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Neuroprotective effects of bone morphogenetic protein 7 (BMP7) treatment after spinal cord injury
Neuroscience Letters 465 (2009) 226–229
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of bone morphogenetic protein 7 (BMP7) treatment after spinal cord injury Juan Pablo de Rivero Vaccari a,b , Alex Marcillo a,b , Doris Nonner c , W. Dalton Dietrich a,b , Robert W. Keane c,∗ a b c
Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, United States Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL 33136, United States Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136, United States
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 31 August 2009 Accepted 11 September 2009 Keywords: Bone morphogenetic protein 7 Spinal cord injury Glutamate excitotoxicity MAPK signaling
a b s t r a c t Bone morphogenetic protein 7 (BMP7) has been shown to ameliorate reduced dendritic growth induced by glutamate excitotoxicity in neuronal tissue cultures and/or provide an enhancement of functional recovery in central nervous system (CNS) injury. BMP7 expression is modulated by spinal cord injury (SCI), but the molecular mechanisms involved in neuroprotection have not been clearly defined. Here, we show that BMP7 treatment of rats subjected to mild cervical SCI significantly increased the prosurvival mitogen-activated protein kinase-38 (MAPK-38) pathway and levels of N-methyl-d-aspartate receptor 1 (NMDAR-1) resulting in a significant increase in neuronal sparing in the ventral horn of the spinal cord. Moreover, BMP7 was neuroprotective against glutamate-mediated excitotoxicity in cultured cortical neurons. These studies show that BMP7 administration may be used as a therapeutic strategy to reduce the damaging excitotoxic effects following SCI. © 2009 Elsevier Ireland Ltd. All rights reserved.
Spinal cord injury (SCI) elicits glutamate excitotoxicity that rapidly induces damage to neuronal dendrites and subsequent neuronal cell death. Recently, bone morphogenetic protein 7 (BMP7) has been reported to selectively stimulate dendritic growth and branching from neurons in culture, but whether BMP7 attenuates neuronal loss following SCI has not been tested. Modulation of BMP expression has been reported in various types of CNS injury including: SCI [32], traumatic brain injury (TBI) [2], and ischemia [19], but whether direct administration of BMPs after SCI leads to neuroprotection has not been evaluated. Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor  (TGF) superfamily. BMPs signal through serine/threonine kinase receptors, composed of type I and II BMP receptors [24,33,35]. These receptors are expressed differently in various tissues, including spinal cord [34]. However, once activated by BMPs, these receptors initiate complex signaling pathways with multiple levels of regulation [13], including the mitogen-activated protein kinase38 (MAPK-p38) pathway [17] involved in synaptic plasticity and neuronal survival [8]. In these studies, adult female Fischer (180–200 g) rats were used. The Institutional Animal Care and Use Committee of the
∗ Corresponding author. Tel.: +1 305 243 5726; fax: +1 305 243 5931. E-mail address: [email protected] (R.W. Keane). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.09.013
University of Miami approved all animal procedures. Rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg). Adequate amounts of anesthesia were determined by monitoring toe-touch. Using aseptic techniques, a midline incision was made at the neck in the skin and musculature to expose the C2–T1 vertebrae. A laminectomy at vertebrae level C5 was performed under the microscope to expose the dorsal surface of the spinal cord. Animals were secured to a spinal frame by clamping the spinous process rostral and caudal to the laminectomy. A moderate contusion injury was performed at cervical level C5 using a circular flap tip of a 4-mm diameter impactor of the Electromagnetic SCI Device (Ohio State University) that transduced a force of ∼3 Kdyn. This injury resulted in slight dimpling of the dura and provided a consistent starting point from which subsequent displacements were measured (0.95 mm displacement injury with a single, brief displacement of <20 ms) upon the exposed dorsal surface of the spinal cord. Body and spinal cord temperature were maintained at 37.5 ◦ C with a feedback-controlled heating blanket monitored by a rectal thermometer. Following injury, the incision was closed with wound clips and sutures, and then each rat was returned to its cage. Animals were sacrificed at different times following SCI. Sham animals were used as controls. Animals were injected intrathecally with 1 g BMP7 (Curis, Inc.), and 1 g intraperitoneally. In addition, animals received a daily injection of 1 g of BMP7 intraperitoneally 20 min after SCI for 2 more days. The control group consisted of animals injected with saline and sham animals. All treatments were performed in a double-blind manner.
