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Keywords:

  • Robo;
  • GTPase-activating protein;
  • dorsal root ganglion;
  • nerve regeneration

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The Slit–Robo GTPase-activating proteins (srGAPs) play an important role in neurite outgrowth and axon guidance; however, little is known about its role in nerve regeneration after injury. Here, we studied the expression of srGAPs in mouse dorsal root ganglia (DRG) following sciatic nerve transection (SNT) using morphometric and immunohistochemical techniques. Reverse transcriptase polymerase chain reaction and Western blot analysis indicated that srGAP1 and srGAP3, but not srGAP2, were expressed in normal adult DRG. Following unilateral SNT, elevated mRNA and protein levels of srGAP1 and srGAP3 were detected in the ipsilateral relative to contralateral L3–4 DRGs from day 3 to day 14. Immunohistochemical results showed that srGAP1 and srGAP3 were largely expressed in subpopulations of DRG neurons in naïve DRGs. However, after SNT, srGAP3 in neurons was significantly increased in the ipsilateral relative to contralateral DRGs, which peaked at day 7 to day 14. Interestingly, DRG neurons with strong srGAP3 labeling also coexpressed Robo2 after peripheral nerve injury. These results suggest that srGAPs are differentially expressed in murine DRG and srGAP3 are the predominant form. Moreover, srGAP3 may participate in Slit–Robo signaling in response to peripheral nerve injury or the course of nerve regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Many molecules and proteins are involved in the complex signaling pathways that mediate neuronal migration and axon guidance in the nervous system. The Slit family proteins play a crucial role in neurite outgrowth and axon pathfinding including serving as a repulsive cue (Brose et al.,1999; Kidd et al.,1999; Li et al.,1999). Slit proteins bind to the extracellular domain of Roundabout (Robo), a transmembrane receptor that determines the cellular response to Slit (Bashaw and Goodman,1999). Rho GTPases are another family of proteins that also modulate dendrite elaboration, neurite outgrowth, and axon guidance (Luo et al.,1994; Threadgill et al.,1997; Yamashita et al.,1999; Li et al.,2000).

Rho GTPases elicit their biological functions by interacting with a variety of intracellular proteins (Yuan et al.,2003; Schmid et al.,2007). When Rho GTPases bind to guanosine triphosphate (GTP), they are in an active state. In contrast, the binding of Rho GTPases to guanosine diphosphate (GDP) induces an inactive enzymatic state (Yuan et al.,2003; Schmid et al.,2007). Therefore, these two functional states allow Rho GTPases to act as signaling switches for downstream effectors. GTPase-activating proteins (GAPs) are a family of proteins that catalyze the intrinsic hydrolysis of GTP, and thus can inactivate GTPases (Wong et al.,2001; Li et al.,2006). Slit–Robo GAPs (srGAPs) contain a Rho GAP domain that regulates the activity of Rho family GTPases and affects actin polymerization (Wong et al.,2001; Schmid et al.,2007), and an SH3 domain that interacts with the CC3 motif of Robo1. Wong et al. (2001) reported that srGAP1 can transduce the repulsion signal of Slit by inactivating Cdc42 in neuronal migration. Moreover, a dose-dependent relationship between srGAP1–Robo interaction and Slit concentration has been demonstrated (Andrews et al.,2008).

srGAPs may play an important biological role during brain development and in neural regeneration. For example, srGAP3 may regulate Rac activity during brain morphogenesis (Soderling et al.,2002). Indeed, loss of srGAP3 in vivo and in vitro results in reduced dendritic spine density as well as impairment in learning and memory. srGAP2 appears to be involved in axon regeneration. For instance, facial nerve transection causes apparent upregulation of srGAP2 in the facial nucleus in adult rats (Madura et al.,2004).

