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Address correspondence to Lisa McKerracher, Université de Montréal, 2900 Edouard-Montpetit, Faculté de médecine, Département de pathologie et biologie cellulaire, Montréal, QC H3T 1J4, Canada. Tel.: (514) 343-6111, ex. 1472. Fax: (514) 282-9990. E-mail: mckerral{at}patho.umontreal.ca
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Abstract |
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Key Words: spinal cord injury; RhoA; apoptosis; MAG; p75NTR
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Introduction |
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Early experiments demonstrated that lysophosphatidic acid causes neurite retraction and cell rounding by activating Rho (Jalink et al., 1994; Tigyi et al., 1996). The use of C3 transferase to inactivate Rho in primary neurons plated on various types of inhibitory proteins and dominant-negative Rho-expressing PC-12 cells provides direct evidence that the inactivation of Rho results in neurite outgrowth on inhibitory substrates. In vivo experiments in rats and mice have shown that inactivation of Rho or of Rho kinase promotes axon regeneration and functional recovery after spinal cord injury (SCI) in rats and mice (Lehmann et al., 1999; Hara et al., 2000; Dergham et al., 2002). However, it is not known how CNS injury may affect Rho expression and activation.
The mechanism where growth inhibitory proteins may affect Rho signaling are beginning to be understood. It has been shown recently that NgR can activate Rho in a p75 neurotrophin receptor (p75NTR) dependent manner (Wang et al., 2002). First, p75NTR-null mutant mice are not inhibited by MAG, showing a key role of p75NTR in growth inhibitory signaling by MAG (Yamashita et al., 2002). Also, Rho binds to p75NTR (Yamashita et al., 1999), and Rho is likely to form part of the membrane raft receptor complex responsible for growth inhibitory signaling (McKerracher and Winton, 2002; Woolf and Bloechlinger, 2002). Although p75NTR has been implicated in apoptosis after SCI (Casha et al., 2001), it is not known to what extent Rho signaling by p75NTR participates in apoptotic events after SCI.
Isoforms of Rho exist, and in neurons RhoA is expressed at higher levels than RhoB and RhoC (Lehmann et al., 1999). Therefore, we have focused on RhoA for our studies in neurons. In nonneuronal cells, Rho family GTPases are best characterized for their effects on organization and regulation of the actin cytoskeleton (Ridley, 2001), but they have also been shown to play a role in the regulation of apoptosis (Jimenez et al., 1995; Aznar and Lacal, 2001; Coleman and Olson, 2002). The extent to which Rho may participate in apoptotic pathways in neuronal cells has yet to be determined. In neurons, Rho is activated in response to chemorepulsive molecules (Jin and Strittmatter, 1997; Wahl et al., 2000) and is important in axon guidance during development. In adult neurons, inhibitory substrates (Lehmann et al., 1999; Niederost et al., 2002; Winton et al., 2002) and secreted factors such as TNF (Neumann et al., 2002) can alter Rho activation levels. Levels of Rho expression are altered in malignant disease (Suwa et al., 1998; Fritz et al., 1999; Clark et al., 2000); however, little is known about how traumatic injury and disease in the CNS alter Rho activation states in vivo.
Rho activation can be studied by probing cell homogenates with the Rho-binding domain (RBD) from the Rho-GTPinteracting protein, rhotekin (Reid et al., 1996). We use this pull-down assay to detect a significant increase in active Rho in CNS tissue homogenates after SCI. We show that SCI causes an increase in active Rho without affecting RhoA expression levels. We made use of an in situ pull-down assay (Li et al., 2002) to determine that neurons and glia in the spinal cord show Rho activation. To test the use of a Rho antagonist to reverse Rho activation, we used a cell-permeable form of C3 transferase (C305) that has a short transport sequence added to the COOH terminal to help entry into cells (Winton et al., 2002). We show that C305 specifically inactivates Rho in vivo and prevents up-regulation of p75NTR. Treatment of injured spinal cord with C305 not only effectively reversed Rho activation but also had cell protective effects.