J.P. de Rivero Vaccari et al. / Neuroscience Letters 465 (2009) 226–229
For detection of BMP7 and activation of the Smad and p38 MAPK signaling pathways, 4–5 mm segments of sham or injured spinal cords (n = 3 per time point: animals were sacrificed at 3 d after SCI) were homogenized in extraction buffer (20 mM Tris–HCl, pH: 7.5, 150 mM NaCl, 1% Triton X-100; 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2.5 mM pyrophosphate, 1 mM -glycerophosphate) with protease and phosphatase inhibitors (Sigma). Proteins were resolved in 10–20% Tris–HCl Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Applied Biosystems) and placed in blocking buffer (PBS, 0.1% Tween-20, 0.4% I-Block (Applied Biosystems) and then incubated for 1 h with: monoclonal antibodies to BMP7 (R&D systems), phospho-Smad-1/5/8, 2 (Cell Signaling), p38 MAPK and phosphorylated p38 MAPK (Cell Signaling), followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling). Visualization of signal was enhanced by chemiluminescence using a phototope-HRP detection kit (Cell Signaling). To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce) and blotted for tubulin (1:5000, BD Biosciences Pharmingen). Quantification of band density was performed using UN-Scan-IT gelTM quantifying software (Silk Scientific), and data was normalized to -tubulin. Antibodies were diluted 1:1000 unless specified otherwise. For stereological cell counting, paraffin-embedded, 10 m transverse sections, taken every 500 m apart from the injury epicenter for 1.5 mm rostral and caudal (7 sections analyzed) were stained with the neuronal antibody NeuN (Chemicon) using diaminobenzidine (DAB) as the chromophore. The total number of NeuN immunoreactive cells per spinal cord was quantified using unbiased stereological procedures. Accordingly, sections collected in the systematic random manner were analyzed by an observer blinded to the treatments using Stereoinvestigator software (MicroBrightfield, Inc., Colchester, VT) and microscopy. Areas of interest were analyzed and determined by the physical fractionator method in which the physical dissector probe was used to estimate the neuronal number in the enclosed volume corresponding to the area of the ventral horn. Immunoreactive cells were those that had degrees of staining greater than background. Statistical comparisons between uninjured and injured groups were made using two-tailed Student’s t-test and a one-way ANOVA followed by Tukey’s multiple comparison tests. P-values of significance used were *P < 0.05. Neuronal cultures were obtained by dissociation of 16–17-day Sprague–Dawley rat embryo brains. The tissue was disrupted into a cell suspension by gentle trituration and the cells were grown on poly-l-lysine coated tissue culture dishes in N5 medium that contained 5% serum fraction that supports the long-term survival of neurons as described [20]. Cultures were treated with 100 M glutamate, or pretreated with 0–200 ng/ml of BMP7 30 min prior to glutamate treatment. Cell viability was assayed by the Alamar blue viability assay, and reading at 595 nm. Growth factors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors in the nucleus. The mitogen-activated protein kinase (MAPK) cascade is one of the best-characterized cascades in this regulatory process and is involved in synaptic plasticity, neuronal survival, and modulation of NMDAR activity [14]. In addition, signaling from ligand-induced heteromeric complexes of type I and type II BMP receptors results in activation of MAPK-p38. In order to determine whether SCI and BMP7 treatment after SCI activated the MAPK pathway, we performed quantitative immunoblot analysis on spinal cord lysates of sham-operated and traumatized rats at 3 days after SCI for phospho-p-38 MAPK expression. Another group of rats were subjected to moderate cervical SCI and treated immediately with 1 g BMP7 intrathecally and 1 g intraperitoneally. As shown in Fig. 1, SCI induced increased levels of phospho-p38
227
Fig. 1. BMP7 treatment after SCI stimulates phosphorylation of MAPK-p38 and increases levels of NMDAR1. Representative immunoblot analysis of spinal cord lysates of sham (Sh), BMP7 treated (BMP7) and injured untreated (C) animals at 3 d after trauma. Spinal cord lysates were immunoblotted with antibodies against NMDAR1 and phospho-p38. Densitometric analysis indicates a significant increase in the levels of NMDAR1 and phospho-p38 following BMP7 treatment. -Tubulin was used as internal standard and control for protein loading. N = 3. Data are presented as mean ± S.E.M. *P < 0.05.