Both srGAP2 and srGAP3 mRNA have been shown in the dorsal root ganglia (DRG) during development (Wong et al.,2001; Yao et al.,2008). Our previous study showed that Robo1 and Robo2 are both expressed in adult rat DRGs, and nerve injury can induce upregulation of these proteins at this location (Yi et al.,2006). Moreover, our in vitro study suggests that Robo2 may promote the outgrowth of neurites in cultured primary DRG neurons (Zhang et al.,2010). However, it remains unclear whether and how members of the srGAPs might be involved in DRG neural regeneration. Therefore, this study determined the srGAP1–3 expression in normal and injured DRG of adult mice, and the role of these proteins in Slit–Robo pathway signaling in murine DRG.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Animals, Surgery, and Tissue Preparation

All experiments were performed in accordance with guidelines for experimental animal use established by the ethics committee of the Hainan Medical College. Sixty C57 mice (30 ± 5 g body weight) were purchased from the Hunan Agricultural University (Changsha, Human, P.R. China). For sciatic nerve transection (SNT), mice were anesthetized using an intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g). The right sciatic nerve around the mid thigh was exposed under sterile conditions and was transected about 3 mm proximal to its division into the tibial and common peroneal nerves. Animals were allowed to survive for 1, 3, 7, and 14 days after surgery (N = 10/time point). Ten age-matched mice without sciatic nerve injury were also used as control for biochemical analysis. For reverse transcriptase polymerase chain reaction (RT-PCR) and western blot (N = 5/time point), ipsilateral and contralateral L3–L4 DRGs, the proximal sciatic nerves stump and spinal cord were collected and fresh–frozen. For anatomical studies, animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.), perfused transcardially with cold 4% paraformaldehyde in 0.1 M phosphate buffer (0.1 M PB, pH 7.2) for 30 min. The aforementioned tissue samples were dissected out, postfixed in the perfusion solution for 3 hrs, and transferred through 10%, 20%, and 30% sucrose for cryoprotection before sectioning.

RT-PCR

The expression of srGAP1–3 mRNAs was determined by RT-PCR, using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a positive control. Total RNA was extracted from L3–4 DRGs of the normal mice (naïve) as well as the sciatic nerve transected mice using TRIzol (Takara, China), and quantified according to optical density measured at 260 nm. cDNAs were synthesized with 1 μg of total RNA from each sample using the RevertAid™ H Minus M-MuLV Reverse Transcriptase kit (Fermentas, Canada). PCR was carried out in a 20 μL volume with the Hot Start TaqMasterMix kit (Takara, China), using an identical amount of cDNA per reaction and 1 μm forward and reverse primers. The primer sequences were as follows (Madura et al.,2004): 5′-CCCCAGGGGCCTGTTGCAGA-3′ and 5′-TGGTGCTCCGTCGAAGCTGC-3′ for srGAP1; 5′-CGCTCCCCTGACTCCACGGC-3′ and 5′-AGACAGATGCGTTGGTTGCC-3′ for srGAP2; 5′-AATCCACCAGGCCCCATCAG-3′ and 5′-GCCGGACTTGTCTGAGGAGC-3′ for srGAP3; and 5′-CCGCAGTCCCAGCGTCGTG-3′ and 5′-AACCATTTTGGGGCTGTACTGC-3′ for HPRT. PCR reaction included 28 cycles of 94°C for 30 sec, 63°C for 30 sec, and 72°C for 1 min. The resulting products were electrophoresed in 1.25% agarose gel containing ethidium bromide. cDNA bands were imaged using IBAS 2.0 (KONTRON, German). Ratio of the integrated optical density of the gene of interest to HPRT was calculated to assess relative levels of srGAP1 and srGAP3 mRNA expression. Experiments were performed using three sets of independently prepared samples.

Western Blot

DRGs were homogenized with ice cold RIPA buffer (1:10 wt/vol) containing 25 mM Tris (pH 7.5), 1 mM EDTA, 2 mM MgCl2, 50 mM KCl, and 5 mM dithiothreitol (DTT) (Boster), centrifuged at 9,000g for 1 min at 4°C. Protein concentration was determined and extracts (50 μg) were loaded on 10% SDS-polyacrylamide gels. Then, proteins were transferred to polyvinylidene difluoride membranes. After blocking in 1% bovine serum albumin solution in Tris Buffered Saline With Tween (i.e., washing buffer containing 10 mM Tris at pH 7.5, 150 mM NaCl, 0.05% Tween-20), the membranes were incubated overnight at 4°C with mouse anti-srGAP1 or srGAP2 antibodies, or rabbit anti-srGAP3 antibodies. Then, membranes were incubated with the appropriate secondary antibodies conjugated to peroxidase for 1 hr at room temperature. Peroxidase enzyme activity was detected using diaminobenzidine.