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Results |
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Treatment with C305 reverses Rho activation after injury
To test if we could reverse the increase in Rho activation in injured spinal cord, we made use of the Rho antagonist C305 (Winton et al., 2002). We injected C305 in a fibrin matrix into the lesion site after spinal cord transection, or C305 alone into contused spinal cord, and the lesion sites were removed 24 h later. Treatment with C305 inactivated Rho, bringing the RhoA activation levels back to the normal basal state (Fig. 3, A and B). To determine if the reversal of Rho activation was sustained after a single injection of the compound, we examined rats 7 d after transection injury and treatment. Even 7 d after C305 treatment, Rho activation still remained at basal levels (Fig. 3, A and B). Next, we asked if C305 remained at the lesion site after treatment. Probing the homogenates with a polyclonal antibody raised against C3 (Winton et al., 2002) demonstrated that C305 was detected at the lesion site at all of the time points tested (Fig. 3 A). To determine if endogenous cells in the spinal cord were able to take up and retain C305 after treatment, we examined sections of rat spinal cord double labeled with an antibody specific for C3 and with cell typespecific markers. We detected intracellular C3 immunoreaction in neurons, astrocytes, and oligodendrocytes after injection of C305 (Fig. 3 C), showing that endogenous cells from the spinal cord take up C305 in vivo.
Rho is active in neurons and glial cells after SCI
Although the location of C305 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 C305 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).
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Treatment with C305 protects cells from apoptosis after injury
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 C305, the number of TUNEL-labeled cells was significantly reduced by 50% after SCI (Fig. 6 B). Not only was C305 present in neurons, astrocytes, and oligodendrocytes (Fig. 3 C), but most cells containing C305 were not TUNEL positive (Fig. 6, C and D). The small number of cells double labeled with C3 and TUNEL (16%) suggests that C305 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).
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Discussion |
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Mechanisms for sustained Rho activation after SCI
Activation of Rho in CNS tissue after SCI injury likely results from changes in the local inhibitory and inflammatory environment. Both neurons and glial cells show increased Rho activation. There is evidence that the inhibitory environment of the CNS contributes to increased Rho activation after SCI. First, we showed that neuronal Rho is activated by MAG and myelin when PC-12 cells are plated on inhibitory substrates (Fig. 1). Soluble Nogo fusion proteins can also activate neuronal Rho (Niederost et al., 2002; Fournier et al., 2003). Other evidence indicates that Rho is activated by NgR-independent growth inhibitory proteins. Inactivation of Rho promotes neurite growth on chondroitin sulfate proteoglycans (Dergham et al., 2002) that are present at glial scars. Also, collapsin and ephrins, chemoreplusive factors that act through different receptors, respectively, both activate Rho (Jin and Strittmatter, 1997; Wahl et al., 2000). Preliminary evidence from our lab indicates that astrocytes plated on inhibitory substrates show Rho activation (unpublished data). Therefore, inhibitory proteins may activate Rho in both neurons and glial cells by NgR and NgR-independent mechanisms.