as compared to sham animals at 3 days after trauma. Moreover, BMP7 treatment resulted in a significant increase in phosphop38 when compared to untreated controls. Since BMP7 signaling is complex and is regulated at multiple levels, we analyzed the influence of BMP7 on the activation of Smad 1/5/8 and Smad 2 pathways (Supplemental Fig. 1). Spinal cords from animals treated with BMP7 after SCI did not show significant alterations in the levels of phospho-Smad 1/5/8 or phospho-Smad 2 when compared to injured, non-treated controls. Since growth factors (glial cell linederived neurotrophic factor (GDNF), fibroblast growth factor (FGF)) modulate NMDAR activity, we analyzed spinal cord lysates from the three experimental groups for expression of NMDAR1. SCI significantly decreased the levels of NMDAR1 in spinal cord lysates at 3 days post-injury. In contrast, spinal cords from BMP7-treated animals showed significant higher levels of NMDAR1 than untreated controls. Thus, SCI activates p38 MAPK and BMP7 treatment causes a further enhancement of this pathway. Moreover, BMP7 administration after SCI resulted in a significant increase in NMDAR1 expression. To determine whether increased phospho-p38 expression was linked to neuronal survival, we counted the number of neurons in the injury epicenter using unbiased stereology. Fig. 2 shows neuronal cell counts of spinal cord sections of the lesion at 3 days after trauma. Spinal cords from animals treated with BMP7 after cervical SCI showed significantly higher number of neurons compared to untreated controls.
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J.P. de Rivero Vaccari et al. / Neuroscience Letters 465 (2009) 226–229
Fig. 2. BMP7 treatment preserves spinal cord neurons following injury. A person blinded to the treatment protocols assessed the number of NeuN positive cells (neurons) in the ventral horn of the spinal cord by unbiased stereological procedures. The number of preserved neurons after injury in BMP7-treated animals (BMP7) was greater than the number of neurons remaining in the ventral horn of the spinal cord of untreated injured animals (C). N = 4. Data are presented as mean ± S.E.M. *P < 0.05 compared to untreated, sham controls (Sh).