Immunofluorescence

Serial frozen sections (15 μm thick) of the DRG and sciatic nerve stump were mounted on poly-lysine-coated microslides. Sections were preincubated in 5% bovine serum albumin in phosphate buffer, and incubated with the primary antibodies at 4°C for 24 hrs, and further reacted with secondary antibodies at RT for 2 hrs. The following pairs of double immunofluorescence were carried out: (1) srGAP1 and neurofilament 200, (2) srGAP3 and S100, (3) srGAP3 and glial fibrillary acidic protein (GFAP), (4) srGAP3 and NF200, and (5) srGAP3 and Robo2. Final dilutions of these primary antibodies were: mouse anti-srGAP1 (1:300) (Abcam, Hong Kong), rabbit anti-srGAP3 (1:400) (Abcam, Hong Kong), rat anti-srGAP3 (Abcam, Hong Kong), mouse anti-GFAP (1:400) (Millipore USA), chicken anti-GFAP (1:400) (Millipore), rat anti-NF200 (1:400; Sigma), mouse anti-Robo2 (1:400) (Abcam, Hong Kong), rabbit anti-srGAP3 (1:400), and mouse anti-S100 (1:400) (Millipore) antibodies (Zheng et al.,2008,2010). Secondary antibodies included fluorescein isothiocyanate (FITC)-goat anti-mouse IgG (1:200), FITC-goat anti-chicken IgG (1:200), Cy3-goat anti-rabbit IgG (1:200) (Boster, China), FITC-goat anti-rat IgG (1:200) (Boster, China). The specificity of the srGAP1 and three antibodies were verified by preabsorption with neutralizing peptides and omission of primary antibody in the incubation buffer, neither yielded specific immunolabeling.

Image Analysis

Immunofluorescent labeling was imaged on a confocal microscope equipped with a digital camera (Nikon 90I). The same exposure was used for capturing immunolabeling signal from comparing groups. For densitometry, five randomly selected images at 20× magnification were taken from each animal. For each image, all neurons with visible nuclei were first counted. Background was defined by sampling the image pixel intensity from neurons with larger diameters that were not labeled by any antibodies. All neurons with visible nuclei displaying fluorescent signal above background by two standard deviations were counted as moderately immunoreactive, whereas neurons with fluorescent signal three standard deviations above background were counted as intensely immunoreactive.

Statistical Analysis

The numerical data of immunoreactivity were expressed as the mean ± SEM. One-way ANOVA and the Dunnett t tests were used for statistical analyses of means between comparing groups using the SPSS 13.0 software. P value less than 0.05 was considered to be statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

srGAPs Messenger Expression in Normal and Lesioned DRG

RT-PCR was carried out to determine mRNA expression of the three family members of the srGAP (1–3) in DRG and the effect of peripheral nerve transection. No srGAP2 mRNA was detected in normal mice DRG and SNT mice DRG (Fig. 1A). In contrast, RT-PCR showed cDNA products of srGAP1 and srGAP3 mRNA in naïve DRGs. The levels of srGAP3 and srGAP1 mRNA were increased in ipsilateral relative to contralateral DRG extracts from nerve-cut mice in a time-dependent manner (Fig. 1A). Densitometry confirmed a statistically significant difference in the amount of srGAP1 and srGAP3 products between the ipsilateral and contralateral DRGs from day 3 to 14 postsurgery (P < 0.01, Dunnett t tests), more dramatic for srGAP3 than srGAP1 (Fig. 1A, B).

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Figure 1. Quantitative analysis of srGAP mRNA and protein expression in DRGs. A: RT-PCR was used to detect the expression of srGAP1, srGAP2srGAP3, and HPRT mRNA 1, 3, 7, and 14 days post-nerve transection relative to naïve DRG and contralateral DRG (con). Little RT-PCR product of srGAP2 is detectable, whereas low and high levels of products are detected for srGAP1 and srGAP3, respectively. B: The quantification of RT-PCR data indicating increases of srGAP3 (filled gray squares) and srGAP1 (filled black diamonds) products in ipsilateral relative to naïve and contralateral DRGs. C: A representative western blot image for srGAP1, srGAP2, and srGAP3. Note the lack of srGAP2 protein expression in DRG homogenates. D: shows that the levels of srGAP1 protein are significantly increased in the ipsilateral DRGs relative to contralateral counterparts at the four surviving time points (P = 0.02, 0.008, 0.012, and 0.058 at day 1, 3, 7, and 14, respectively). Difference of srGAP1 protein levels between the ipsilateral and contralateral sides is no statistically significance (P = 0.160, 0.089, 0.068, and 0.075 at day 1, 3, 7, and 14 days postlesion).