The inflammatory environment may contribute to Rho activation after SCI. Reactive astrocytes secrete TNF, and TNF has been shown to activate Rho in neurons expressing TNF receptors (Neumann et al., 2002). Inflammation after injury is considered to cause secondary damage because it progresses with time and causes continued cell death after the primary traumatic insult (Schwartz and Fehlings, 2002; Popovich and Jones, 2003). Our failure to detect Rho activation in p75NTR-null mutant mice early after SCI (24 h) suggests activation of NgR signaling to p75NTR is an early event in CNS injury. However, 3 d after injury in these mice Rho activation was observed, a finding that indicates that at later time points Rho activation is p75NTR independent. Many factors activate Rho independently of p75NTR such as semaphorins, ephrins, and thrombin that are known to be present after SCI (Donovan et al., 1997; Wahl et al., 2000; De Winter et al., 2002; Shirvan et al., 2002; Swiercz et al., 2002). The p75NTR dependence of early Rho activation is interesting because thrombin and TNF, both known to activate Rho, are p75NTR independent and are present early after SCI (Donovan et al., 1997; Citron et al., 2000; Lee et al., 2000). Our experiments with the p75NTR knockout mice were with whole tissue homogenates and do not address significant changes in individual cell types early after injury. The massive Rho activation we observe in normal mice and rats after SCI likely represents the combined effects of the many different Rho-activating factors. Secondary damage by inflammation may also contribute to activation of Rho, and if this is the case, then Rho may be an important target to prevent secondary inflammatory damage. Our results after treatment with C305 show that inactivation of Rho reduces cell death that follows injury. Further, the massive activation of Rho that we observed after injury was sustained for at least 7 d. Therefore, multiple local signals may activate Rho in CNS cells. We speculate that continued presence of growth inhibitory molecules at the site of a CNS lesion contributes to sustained activation of Rho in neurons and glia after SCI.
Rho activation leads to apoptosis after SCI
Rho-GTPases are known regulators of apoptosis in various cell types. In nonneuronal cells, such as epithelial cells (Fiorentini et al., 1998a, b), endothelial cells (Hippenstiel et al., 2002), T cells (Moorman et al., 1996; Gomez et al., 1997), and some fibroblasts (Bobak et al., 1997), inactivation of Rho causes apoptosis through a Bcl-2dependent mechanism. In other cells, such as PC-12 cells (Mills et al., 1998) and endogenous cells of the spinal cord, as we show here, inactivation of Rho protects cells from apoptosis. In NIH 3T3 fibroblasts, overexpression of active Rho induces cell death upon serum withdrawal (Jimenez et al., 1995). Thus, the cell background is critical in the effect of Rho signaling and cell death. In PC-12 cells, a neural cell line, Rho proteins have been shown to induce Rho-dependent membrane blebbing (Mills et al., 1998), a morphological characteristic of apoptosis. In cultured astrocytes and hippocampal neurons, treatment with thrombin, a protease found after CNS trauma, causes Rho-dependent apoptosis. Treatment with C3 reversed the thrombin-induced apoptosis of astrocytes and neurons by 50% (Donovan et al., 1997). In neurons, TNF activates Rho (Neumann et al., 2002), and antibody-mediated blocking of TNF reduces apoptosis after SCI (Lee et al., 2000). These data support our direct evidence that Rho activation contributes to apoptosis after traumatic SCI. Rho activation in neurons alone may not be sufficient to cause cell death, such as when neurons are plated on inhibitory substrates in culture. Our data suggest that in vivo the combination of multiple Rho-activating factors, including the myelin-derived inhibitory factors, contribute to apoptotic signaling cascades.
Mechanism of Rho activation
Our results show that blocking the activation of Rho after SCI prevents an increased synthesis of p75NTR protein (Fig. 8 C), implicating Rho activation in the transcriptional changes in p75NTR expression. This result of C305 can be explained by a mechanism in which the change in transcriptional factor activation is Rho dependent (Fig. 9). It is known that Rho is involved in the activation of transcription factors in the nucleus that control synthesis of proapoptotic mRNAs such as c-jun and NFß, members of the p75NTR apoptotic cascades (Aznar and Lacal, 2001; Huang and Reichardt, 2001). Therefore, we speculate that treatment with C305 to block Rho activation after injury suppresses apoptosis by preventing the synthesis of proapoptotic proteins such as p75NTR.
