. Beads incubated with buffer only show no active Rho; only GST-RBD band is detected when blot is overexposed.
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fig1: RhoA is activated when cells are plated on growth inhibitory substrates. (A) Rho activation levels were examined in vitro in PC-12 cells plated on either poly-l-lysine, myelin (8 μg), or MAG (8 μg). Active GTP-bound RhoA was isolated by pull-down assay 24 h after the cells were plated on substrates and detected by immunoblotting with anti-RhoA antibody. Total Rho levels were determined from whole cell lysates as shown in the bottom panel. (B) Reversal of Rho activation by treatment of cells with C3–05. PC-12 cells plated on myelin were treated with C3–05 (1 μg/ml), and Rho-GTP levels were detected by pull-down assay. The middle panel shows total Rho levels, and the bottom panel shows whole cell lysates probed with an anti-C3 antibody. Samples for pull-down assays and total Rho and C3 blots were from the same homogenates. (C) Pull-down assay with GST-RBD without lysate. Beads incubated with buffer only show no active Rho; only GST-RBD band is detected when blot is overexposed.

Mentions: To examine the effect of growth-inhibitory proteins on Rho activation, we plated PC-12 cells on myelin, MAG, or poly-l-lysine substrates. We measured amounts of GTP-Rho in cell lysates by precipitation with RBD from rhotekin that binds only GTP-bound Rho (Reid et al., 1996). Cells plated on inhibitory substrates had high endogenous Rho-GTP levels compared with poly-l-lysine controls (Fig. 1 A). The activation of Rho in cells plated on myelin or MAG was reversed by treatment with the Rho antagonist C3–05 (Fig. 1 B). The RBD beads incubated without lysate (buffer only) show no active Rho when overexposed GST-RBD is detectable (Fig. 1 C), showing the specificity of the assay for Rho. Treatment of neuronal cells with C3–05 promotes neurite outgrowth on MAG or myelin substrates (Winton et al., 2002). Our results with MAG and myelin are consistent with recent studies showing Rho activation in the presence of Nogo (Niederost et al., 2002) or upon activation of Nogo receptor (Wang et al., 2002).


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)

RhoA is activated when cells are plated on growth inhibitory substrates. (A) Rho activation levels were examined in vitro in PC-12 cells plated on either poly-l-lysine, myelin (8 μg), or MAG (8 μg). Active GTP-bound RhoA was isolated by pull-down assay 24 h after the cells were plated on substrates and detected by immunoblotting with anti-RhoA antibody. Total Rho levels were determined from whole cell lysates as shown in the bottom panel. (B) Reversal of Rho activation by treatment of cells with C3–05. PC-12 cells plated on myelin were treated with C3–05 (1 μg/ml), and Rho-GTP levels were detected by pull-down assay. The middle panel shows total Rho levels, and the bottom panel shows whole cell lysates probed with an anti-C3 antibody. Samples for pull-down assays and total Rho and C3 blots were from the same homogenates. (C) Pull-down assay with GST-RBD without lysate. Beads incubated with buffer only show no active Rho; only GST-RBD band is detected when blot is overexposed.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: RhoA is activated when cells are plated on growth inhibitory substrates. (A) Rho activation levels were examined in vitro in PC-12 cells plated on either poly-l-lysine, myelin (8 μg), or MAG (8 μg). Active GTP-bound RhoA was isolated by pull-down assay 24 h after the cells were plated on substrates and detected by immunoblotting with anti-RhoA antibody. Total Rho levels were determined from whole cell lysates as shown in the bottom panel. (B) Reversal of Rho activation by treatment of cells with C3–05. PC-12 cells plated on myelin were treated with C3–05 (1 μg/ml), and Rho-GTP levels were detected by pull-down assay. The middle panel shows total Rho levels, and the bottom panel shows whole cell lysates probed with an anti-C3 antibody. Samples for pull-down assays and total Rho and C3 blots were from the same homogenates. (C) Pull-down assay with GST-RBD without lysate. Beads incubated with buffer only show no active Rho; only GST-RBD band is detected when blot is overexposed.
Mentions: To examine the effect of growth-inhibitory proteins on Rho activation, we plated PC-12 cells on myelin, MAG, or poly-l-lysine substrates. We measured amounts of GTP-Rho in cell lysates by precipitation with RBD from rhotekin that binds only GTP-bound Rho (Reid et al., 1996). Cells plated on inhibitory substrates had high endogenous Rho-GTP levels compared with poly-l-lysine controls (Fig. 1 A). The activation of Rho in cells plated on myelin or MAG was reversed by treatment with the Rho antagonist C3–05 (Fig. 1 B). The RBD beads incubated without lysate (buffer only) show no active Rho when overexposed GST-RBD is detectable (Fig. 1 C), showing the specificity of the assay for Rho. Treatment of neuronal cells with C3–05 promotes neurite outgrowth on MAG or myelin substrates (Winton et al., 2002). Our results with MAG and myelin are consistent with recent studies showing Rho activation in the presence of Nogo (Niederost et al., 2002) or upon activation of Nogo receptor (Wang et al., 2002).

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