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srGAP1 and srGAP3 Protein Expression in Normal and Lesioned DRG

Consistent with the RT-PCR data, western blot analysis failed to detect srGAP2 protein products in normal DRG and SNT DRG, whereas srGAP1 and srGAP3 proteins were detected in DRG homogenates (Fig. 1C). Levels of srGAP1 and srGAP3 proteins showed a trend of increase in ipsilateral relative to contralateral DRG from sciatic nerve transected mice during the first weeks post-nerve cut (Fig. 1C), with the levels of srGAP3 higher in the ipsilateral relative to the contralateral size at all surviving points (*P < 0.01, student's t test). The difference in the levels of srGAP1 protein between the two sides was no statistically difference (P = 0.089, 0.068, and 0.075 at day 3, 7, and 14 days post-nerve cut) (Fig. 1D).

Cellular Localization of srGAP1 and srGAP3 in Normal Mouse DRG

Because both RT-PCR and western blot revealed little expression of srGAP2, we assessed the expression of srGAP1 and srGAP3 in the DRGs from normal mice and from the contralateral side of the operated mice, using mouse anti-srGAP1, rabbit anti-srGAP3, and rat anti-srGAP3. To test the specificity of these antibodies, we used cultured DRG neurons (Zhang et al.,2010) as positive control, and primary antibody omission and preabsorption as negative controls. The antibodies exhibited positive labeling in cultured DRG neurons (Supporting Information, Fig. S1A, D, G). In contrast, no labelings were found when the primary antibodies were omitted (Supporting Information, Fig. S1B, E, H) or neutralized with corresponding antigen peptides in the incubation buffer (Supporting Information, Fig. S1C, F, I).

srGAP1 immunofluorescence was present in the cytoplasm of a subset of relatively large cells in the DRG (Fig. 2A, D). In these cells, srGAP1 immunoreactivity partially colocalized with the neuronal marker N200 (Fig. 2A–C). It should be noted that small cellular and process-like profiles were seen around large neuron-like perikarya with and without cytoplasmic srGAP1 reactivity. Double immunofluorescence showed a colocalization of srGAP1 with GFAP labeling among potential satellite glial cells (Fig. 2D–F). srGAP3 was expressed in a large subpopulation of DRG cells, with varying staining intensity among cells (Fig. 2G, J). Double immunofluorescence showed a very frequent, if not complete, colocalization of srGAP3 and N200 (Fig. 2G–I). As with srGAP1, srGAP3 rarely colocalized with GFAP (Fig. 2J–L). Additional double immunofluorescent characterization demonstrated a colocalization of srGAP1 and srGAP3 among DRG cells (Fig. 2M–O).

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Figure 2. Representative confocal double-immunofluorescence of srGAP1, srGAP3, N200, and GFAP in normal adult mouse DRG. Panels (A–C) show double labeling of srGAP1 and N200 in a subpopulation of DRG neurons (small arrow). Note srGAP1 expression in small satellite-like cellular profiles (large arrow head). Panels (D–F) show colocalization of srGAP1 with the glial marker GFAP around large DRG neuronal perikarya that can be srGAP1 positive or negative. Panels (G–I) show a large number of DRG neurons coexpressing srGAP3 and N200. In contrast, little colocalization exists for srGAP3 and GFAP (J–L). Colocalization of srGAP1 and srGAP3 occurs in some DRG neuronal perikarya (M–O). Scale bar = 40 μm in (A–L) and = 20μm in (M–O).