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Materials and methods |
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To treat rats with a Rho antagonist, 50 µg of C305 was injected in a fibrin matrix (Tisseel kit; Baxter) into transected spinal cord as described (Dergham et al., 2002). In mice, 10 µg of C305 in fibrin was injected, except for the experiment for TUNEL labeling where 1 µg in fibrin was injected. C305 (50 µg) in PBS without fibrin was injected into rat contusion injury sites. All animal procedures followed guidelines from the Canadian Council of Animal Care.
Cell culture
PC-12 cells were grown in DME with 10% horse serum, 5% FBS, 1% penicillin-streptomycin. PC-12 cells were grown on poly-L-lysine (0.1 µg/ml) (Sigma-Aldrich) or myelin (8 µg per well) or MAG- (8 µg) coated 6-well culture dishes. After the cells settled (36 h at 37°C), the media was aspirated, and fresh media containing the C305 (1 µg/ml) was added to the undifferentiated cultures. The cells were harvested 24 h later, washed with ice cold TBS, and lysed in modified RIPA buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF). Cell lysates were clarified by centrifugation at 13,000 g for 10 min at 4°C and kept at -80°C.
Pull-down assays and immunoblotting
Purification of GST-RBD was performed as described previously (Ren and Schwartz, 2000). Bacteria expressing GST-RBD in a pGEX vector (a gift from John Collard, Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands) were grown in L-broth with 100 µl/ml ampicillin. Overnight cultures were diluted 1:10 into 3,600 ml L-broth and incubated in a shaking bacterial incubator at 37°C for 2 h. Isopropyl-ß-D-thiogalactopyranoside (0.5 mM) was then added to the incubating cultures for 2 h. Bacteria were collected by centrifugation at 5,000 g for 15 min. The pellets were resuspended in 40 ml lysis buffer (50 mM Tris, pH 7.5, 1% Triton X, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF). After sonication, the lysates were spun at 14,000 rpm for 30 min at 4°C. The clarified bacterial lysate was then incubated with glutathione agarose beads (0.6 ml wet volume; preswelled with water) (Sigma-Aldrich) for 60 min at 4°C. The coupled beads were then washed six times in wash buffer (50 mM Tris, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 µg /ml aprotinin, 1 µg/ml leupeptin, and 0.1 mM PMSF) and once in wash buffer containing 10% glycerol. Beads were then resuspended in 8 ml of the wash buffer containing 10% glycerol and stored overnight at -80°C. Frozen tissue was homogenized in modified RIPA buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF). The homogenates and cell lysates were clarified by two 10-min centrifugations at 13,000 g at 4°C. They were then incubated for 50 min at 4°C with GST-RBD (a gift from John Collard, Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands) coupled beads (2030 µg/sample). The beads were then washed four times and eluted in sample buffer. GTP-bound Rho and total Rho present in tissue homogenates were detected by Western blot. The proteins were transferred to nitrocellulose and probed using a monoclonal RhoA antibody (Santa Cruz Biotechnology, Inc.). Bands were visualized with peroxidase-linked secondary antibodies (Promega) and an HRP-based chemiluminescence reaction (Pierce Chemical Co.). C305 was detected using a C3-specific polyclonal antibody (Winton et al., 2002). P75NTR was detected with a polyclonal antibody raised against p75NTR (Promega). For all blots, 20 µg of protein was loaded into each lane. Blots were scanned for densitometry using an Epson perfection 1200U scanner, transferred to Adobe Photoshop® 6.0, and the images were the analyzed with the densitometry IQ MAC 1.2 software (Molecular Dynamics). The software measures the pixel density in the band image after background subtraction, and the densitometry value is in arbitrary units. Statistical tests were performed using In Stat (Graph Pad).
For in situ pull-down assays, rat spinal cord cryosections (16-µm thickness, fresh) were postfixed with 4% PFA and incubated with the clarified bacterial lysate, prepared from bacteria expressing GST-RBD or GST alone, as described above, overnight at 4°C. The sections were then washed three times in TBS, blocked in 3% BSA for 1 h at room temperature, and incubated with anti-GST antibody (New England Biolabs, Inc.) and with cell typespecific antibodies (NeuN, GFAP, and MAB328; Chemicon) or with antibody raised against p75NTR (Promega) overnight at 4°C. Sections were washed in TBS and incubated for 2 h at room temperature with FITC, Texas red, or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).