Glutamate excitotoxicity contributes to neuronal cell death in primary neuronal cultures [28] and following CNS injury [12]. To determine whether BMP7 blocked glutamate excitotoxicity, we pretreated primary neuronal cultures with 0–200 ng/ml of BMP7 followed by glutamate treatment (100 M). Fig. 3 shows that at a concentration of 50 or 100 ng/ml, BMP7 significantly blocked neuronal death. Thus, BMP7 significantly blocks glutamate excitotoxicity of primary neurons. In this study, we have shown for the first time that BMP7 treatment of rats subjected to mild cervical SCI significantly increased the MAPK-p38 pathway and levels of NMDAR1, resulting in a significant increase in neuronal sparing. Both BMP7 and its receptor are upregulated after CNS injury and stroke [4,5,16,22,32]. Neuroprotective effects of BMP7 have been reported in that post-stroke BMP7 administration partially improves motor function at 2 weeks after ischemia in rats [7,19,29]. Moreover, neuroprogenitor implants that secrete Noggin, a BMP7 antagonist, in the damage spinal cord show a significant increase in lesion volume and the number of infiltrating macrophages [10]. In contrast, intracisternal BMP7 injection
after TBI did not result in improved functional outcomes [2]. Since BMP7 has a short half-life (about 10–30 min) and works best within a short range, it is possible that discrepancies in outcome measures after BMP7 treatment in the various CNS injury models are due to lack of consistent and efficient BMP7 delivery. BMP7 has been shown to selectively enhance dendrite growth of sympathetic, cerebral cortical and hippocampal neurons in culture. Our novel finding is that BMP7 can prevent excitotoxic cell death of cortical neurons associated with excess glutamate in a dose dependent manner. BMP7 dosage in the range of 50–100 ng/ml was more effective than other dosages tested. Esquenazi and co-workers [11] have demonstrated that BMP7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro. Although our present experiments do not address the mechanism by which BMP7 antagonizes the effects of glutamate, our findings suggest that stimulation of the pro-survival MAPK-p38 pathway may be involved. This idea is consistent with other reports demonstrating MAPK signaling as neuroprotective [1,18,30]. Various types of CNS injuries result in significant loss of NMDAR1 and NR2A/B subunits. The loss is due to nontranscriptional changes in the NMDAR, since mRNA levels remain unchanged during the post-injury period [21]. Our studies demonstrate a loss of NMDAR1 in the spinal cord at 3 days after trauma. However, BMP7 treatment resulted in a significant increase in the levels of NMDAR1 in spinal cord lysates. The increase in NMDAR1 expression was associated with increased stimulation of the MAPK-p38 pathway. Other growth factors such as insulin-like growth factor [31], fibroblast growth factor [14], neurotrophin like glial cell line-derived neurotrophic factor [26] induced activation of the MAPK pathway and may modulate NMDAR activity, that may be responsible for the neuroprotective effects in CNS injury models [14]. Moreover, brain-derived neurotrophic factor (BDNF) has been shown to activate BMP7 signaling [6,27]. Excitotoxic mechanisms have been shown to participate in the pathophysiology of SCI [9]. Microdialysis studies have demonstrated elevations in the extracellular concentrations of glutamate after experimental SCI [23]. Other studies have reported alterations in specific glutamate receptor populations [3] that emphasize the potential role of this neurotransmitter system in SCI in terms of neuronal vulnerability. Our results show that SCI increases MAPKp38. Moreover, BMP7 treatment further significantly increased levels of MAPK-p38 that were associated with increased NMDAR1 expression. Since growth factors decrease NMDAR activity, it is possible that neurons stimulated by BMP7 treatment downregulate NMDAR1 that renders them refractory to excessive glutamate after trauma. The increased MAPK-p38 induced by SCI may not be sufficient to rescue these neurons, thus levels of NMDAR1 decrease after SCI even though MAPK-p38 levels increase. Although previous preclinical studies have reported the beneficial effect of several glutamate receptor blockers on histopathological and behavioral outcomes after experimental SCI [15,25] no clinical studies have shown efficacy with this therapeutic approach. More basic research is therefore needed to investigate novel targets for preventing the damage induced by excitotoxicity after SCI. Thus, as excitotoxicity impacts on outcomes of various CNS injury and neurodegenerative diseases, BMP7 may play an important role in modulating CNS responses and neuronal survival.
Acknowledgments Fig. 3. BMP7 blocks glutamate excitotoxicity in primary neuronal cultures. Primary cortical neurons were treated with 100 M glutamate for 8 h resulting in cell death as assayed by Alamar blue viability assay. BMP7 (10 ng/ml to 25 g/ml) administered 30 min prior to glutamate treatment significantly decreased glutamate-induced cell death. Values are normalized to non-treated controls (C). N = 16 wells.