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Densitometric analyses were carried out to assess the levels of srGAP1 and srGAP3 expression among DRG neurons using a threshold cutoff method (N = 5). Overall, about 20% DRG neurons exhibited srGAP1 immunolabeling, among which 17.0 ± 3.5% showed moderate level of immunofluorescence, whereas the minor remainder exhibited strong staining (Fig. 3). However, up to 72.2 ± 3.5% of DRG neurons were immunoreactive for srGAP3, and they exhibited moderate (∼ 60%) to intense (∼ 40%) immunofluorescence (Fig. 3).

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Figure 3. Densitometric analysis of srGAP1 and srGAP3 in normal adult DRG neurons. Overall, a larger amount of DRG neurons exhibits labeling for srGAP3 relative to srGAP1. Most srGAP1 expressing neurons show moderate immunolabeling intensity, whereas a relatively large fraction of srGAP3 expressing neurons show strong labeling intensity.

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Effect of Peripheral Nerve Cut on srGAP1 and srGAP3 Immunolabeling in DRG

The distribution and amount of neurons immunoreactive for srGAP1 antibodies did not exhibit apparent difference between the ipsilateral and contralateral DRGs from day 1 to day 14 postsurgery (Fig. 4A–E). A densitometric analysis on neurons with moderate and intense fluorescence staining failed to find statistically significant difference between the two sides of DRGs (Fig. 4F).

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Figure 4. Immunofluorescence srGAP1 in ipsilateral relative to contralateral DRG following SNT surviving from day 1 to day 14. The pattern of immunolabeling remains largely unchanged in the lesioned DRG relative to control (A–E). Densitometry indicates no significant increase of labeling in the total number of labeled cells (mean ± SEM) or the number of cells with intense labeling (F). The scale bar = 40 μm for image panels.

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In contrast, a significant increase in the total number of neurons stained for srGAP3 (with moderate and intense levels of immunofluorescence) was observed in the ipsilateral DRG relative to contralateral counterpart (Fig. 5A–E). Densitometric data indicated that the increase was statistically significant at day 1, further enhanced through day 7 and maintained at day 14, after nerve transection (Fig. 5F). Both the densities of the moderate and strongly labeled neurons were increased in the lesioned relative control sides of the DRG from day 1 to day 14 postsurgery (Fig. 5F) (P < 0.001 at each time point).

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Figure 5. Immunofluorescence srGAP3 in ipsilateral relative to contralateral DRG following SNT surviving from day 1 to day 14. The pattern of immunolabeling remains largely unchanged in the lesioned DRG relative to control, involving neuronal profiles (A-E). However, densitometry indicates an increase of labeling in the total number of immunoreactive cells (mean ± SEM) as well as the number of cells with intense labeling (F) relative to control. * and # indicate a statistically significant difference of P < 0.01 compared to normal DRG; + indicates a statistically significant difference of P < 0.01 compared to 1 and 3 days. Con: normal DRG. One-way ANOVA and Dunnett t tests, N = 5/time point. The scale bar = 20 μm for image panels.

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Colocalization of srGAP3 and Robo2 in Lesioned DRG

Because Robo2 is up-regulated in DRG following peripheral nerve injury (Yi et al.,2006), we explored potential colocalization of the srGAP3 and Robo2 in the lesioned DRG. As expected, there existed a great extent of colocalization of srGAP3 and Robo2, and a parallel increase in the expression of the two proteins in L3–4 DRG after sciatic nerve cut (Fig. 6). In brief, densitometric data indicated that the highest expression of the two proteins in larger neurons occurred in the lesioned DRG at day 7 and day 14 after nerve transection, reaching an average of 18 ± 2.5 cells per section, as compared to 5 ± 2.1 cells per section in the control DRG (P < 0.01, N = 5).

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Figure 6. Representative confocal immunofluorescence illustrating colocalization of srGAP3 Robo2 and srGAP3 in DRG after SNT. Strong coexpression of the two markers are detected at day 1 (A–C), 7 (D–F), and 14 (G–I). Arrows and arrowheads point to colocalized DRG neuronal profiles. Scale bar = 20 μm.