TUNEL labeling and immunohistochemistry
Spinal cord samples of 3 and 4 mm spanning the lesion sites of mice and rats, respectively, were dissected. Normal spinal cord was a 4-mm section from sham control cords. All spinal cord pieces were postfixed in 4% PFA, washed, and embedded in paraffin. Transverse sections of 6-µm thickness were cut, deparaffinized in xylene, and rehydrated by ethanol washes. TUNEL labeling was performed using the Fluorescein-FragEL DNA Fragmentation kit (Oncogene Research Products). The sections were costained with Hoechst 33342 (Sigma-Aldrich), and only TUNEL-positive cells that correlated with Hoechst 33342stained nuclei were counted. To quantitatively examine the numbers of apoptotic cells, TUNEL-positive cells were counted on sections from control, lesion, and C305treated animals. A blinded researcher counted the total number of TUNEL-positive cells located in the entire transverse section. The average number of TUNEL-positive cells per section was calculated from values obtained by counting 4050 random sections throughout the lesion site of each animal, with three animals examined per group. The TUNEL-positive cells (green) were distinguished from autofluorescent macrophages (red) through the use of a merge red/green filter. Cells labeled with both TUNEL (green) and p75 (red) were counted in a merge red/green filter after verifying colocalization with Hoechst stain. Values were obtained by counting 20 random sections throughout the lesion site of each animal, with three animals examined per group. Immunohistochemistry with cell typespecific antibodies (NeuN, GFAP, and MAB328; Chemicon) or with a polyclonal C3 antibody (Winton et al., 2002) was performed on paraffin sections. After deparaffinization, transverse sections were treated with 2x saline sodium citrate at 80°C for 20 min. Sections were blocked in TBS containing 3% BSA and 2% goat serum and incubated overnight with primary antibody at 4°C followed by a 2-h incubation with FITC or Texas redconjugated secondary antibodies (Jackson ImmunoResearch Laboratories). All pictures were taken with northern eclipse software and transferred to Adobe Illustrator® 9.0.
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Acknowledgments |
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This work was funded by the Canadian Institute of Health Research and the Natural Sciences and Engineering Research Council.
Submitted: 21 January 2003
Revised: 7 May 2003
Accepted: 7 May 2003
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References |
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Aznar, S., and J.C. Lacal. 2001. Rho signals to cell growth and apoptosis. Cancer Lett. 165:110.[CrossRef][Medline]
Beattie, M.S., A.W. Harrington, R. Lee, J.Y. Kim, S.L. Boyce, F.M. Longo, J.C. Bresnahan, B.L. Hempstead, and S.O. Yoon. 2002. ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron. 36:375386.[Medline]
Bobak, D., J. Moorman, A. Guanzon, L. Gilmer, and C. Hahn. 1997. Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis. Oncogene. 15:21792189.[CrossRef][Medline]
Brandoli, C., B. Shi, B. Pflug, P. Andrews, J.R. Wrathall, and I. Mocchetti. 2001. Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord. Brain Res. Mol. Brain Res. 87:6170.[Medline]
Casha, S., W.R. Yu, and M.G. Fehlings. 2001. Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat. Neuroscience. 103:203218.[CrossRef][Medline]
Cheema, S.S., G.L. Barrett, and P.F. Bartlett. 1996. Reducing p75 nerve growth factor receptor levels using antisense oligonucleotides prevents the loss of axotomized sensory neurons in the dorsal root ganglia of newborn rats. J. Neurosci. Res. 46:239245.[CrossRef][Medline]
Citron, B.A., I.V. Smirnova, P.M. Arnold, and B.W. Festoff. 2000. Upregulation of neurotoxic serine proteases, prothrombin, and protease-activated receptor 1 early after spinal cord injury. J. Neurotrauma. 17:11911203.[Medline]
Clark, E.A., T.R. Golub, E.S. Lander, and R.O. Hynes. 2000. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 406:532535.[CrossRef][Medline]
Coleman, M.L., and M.F. Olson. 2002. Rho GTPase signalling pathways in the morphological changes associated with apoptosis. Cell Death Differ. 9:493504.[CrossRef][Medline]
David, S., and S. Lacroix. 2003. Molecular approaches to spinal cord repair. Annu. Rev. Neurosci. 26:411440.[CrossRef][Medline]
Dechant, G., and Y.A. Barde. 2002. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat. Neurosci. 5:11311136.[CrossRef][Medline]
Dergham, P., B. Ellezam, C. Essagian, H. Avedissian, W.D. Lubell, and L. McKerracher. 2002. Rho signaling pathway targeted to promote spinal cord repair. J. Neurosci. 22:65706577.