This work was supported by Department of Defense grant: USAMRMC X81XWH-05-1-0001. We thank David Molnar and Dr. Juan Carlos de Rivero Vaccari for technical assistance.
J.P. de Rivero Vaccari et al. / Neuroscience Letters 465 (2009) 226–229
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of bone morphogenetic protein 7 (BMP7) treatment after spinal cord injury Juan Pablo de Rivero Vaccari a,b , Alex Marcillo a,b , Doris Nonner c , W. Dalton Dietrich a,b , Robert W. Keane c,∗ a b c
Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, United States Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL 33136, United States Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136, United States
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 31 August 2009 Accepted 11 September 2009 Keywords: Bone morphogenetic protein 7 Spinal cord injury Glutamate excitotoxicity MAPK signaling
a b s t r a c t Bone morphogenetic protein 7 (BMP7) has been shown to ameliorate reduced dendritic growth induced by glutamate excitotoxicity in neuronal tissue cultures and/or provide an enhancement of functional recovery in central nervous system (CNS) injury. BMP7 expression is modulated by spinal cord injury (SCI), but the molecular mechanisms involved in neuroprotection have not been clearly defined. Here, we show that BMP7 treatment of rats subjected to mild cervical SCI significantly increased the prosurvival mitogen-activated protein kinase-38 (MAPK-38) pathway and levels of N-methyl-d-aspartate receptor 1 (NMDAR-1) resulting in a significant increase in neuronal sparing in the ventral horn of the spinal cord. Moreover, BMP7 was neuroprotective against glutamate-mediated excitotoxicity in cultured cortical neurons. These studies show that BMP7 administration may be used as a therapeutic strategy to reduce the damaging excitotoxic effects following SCI. © 2009 Elsevier Ireland Ltd. All rights reserved.
Spinal cord injury (SCI) elicits glutamate excitotoxicity that rapidly induces damage to neuronal dendrites and subsequent neuronal cell death. Recently, bone morphogenetic protein 7 (BMP7) has been reported to selectively stimulate dendritic growth and branching from neurons in culture, but whether BMP7 attenuates neuronal loss following SCI has not been tested. Modulation of BMP expression has been reported in various types of CNS injury including: SCI [32], traumatic brain injury (TBI) [2], and ischemia [19], but whether direct administration of BMPs after SCI leads to neuroprotection has not been evaluated. Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor  (TGF) superfamily. BMPs signal through serine/threonine kinase receptors, composed of type I and II BMP receptors [24,33,35]. These receptors are expressed differently in various tissues, including spinal cord [34]. However, once activated by BMPs, these receptors initiate complex signaling pathways with multiple levels of regulation [13], including the mitogen-activated protein kinase38 (MAPK-p38) pathway [17] involved in synaptic plasticity and neuronal survival [8]. In these studies, adult female Fischer (180–200 g) rats were used. The Institutional Animal Care and Use Committee of the
∗ Corresponding author. Tel.: +1 305 243 5726; fax: +1 305 243 5931. E-mail address: [email protected] (R.W. Keane). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.09.013
University of Miami approved all animal procedures. Rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg). Adequate amounts of anesthesia were determined by monitoring toe-touch. Using aseptic techniques, a midline incision was made at the neck in the skin and musculature to expose the C2–T1 vertebrae. A laminectomy at vertebrae level C5 was performed under the microscope to expose the dorsal surface of the spinal cord. Animals were secured to a spinal frame by clamping the spinous process rostral and caudal to the laminectomy. A moderate contusion injury was performed at cervical level C5 using a circular flap tip of a 4-mm diameter impactor of the Electromagnetic SCI Device (Ohio State University) that transduced a force of ∼3 Kdyn. This injury resulted in slight dimpling of the dura and provided a consistent starting point from which subsequent displacements were measured (0.95 mm displacement injury with a single, brief displacement of <20 ms) upon the exposed dorsal surface of the spinal cord. Body and spinal cord temperature were maintained at 37.5 ◦ C with a feedback-controlled heating blanket monitored by a rectal thermometer. Following injury, the incision was closed with wound clips and sutures, and then each rat was returned to its cage. Animals were sacrificed at different times following SCI. Sham animals were used as controls. Animals were injected intrathecally with 1 g BMP7 (Curis, Inc.), and 1 g intraperitoneally. In addition, animals received a daily injection of 1 g of BMP7 intraperitoneally 20 min after SCI for 2 more days. The control group consisted of animals injected with saline and sham animals. All treatments were performed in a double-blind manner.