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srGAP1 and srGAP3 Immunolabeling in Sciatic Nerve Stump

Double immunofluorescence for srGAP3 and S100 and for srGAP1 and srGAP3 were performed on longitudinal sections of normal control sciatic nerve as well as the proximal sciatic nerve stump at all surviving time points. Overall, there were no apparent alterations in the staining pattern in the nerve stamps relative to normal nerve (data not shown). Of note, the pattern of srGAP1 immunolabeling appeared different from that of srGAP3 (Supporting Information, Fig. S2A–C). Expression of srGAP1 was detected around axonal bundles (Supporting Information, Fig. S2A), whereas srGAP3 labeling was low but present in the center of the axons as punctuate elements, suggestive of a localization in the axoplasm (Supporting Information, Fig. S2B). Thus, the labeling pattern of srGAP1 was comparable to that of S100 in the nerve stump (Supporting Information, Fig. S2D–F).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

srGAPs are Differentially Expressed in Adult Mouse DRG

In this study, we used RT-PCR, western blot, and immunohistochemistry to determine the expression of the three srGAP members in adult mouse DRG. RT-PCR analysis detects little expression of srGAP2 in DRG, which is consistent with a lack of signal in western blot and immunohistochemistry (data not shown). In contrast, RT-PCR and western blot indicate that both srGAP1 and srGAP3 are expressed in the DRG, with srGAP3 levels higher relative to srGAP1. Consistent with these biochemical data, srGAP1 and srGAP3 immunolabeling was present in the DRG. A relatively small subpopulation of DRG cells is immunoreactive for srGAP1, whereas a lot of DRG cells express srGAP3. Both srGAP1 and srGAP3 immunoreactive cells show colocalization with the neuronal marker N200. Thus, these observations suggest that srGAP1 and srGAP3 are expressed in DRG neurons. It should be noted that srGAP1 labeling may colocalize with GFAP in satellite glial cells in the DRG, whereas srGAP3 appears to rarely coexist with GFAP. Moreover, these two members of srGAPs may be coexpressed in the same DRG neuron.

Spinal Nerve Injury Enhances Largely srGAP3 Expression in DRG

Following SNT, elevated levels of srGAP1 and srGAP3 messenger expression are detected in the ipsilateral relative to contralateral L3–4 DRGs, although the upregulation is apparently more robust for srGAP3 relative to srGAP1. Consistent with the messenger changes, levels of srGAP3 proteins are increased in the lesioned DRGs relative to control. Moreover, densitometric analysis demonstrates increased immunolabeling for srGAP3 in DRG neurons following spinal nerve injury, from day 1 up to at least day 14 post-nerve cut. This finding suggests that srGAP3 is the major member of this protein family in response to spinal nerve injury. However, it is noteworthy that srGAP1 immunolabeling is fairly impressive in the proximal stump of the sciatic nerve. We cannot exclude a possibility that srGAP1 may be largely transported into the nerve trunk or perhaps unregulated in regenerating spinal nerve. It should be noted that, although we have failed to detect significant srGAP2 expression in the primary spinal neurons, a previous study shows an upregulation of srGAP2 in the facial nucleus following facial nerve cut in the adult rats (Madura et al.,2004). Together, the present and early studies indicate that the srGAP family proteins are upregulated in spinal and cranial relay neurons after peripheral nerve injury.

Involvement of Slit–Robo Network Proteins in Spinal Nerve Injury

Injury to the peripheral process of the DRG may impact the spinal sensory neurons on various aspects (Woolf et al.,1990; Madura et al.,2004). Change in gene and protein expression occurs following the acute insult and during the course of spinal nerve regeneration. The neuronal repair mechanism often involves molecules that govern early neural development (Costigan et al.,2002). The expression of the three members of the srGAP family in the brain is generally overlapped with that of Robo1 and/or Robo2 receptors in the rat brain, as revealed by in situ hybridization (Marillat et al.,2002; Whitford et al.,2002). In the previous study, increased Robo2 expression was detected in DRG neurons after injury (Yi et al.,2006). Here, we further demonstrate a potential correlative upregulation of srGAP3 with Robo2 in DRG neurons in response to spinal nerve cut. Because the srGAP and Robo system plays an essential role in neuritic growth and network formation, it is perhaps not surprising that srGAP3 and Robo2 are upregulated concurrently in the DRG after nerve injury. In general, the induction of srGAP3 and Robo2 could represent an attempt by the injured primary sensory neurons to initiate a regenerative program.