De Winter, F., M. Oudega, A.J. Lankhorst, F.P. Hamers, B. Blits, M.J. Ruitenberg, R.J. Pasterkamp, W.H. Gispen, and J. Verhaagen. 2002. Injury-induced class 3 semaphorin expression in the rat spinal cord. Exp. Neurol. 175:6175.[CrossRef][Medline]
Donovan, F.M., C.J. Pike, C.W. Cotman, and D.D. Cunningham. 1997. Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities. J. Neurosci. 17:53165326.
Fiorentini, C., A. Fabbri, L. Falzano, A. Fattorossi, P. Matarrese, R. Rivabene, and G. Donelli. 1998a. Clostridium difficile toxin B induces apoptosis in intestinal cultured cells. Infect. Immun. 66:26602665.
Fiorentini, C., P. Matarrese, E. Straface, L. Falzano, A. Fabbri, G. Donelli, A. Cossarizza, P. Boquet, and W. Malorni. 1998b. Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis. Exp. Cell Res. 242:341350.[CrossRef][Medline]
Fournier, A.E., B.T. Takizawa, and S.M. Strittmatter. 2003. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J. Neurosci. 23:14161423.
Frade, J.M., and Y.A. Barde. 1999. Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord. Development. 126:683690.
Fritz, G., I. Just, and B. Kaina. 1999. Rho GTPases are over-expressed in human tumors. Int. J. Cancer. 81:682687.[CrossRef][Medline]
Gomez, J., C. Martinez, M. Giry, A. Garcia, and A. Rebollo. 1997. Rho prevents apoptosis through Bcl-2 expression: implications for interleukin-2 receptor signal transduction. Eur. J. Immunol. 27:27932799.[Medline]
Grossman, S.D., L.J. Rosenberg, and J.R. Wrathall. 2001. Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp. Neurol. 168:273282.[CrossRef][Medline]
Hara, M., M. Takayasu, K. Watanabe, A. Noda, T. Takagi, Y. Suzuki, and J. Yoshida. 2000. Protein kinase inhibition by fasudil hydrochloride promotes neurological recovery after spinal cord injury in rats. J. Neurosurg. 93:94101.[Medline]
Hippenstiel, S., B. Schmeck, P.D. N'Guessan, J. Seybold, M. Krull, K. Preissner, C.V. Eichel-Streiber, and N. Suttorp. 2002. Rho protein inactivation induced apoptosis of cultured human endothelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 283:L830L838.