J.P. de Rivero Vaccari et al. / Neuroscience Letters 465 (2009) 226–229
For detection of BMP7 and activation of the Smad and p38 MAPK signaling pathways, 4–5 mm segments of sham or injured spinal cords (n = 3 per time point: animals were sacrificed at 3 d after SCI) were homogenized in extraction buffer (20 mM Tris–HCl, pH: 7.5, 150 mM NaCl, 1% Triton X-100; 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2.5 mM pyrophosphate, 1 mM -glycerophosphate) with protease and phosphatase inhibitors (Sigma). Proteins were resolved in 10–20% Tris–HCl Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Applied Biosystems) and placed in blocking buffer (PBS, 0.1% Tween-20, 0.4% I-Block (Applied Biosystems) and then incubated for 1 h with: monoclonal antibodies to BMP7 (R&D systems), phospho-Smad-1/5/8, 2 (Cell Signaling), p38 MAPK and phosphorylated p38 MAPK (Cell Signaling), followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling). Visualization of signal was enhanced by chemiluminescence using a phototope-HRP detection kit (Cell Signaling). To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce) and blotted for tubulin (1:5000, BD Biosciences Pharmingen). Quantification of band density was performed using UN-Scan-IT gelTM quantifying software (Silk Scientific), and data was normalized to -tubulin. Antibodies were diluted 1:1000 unless specified otherwise. For stereological cell counting, paraffin-embedded, 10 m transverse sections, taken every 500 m apart from the injury epicenter for 1.5 mm rostral and caudal (7 sections analyzed) were stained with the neuronal antibody NeuN (Chemicon) using diaminobenzidine (DAB) as the chromophore. The total number of NeuN immunoreactive cells per spinal cord was quantified using unbiased stereological procedures. Accordingly, sections collected in the systematic random manner were analyzed by an observer blinded to the treatments using Stereoinvestigator software (MicroBrightfield, Inc., Colchester, VT) and microscopy. Areas of interest were analyzed and determined by the physical fractionator method in which the physical dissector probe was used to estimate the neuronal number in the enclosed volume corresponding to the area of the ventral horn. Immunoreactive cells were those that had degrees of staining greater than background. Statistical comparisons between uninjured and injured groups were made using two-tailed Student’s t-test and a one-way ANOVA followed by Tukey’s multiple comparison tests. P-values of significance used were *P < 0.05. Neuronal cultures were obtained by dissociation of 16–17-day Sprague–Dawley rat embryo brains. The tissue was disrupted into a cell suspension by gentle trituration and the cells were grown on poly-l-lysine coated tissue culture dishes in N5 medium that contained 5% serum fraction that supports the long-term survival of neurons as described [20]. Cultures were treated with 100 M glutamate, or pretreated with 0–200 ng/ml of BMP7 30 min prior to glutamate treatment. Cell viability was assayed by the Alamar blue viability assay, and reading at 595 nm. Growth factors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors in the nucleus. The mitogen-activated protein kinase (MAPK) cascade is one of the best-characterized cascades in this regulatory process and is involved in synaptic plasticity, neuronal survival, and modulation of NMDAR activity [14]. In addition, signaling from ligand-induced heteromeric complexes of type I and type II BMP receptors results in activation of MAPK-p38. In order to determine whether SCI and BMP7 treatment after SCI activated the MAPK pathway, we performed quantitative immunoblot analysis on spinal cord lysates of sham-operated and traumatized rats at 3 days after SCI for phospho-p-38 MAPK expression. Another group of rats were subjected to moderate cervical SCI and treated immediately with 1 g BMP7 intrathecally and 1 g intraperitoneally. As shown in Fig. 1, SCI induced increased levels of phospho-p38
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Fig. 1. BMP7 treatment after SCI stimulates phosphorylation of MAPK-p38 and increases levels of NMDAR1. Representative immunoblot analysis of spinal cord lysates of sham (Sh), BMP7 treated (BMP7) and injured untreated (C) animals at 3 d after trauma. Spinal cord lysates were immunoblotted with antibodies against NMDAR1 and phospho-p38. Densitometric analysis indicates a significant increase in the levels of NMDAR1 and phospho-p38 following BMP7 treatment. -Tubulin was used as internal standard and control for protein loading. N = 3. Data are presented as mean ± S.E.M. *P < 0.05.