Currently, it is neither clear about the signal that may trigger srGAP/Robo2 overexpression nor about the downstream effectors of this pathway in the injury spinal nerve system. Data suggest that srGAP3 and Robo2 expression can be influenced by the target tissues that provide trophic support (Bomze et al.,2001; Bloechlinger et al.,2004). srGAP1 appears to transduce the Slit repulsion signal through inactivation of Cdc42, as identified during neuronal migration (Wong et al.,2001). srGAP3 may transduce Slit–Robo signaling at the onset of spinogenesis on dendritic filopodia (Yao et al.,2008). Besides serving a repulsive guidance cue, Slit has also been shown to promote axonal elongation and branching, including in DRG (Wang et al.,1999; Li et al.,2006) and trigeminal ganglia neurons (Ozdinler and Erzurumlu,2002). Robo2 is expressed in DRG neurons (Yi et al.,2006) and may serve permissive and promotive roles for neurite outgrowth via Robo1–Robo2 and Robo2–Slit interactions (Hivert et al.,2002; Lin et al.,2005). In vitro studies have demonstrated that Robo2 receptors can mediate the positive branching activity of Slit proteins (Yuan et al.,2003; Ma and Tessier-Lavigne,2007).

In summary, this study shows a differential messenger, protein level, and in situ expression of the srGAP members in adult murine DRG. We observe a robust upregulation of srGAP3 in DRG neurons following peripheral spinal nerve injury. This injury-induced upregulation of srGAP3 appears concomitant with that of Robo2. These findings suggest that the Slit–Robo-srGAP3 pathway plays a role in the primary spinal nerve system, and members of this molecular pathway may synergistically participate in response to nerve injury.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information
  • Andrews W, Barber M, Hernadez-Miranda LR, Xian J, Rakic S, Sundaresan V, Rabbitts TH, Pannell R, Rabbitts P, Thompson H, Erskine L, Murakami F, Parnavelas JG. 2008. The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons. Dev Biol 313: 648658.
  • Bashaw GJ, Goodman CS. 1999. Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion. Cell 97: 917926.
  • Bloechlinger S, Karchewski LA, Woolf CJ. 2004. Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury. Eur J Neurosci 19: 11191132.
  • Bomze HM, Bulsara KR, Iskandar BJ, Caroni P, Skene JH. 2001. Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons. Nat Neurosci 4: 3843.
  • Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T. 1999. Slit proteins bind robe receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96: 795806.
  • Costigan M, Befort K, Karchewski L, Griffin RS, D'Urso D, Allchorne A, Sitarski J, Mannion JW, Pratt RE, Woolf CJ. 2002. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci 3: 16.
  • Hivert B, Liu Z, Chuang CY, Doherty P, Sundaresan V. 2002. Robo1 and Robo2 are homophilic binding molecules that promote axonal growth. Mol Cell Neurosci 21: 534545.
  • Kidd T, Bland KS, Goodman CS. 1999. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96: 785794.
  • Li HS, Chen JH, Wu W, Fagaly T, Zhou LJ, Yuan WL, Dupuis S, Jiang ZH, Nash W, Gick C, Ornitz DM, Wu JY, Rao Y. 1999. Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96: 807818.
  • Li X, Chen Y, Liu Y, Gao J, Gao F, Bartlam M, Wu JY, Rao Z. 2006. Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway. J Biol Chem 281: 2843028437.
  • Li Z, Van Aelst L, Cline HT. 2000. Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo. Nat Neurosci 3: 217225.
  • Lin L, Rao Y, Isacson O. 2005. Netrin-1 and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons. Mol Cell Neurosci 28: 547555.
  • Luo L, Liao YJ, Jan LY, Jan YN. 1994. Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev 8: 17871802.
  • Ma L, Tessier-Lavigne M. 2007. Dual branch-promoting and branch-repelling actions of Slit/Robo signaling on peripheral and central branches of developing sensory axons. J Neurosci 27: 68436851.
  • Madura T, Yamashita T, Kubo T, Tsuji L, Hosokawa K, Tohyama M. 2004. Changes in mRNA of Slit-Robo GTPase-activating protein 2 following facial nerve transection. Brain Res Mol Brain Res 123: 7680.