Huang, E.J., and L.F. Reichardt. 2001. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24:677736.[CrossRef][Medline]
Hutton, L.A., J. deVellis, and J.R. Perez-Polo. 1992. Expression of p75NGFR TrkA, and TrkB mRNA in rat C6 glioma and type I astrocyte cultures. J. Neurosci. Res. 32:375383.[Medline]
Jalink, K., E.J. van Corven, T. Hengeveld, N. Morii, S. Narumiya, and W.H. Moolenaar. 1994. Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J. Cell Biol. 126:801810.[Abstract]
Jimenez, B., M. Arends, P. Esteve, R. Perona, R. Sanchez, S. Ramon y Cajal, A. Wyllie, and J.C. Lacal. 1995. Induction of apoptosis in NIH3T3 cells after serum deprivation by overexpression of rho-p21, a GTPase protein of the ras superfamily. Oncogene. 10:811816.[Medline]
Jin, Z., and S.M. Strittmatter. 1997. Rac1 mediates collapsin-1-induced growth cone collapse. J. Neurosci. 17:62566263.
Kaplan, D.R., and F.D. Miller. 2000. Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10:381391.[CrossRef][Medline]
Lee, K.F., E. Li, L.J. Huber, S.C. Landis, A.H. Sharpe, M.V. Chao, and R. Jaenisch. 1992. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell. 69:737749.[Medline]
Lee, Y.B., T.Y. Yune, S.Y. Baik, Y.H. Shin, S. Du, H. Rhim, E.B. Lee, Y.C. Kim, M.L. Shin, G.J. Markelonis, and T.H. Oh. 2000. Role of tumor necrosis factor-alpha in neuronal and glial apoptosis after spinal cord injury. Exp. Neurol. 166:190195.[CrossRef][Medline]
Lehmann, M., A. Fournier, I. Selles-Navarro, P. Dergham, A. Sebok, N. Leclerc, G. Tigyi, and L. McKerracher. 1999. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J. Neurosci. 19:75377547.
Li, Z., C.D. Aizenman, and H.T. Cline. 2002. Regulation of Rho GTPases by crosstalk and neuronal activity in vivo. Neuron. 33:741750.[Medline]
Liu, X.Z., X.M. Xu, R. Hu, C. Du, S.X. Zhang, J.W. McDonald, H.X. Dong, Y.J. Wu, G.S. Fan, M.F. Jacquin, et al. 1997. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17:53955406.
McKerracher, L., and M.J. Winton. 2002. Nogo on the go. Neuron. 36:345348.[Medline]
Mills, J.C., N.L. Stone, J. Erhardt, and R.N. Pittman. 1998. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J. Cell Biol. 140:627636.
Moorman, J.P., D.A. Bobak, and C.S. Hahn. 1996. Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4. J. Immunol. 156:41464153.[Abstract]
Neumann, H., R. Schweigreiter, T. Yamashita, K. Rosenkranz, H. Wekerle, and Y.A. Barde. 2002. Tumor necrosis factor inhibits neurite outgrowth and branching of hippocampal neurons by a rho-dependent mechanism. J. Neurosci. 22:854862.[CrossRef][Medline]
Niederost, B., T. Oertle, J. Fritsche, R.A. McKinney, and C.E. Bandtlow. 2002. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1. J. Neurosci. 22:1036810376.
Popovich, P.G., and T.B. Jones. 2003. Manipulating neuroinflammatory reactions in the injured spinal cord: back to basics. Trends Pharmacol. Sci. 24:1317.[CrossRef][Medline]
Reid, T., T. Furuyashiki, T. Ishizaki, G. Watanabe, N. Watanabe, K. Fujisawa, N. Morii, P. Madaule, and S. Narumiya. 1996. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J. Biol. Chem. 271:1355613560.