as compared to sham animals at 3 days after trauma. Moreover, BMP7 treatment resulted in a significant increase in phosphop38 when compared to untreated controls. Since BMP7 signaling is complex and is regulated at multiple levels, we analyzed the influence of BMP7 on the activation of Smad 1/5/8 and Smad 2 pathways (Supplemental Fig. 1). Spinal cords from animals treated with BMP7 after SCI did not show significant alterations in the levels of phospho-Smad 1/5/8 or phospho-Smad 2 when compared to injured, non-treated controls. Since growth factors (glial cell linederived neurotrophic factor (GDNF), fibroblast growth factor (FGF)) modulate NMDAR activity, we analyzed spinal cord lysates from the three experimental groups for expression of NMDAR1. SCI significantly decreased the levels of NMDAR1 in spinal cord lysates at 3 days post-injury. In contrast, spinal cords from BMP7-treated animals showed significant higher levels of NMDAR1 than untreated controls. Thus, SCI activates p38 MAPK and BMP7 treatment causes a further enhancement of this pathway. Moreover, BMP7 administration after SCI resulted in a significant increase in NMDAR1 expression. To determine whether increased phospho-p38 expression was linked to neuronal survival, we counted the number of neurons in the injury epicenter using unbiased stereology. Fig. 2 shows neuronal cell counts of spinal cord sections of the lesion at 3 days after trauma. Spinal cords from animals treated with BMP7 after cervical SCI showed significantly higher number of neurons compared to untreated controls.
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Fig. 2. BMP7 treatment preserves spinal cord neurons following injury. A person blinded to the treatment protocols assessed the number of NeuN positive cells (neurons) in the ventral horn of the spinal cord by unbiased stereological procedures. The number of preserved neurons after injury in BMP7-treated animals (BMP7) was greater than the number of neurons remaining in the ventral horn of the spinal cord of untreated injured animals (C). N = 4. Data are presented as mean ± S.E.M. *P < 0.05 compared to untreated, sham controls (Sh).