  • Marillat V, Cases O, Nguyen-Ba-Charvet KT, Tessier-Lavigne M, Sotelo C, CheDotal A. 2002. Spatiotemporal expression patterns of slit and robo genes in the rat brain. J Comp Neurol 442: 130155.
  • Ozdinler PH, Erzurumlu RS. 2002. Slit2, a branching-arborization factor for sensory axons in the Mammalian CNS. J Neurosci 22: 45404549.
  • Schmid BC, Rezniczek GA, Fabjani G, Yoneda T, Leodolter S, Zeillinger R. 2007. The neuronal guidance cue Slit2 induces targeted migration and may play a role in brain metastasis of breast cancer cells. Breast Cancer Res Treat 106: 333342.
  • Soderling SH, Binns KL, Wayman GA, Davee SM, Ong SH, Pawson T, Scott JD. 2002. The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Nat Cell Biol 4: 970975.
  • Threadgill R, Bobb K, Ghosh A. 1997. Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42. Neuron 19: 625634.
  • Wang KH, Brose K, Arnott D, Kidd T, Goodman CS, Henzel W, Tessier-Lavigne M. 1999. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96: 771784.
  • Whitford KL, Marillat V, Stein E, Goodman CS, Tesssier-Lavigne M, Chedotal A, Ghosh A. 2002. Regulation of cortical dendrite development by Slit-Robo interactions. Neuron 33: 4761.
  • Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, Tang H, Wen L, Brady-Kalnay SM, Mei L, Wu JY, Xiong WC, Rao Y. 2001. Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107: 209221.
  • Woolf CJ, Reynolds ML, Molander C, O'Brien C, Lindsay RM, Benowitz LI. 1990. The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury. Neuroscience 34: 465478.
  • Yamashita T, Tucker KL, Barde YA. 1999. Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron 24: 585593.
  • Yao Q, Jin WL, Wang Y, Ju G. 2008. Regulated shuttling of Slit-Robo-GTPase activating proteins between nucleus and cytoplasm during brain development. Cell Mol Neurobiol 28: 205221.
  • Yi XN, Zheng LF, Zhang JW, Zhang LZ, Xu YZ, Luo G, Luo XG. 2006. Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury. Neurosci Res 56: 314321.
  • Yuan XB, Jin M, Xu X, Song YQ, Wu CP, Poo MM, Duan S. 2003. Signalling and crosstalk of Rho GTPases in mediating axon guidance. Nat Cell Biol 5: 3845.
  • Zhang HY, Zheng LF, Yi XN, Chen ZB, He ZP, Zhao D, Zhang XF, Ma ZJ. 2010. Slit1 promotes regenerative neurite outgrowth of adult dorsal root ganglion neurons in vitro via binding to the Robo receptor. J Chem Neuroanat 39: 256261.
  • Zheng LF, Wang R, Xu YZ, Yi XN, Zhang JW, Zeng ZC. 2008. Calcitonin gene-related peptide dynamics in rat dorsal root ganglia and spinal cord following different sciatic nerve injuries. Brain Res 1187: 2032.
  • Zheng LF, Wang R, Yu QP, Wang H, Yi XN, Wang QB, Zhang JW, Zhang GX, Xu YZ. 2010. Expression of HGF/c-Met is dynamically regulated in the dorsal root ganglions and spinal cord of adult rats following sciatic nerve ligation. Neurosignals 18: 4956.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
AR_22419_sm_SuppFig1.tif7206KSupplemental Figure 1: Characterization of srGAP1 and srGAP3 antibody specificity using primary culture of DRG as positive control and antibody omission and preabsorption as negative controls. The left panels show positive labeling for the mouse anti-srGAP1 antibody and the rabbit and rat antibodies against srGAP3. In the absence of the primary antibody (middle panels) or the addition of excessive immunogenic peptides (right panels) in the incubation buffer, no specific immunolabeling is found in the DRG sections. Scale bar = 100 µm.
AR_22419_sm_SuppFig2.tif2007KSupplemental Figure. 2: Expression of srGAP1 and srGAP3 in the proximal stump of the sciatic nerve. Fairly distinct srGAP1 immunofluorescence is present in the nerve trunk (A), with the labeled profiles appearing as longitudinal bundles and arranging in a similar fashion as that of S100 labeling (E). srGAP3 labeling is fairly low in the proximal nerve stump (B, D). Scale bar = 40 µm.

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