Ren, X.D., and M.A. Schwartz. 2000. Determination of GTP loading on Rho. Methods Enzymol. 325:264272.[Medline]
Ridley, A.J. 2001. Rho family proteins: coordinating cell responses. Trends Cell Biol. 11:471477.[CrossRef][Medline]
Schwab, M.E. 2002. Increasing plasticity and functional recovery of the lesioned spinal cord. Prog. Brain Res. 137:351359.[Medline]
Schwab, M.E., J.P. Kapfhammer, and C.E. Bandtlow. 1993. Inhibitors of neurite growth. Annu. Rev. Neurosci. 16:565595.[CrossRef][Medline]
Schwartz, G., and M.G. Fehlings. 2002. Secondary injury mechanisms of spinal cord trauma: a novel therapeutic approach for the management of secondary pathophysiology with the sodium channel blocker riluzole. Prog. Brain Res. 137:177190.[Medline]
Semkova, I., and J. Krieglstein. 1999. Ciliary neurotrophic factor enhances the expression of NGF and p75 low-affinity NGF receptor in astrocytes. Brain Res. 838:184192.[CrossRef][Medline]
Shirvan, A., M. Kimron, V. Holdengreber, I. Ziv, Y. Ben-Shaul, S. Melamed, E. Melamed, A. Barzilai, and A.S. Solomon. 2002. Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy. J. Biol. Chem. 277:4979949807.
Shuman, S.L., J.C. Bresnahan, and M.S. Beattie. 1997. Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J. Neurosci. Res. 50:798808.[CrossRef][Medline]
Steward, O., P.E. Schauwecker, L. Gutth, Z. Zhang, M. Fujiki, D. Inman, J. Wrathall, G. Kempermann, F.H. Gage, K.E. Saatman, et al. 1999. Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models. Exp. Neurol. 157:1942.[CrossRef][Medline]
Suwa, H., G. Ohshio, T. Imamura, G. Watanabe, S. Arii, M. Imamura, S. Narumiya, H. Hiai, and M. Fukumoto. 1998. Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br. J. Cancer. 77:147152.[Medline]
Swiercz, J.M., R. Kuner, J. Behrens, and S. Offermanns. 2002. Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron. 35:5163.[Medline]
Tang, S., J. Qiu, E. Nikulina, and M.T. Filbin. 2001. Soluble myelin-associated glycoprotein released from damaged white matter inhibits axonal regeneration. Mol. Cell. Neurosci. 18:259269.[CrossRef][Medline]
Tigyi, G., D.J. Fischer, A. Sebok, F. Marshall, D.L. Dyer, and R. Miledi. 1996. Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of cyclic AMP signaling. J. Neurochem. 66:549558.[Medline]
Vinson, M., O. Rausch, P.R. Maycox, R.K. Prinjha, D. Chapman, R. Morrow, A.J. Harper, C. Dingwall, F.S. Walsh, S.A. Burbidge, and D.R. Riddell. 2003. Lipid rafts mediate the interaction between myelin-associated glycoprotein (MAG) on myelin and MAG-receptors on neurons. Mol. Cell. Neurosci. 22:344352.[CrossRef][Medline]
Wahl, S., H. Barth, T. Coiossek, K. Akoriess, and B.K. Mueller. 2000. Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J. Cell Biol. 149:263270.
Wang, K.C., J.A. Kim, R. Sivasankaran, R. Segal, and Z. He. 2002. P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature. 420:7478.[CrossRef][Medline]
Widenfalk, J., K. Lundstromer, M. Jubran, S. Brene, and L. Olson. 2001. Neurotrophic factors and receptors in the immature and adult spinal cord after mechanical injury or kainic acid. J. Neurosci. 21:34573475.
Winton, M.J., C.I. Dubreuil, D. Lasko, N. Leclerc, and L. McKerracher. 2002. Characterization of new cell permeable C3-like proteins that inactivate rho and stimulate neurite outgrowth on inhibitory substrates. J. Biol. Chem. 277:3282032829.
Woolf, C.J., and S. Bloechlinger. 2002. Neuroscience. It takes more than two to Nogo. Science. 297:11321134.
Yamashita, T., K.L. Tucker, and Y.A. Barde. 1999. Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron. 24:585593.[Medline]
Yamashita, T., H. Higuchi, and M. Tohyama. 2002. The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J. Cell Biol. 157:565570.