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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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Treatment with the Rho antagonist C3–05 after contusion or transection of the spinal cord reverses RhoA activation after injury. (A) Injection of C3–05 into the injury site reversed RhoA activation to basal levels after SCI. Active GTP-RhoA was isolated by pull-down assay and detected with antibodies specific for RhoA. Total RhoA from the same animals was detected by immunoblot. Anti-C3 antibody immunoblot of the same homogenate showed C3–05 was detected at the lesion site for 7 d (C3–05). The same homogenates were used to determine levels of Rho and C3. (B) Densitometric analysis of the reversal of Rho activation by C3–05 after mouse hemisection (n = 2); rat contusion (n = 3); rat transection after 24 h (n = 3); rat transection after 3 d (n = 3); and rat transection after 7 d (n = 2). n represents the number of animals. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Double immunocytochemistry with cell-type specific markers (red) and a specific antibody against C3 (green). Neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) show C3 immunoreactivity within cells in injured rat spinal cord treated with C3–05. Bars, 50 μm.
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fig3: Treatment with the Rho antagonist C3–05 after contusion or transection of the spinal cord reverses RhoA activation after injury. (A) Injection of C3–05 into the injury site reversed RhoA activation to basal levels after SCI. Active GTP-RhoA was isolated by pull-down assay and detected with antibodies specific for RhoA. Total RhoA from the same animals was detected by immunoblot. Anti-C3 antibody immunoblot of the same homogenate showed C3–05 was detected at the lesion site for 7 d (C3–05). The same homogenates were used to determine levels of Rho and C3. (B) Densitometric analysis of the reversal of Rho activation by C3–05 after mouse hemisection (n = 2); rat contusion (n = 3); rat transection after 24 h (n = 3); rat transection after 3 d (n = 3); and rat transection after 7 d (n = 2). n represents the number of animals. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Double immunocytochemistry with cell-type specific markers (red) and a specific antibody against C3 (green). Neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) show C3 immunoreactivity within cells in injured rat spinal cord treated with C3–05. Bars, 50 μm.

Mentions: To investigate RhoA activation states after traumatic CNS injury, we measured active RhoA levels in rodent tissue homogenates by Rho pull-down assay. We studied tissue isolated from regions of traumatically injured spinal cords of both rats and mice because of their different responses to injury. Rats develop an extensive necrotic lesion cavity after SCI, whereas mice do not (Steward et al., 1999). In rats, we examined spinal cord regions after transection or contusion injury. Experiments are shown as paired control and injured CNS samples (Figs. 2 and 3). Homogenates from different animals were not pooled, and each gel lane represents results from one animal. In uninjured CNS tissue, GTP-Rho levels were consistently low (Fig. 2, controls). By contrast, Rho activation is dramatically increased after injury (Fig. 2, A and B), increasing over 10-fold (Fig. 2 C). Expression levels of total RhoA, as detected by Western blots from tissue homogenates used for isolation of GTP-Rho, did not change (Fig. 2, A and B). These results show that Rho is massively activated in CNS tissue of rats and mice after SCI compared with uninjured spinal cord. To examine if the activation of Rho after SCI injury was sustained or transient, we prepared homogenates from transected spinal cord 1.5 h, 24 h, 3 d, and 7 d after lesion. Interestingly, we found that Rho was active as early as 1.5 h after injury. The significant increase in activation observed by 24 h was sustained for at least 7 d (Fig. 2, B and C).


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)

Treatment with the Rho antagonist C3–05 after contusion or transection of the spinal cord reverses RhoA activation after injury. (A) Injection of C3–05 into the injury site reversed RhoA activation to basal levels after SCI. Active GTP-RhoA was isolated by pull-down assay and detected with antibodies specific for RhoA. Total RhoA from the same animals was detected by immunoblot. Anti-C3 antibody immunoblot of the same homogenate showed C3–05 was detected at the lesion site for 7 d (C3–05). The same homogenates were used to determine levels of Rho and C3. (B) Densitometric analysis of the reversal of Rho activation by C3–05 after mouse hemisection (n = 2); rat contusion (n = 3); rat transection after 24 h (n = 3); rat transection after 3 d (n = 3); and rat transection after 7 d (n = 2). n represents the number of animals. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Double immunocytochemistry with cell-type specific markers (red) and a specific antibody against C3 (green). Neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) show C3 immunoreactivity within cells in injured rat spinal cord treated with C3–05. Bars, 50 μm.
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fig3: Treatment with the Rho antagonist C3–05 after contusion or transection of the spinal cord reverses RhoA activation after injury. (A) Injection of C3–05 into the injury site reversed RhoA activation to basal levels after SCI. Active GTP-RhoA was isolated by pull-down assay and detected with antibodies specific for RhoA. Total RhoA from the same animals was detected by immunoblot. Anti-C3 antibody immunoblot of the same homogenate showed C3–05 was detected at the lesion site for 7 d (C3–05). The same homogenates were used to determine levels of Rho and C3. (B) Densitometric analysis of the reversal of Rho activation by C3–05 after mouse hemisection (n = 2); rat contusion (n = 3); rat transection after 24 h (n = 3); rat transection after 3 d (n = 3); and rat transection after 7 d (n = 2). n represents the number of animals. *P < 0.05 compared with lesion without treatment; P value determined by unpaired t test. (C) Double immunocytochemistry with cell-type specific markers (red) and a specific antibody against C3 (green). Neurons (NeuN), astrocytes (GFAP), and oligodendrocytes (MAB328) show C3 immunoreactivity within cells in injured rat spinal cord treated with C3–05. Bars, 50 μm.
Mentions: To investigate RhoA activation states after traumatic CNS injury, we measured active RhoA levels in rodent tissue homogenates by Rho pull-down assay. We studied tissue isolated from regions of traumatically injured spinal cords of both rats and mice because of their different responses to injury. Rats develop an extensive necrotic lesion cavity after SCI, whereas mice do not (Steward et al., 1999). In rats, we examined spinal cord regions after transection or contusion injury. Experiments are shown as paired control and injured CNS samples (Figs. 2 and 3). Homogenates from different animals were not pooled, and each gel lane represents results from one animal. In uninjured CNS tissue, GTP-Rho levels were consistently low (Fig. 2, controls). By contrast, Rho activation is dramatically increased after injury (Fig. 2, A and B), increasing over 10-fold (Fig. 2 C). Expression levels of total RhoA, as detected by Western blots from tissue homogenates used for isolation of GTP-Rho, did not change (Fig. 2, A and B). These results show that Rho is massively activated in CNS tissue of rats and mice after SCI compared with uninjured spinal cord. To examine if the activation of Rho after SCI injury was sustained or transient, we prepared homogenates from transected spinal cord 1.5 h, 24 h, 3 d, and 7 d after lesion. Interestingly, we found that Rho was active as early as 1.5 h after injury. The significant increase in activation observed by 24 h was sustained for at least 7 d (Fig. 2, B and C).

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