Glutamate excitotoxicity contributes to neuronal cell death in primary neuronal cultures [28] and following CNS injury [12]. To determine whether BMP7 blocked glutamate excitotoxicity, we pretreated primary neuronal cultures with 0–200 ng/ml of BMP7 followed by glutamate treatment (100 M). Fig. 3 shows that at a concentration of 50 or 100 ng/ml, BMP7 significantly blocked neuronal death. Thus, BMP7 significantly blocks glutamate excitotoxicity of primary neurons. In this study, we have shown for the first time that BMP7 treatment of rats subjected to mild cervical SCI significantly increased the MAPK-p38 pathway and levels of NMDAR1, resulting in a significant increase in neuronal sparing. Both BMP7 and its receptor are upregulated after CNS injury and stroke [4,5,16,22,32]. Neuroprotective effects of BMP7 have been reported in that post-stroke BMP7 administration partially improves motor function at 2 weeks after ischemia in rats [7,19,29]. Moreover, neuroprogenitor implants that secrete Noggin, a BMP7 antagonist, in the damage spinal cord show a significant increase in lesion volume and the number of infiltrating macrophages [10]. In contrast, intracisternal BMP7 injection
after TBI did not result in improved functional outcomes [2]. Since BMP7 has a short half-life (about 10–30 min) and works best within a short range, it is possible that discrepancies in outcome measures after BMP7 treatment in the various CNS injury models are due to lack of consistent and efficient BMP7 delivery. BMP7 has been shown to selectively enhance dendrite growth of sympathetic, cerebral cortical and hippocampal neurons in culture. Our novel finding is that BMP7 can prevent excitotoxic cell death of cortical neurons associated with excess glutamate in a dose dependent manner. BMP7 dosage in the range of 50–100 ng/ml was more effective than other dosages tested. Esquenazi and co-workers [11] have demonstrated that BMP7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro. Although our present experiments do not address the mechanism by which BMP7 antagonizes the effects of glutamate, our findings suggest that stimulation of the pro-survival MAPK-p38 pathway may be involved. This idea is consistent with other reports demonstrating MAPK signaling as neuroprotective [1,18,30]. Various types of CNS injuries result in significant loss of NMDAR1 and NR2A/B subunits. The loss is due to nontranscriptional changes in the NMDAR, since mRNA levels remain unchanged during the post-injury period [21]. Our studies demonstrate a loss of NMDAR1 in the spinal cord at 3 days after trauma. However, BMP7 treatment resulted in a significant increase in the levels of NMDAR1 in spinal cord lysates. The increase in NMDAR1 expression was associated with increased stimulation of the MAPK-p38 pathway. Other growth factors such as insulin-like growth factor [31], fibroblast growth factor [14], neurotrophin like glial cell line-derived neurotrophic factor [26] induced activation of the MAPK pathway and may modulate NMDAR activity, that may be responsible for the neuroprotective effects in CNS injury models [14]. Moreover, brain-derived neurotrophic factor (BDNF) has been shown to activate BMP7 signaling [6,27]. Excitotoxic mechanisms have been shown to participate in the pathophysiology of SCI [9]. Microdialysis studies have demonstrated elevations in the extracellular concentrations of glutamate after experimental SCI [23]. Other studies have reported alterations in specific glutamate receptor populations [3] that emphasize the potential role of this neurotransmitter system in SCI in terms of neuronal vulnerability. Our results show that SCI increases MAPKp38. Moreover, BMP7 treatment further significantly increased levels of MAPK-p38 that were associated with increased NMDAR1 expression. Since growth factors decrease NMDAR activity, it is possible that neurons stimulated by BMP7 treatment downregulate NMDAR1 that renders them refractory to excessive glutamate after trauma. The increased MAPK-p38 induced by SCI may not be sufficient to rescue these neurons, thus levels of NMDAR1 decrease after SCI even though MAPK-p38 levels increase. Although previous preclinical studies have reported the beneficial effect of several glutamate receptor blockers on histopathological and behavioral outcomes after experimental SCI [15,25] no clinical studies have shown efficacy with this therapeutic approach. More basic research is therefore needed to investigate novel targets for preventing the damage induced by excitotoxicity after SCI. Thus, as excitotoxicity impacts on outcomes of various CNS injury and neurodegenerative diseases, BMP7 may play an important role in modulating CNS responses and neuronal survival.
Acknowledgments Fig. 3. BMP7 blocks glutamate excitotoxicity in primary neuronal cultures. Primary cortical neurons were treated with 100 M glutamate for 8 h resulting in cell death as assayed by Alamar blue viability assay. BMP7 (10 ng/ml to 25 g/ml) administered 30 min prior to glutamate treatment significantly decreased glutamate-induced cell death. Values are normalized to non-treated controls (C). N = 16 wells.
This work was supported by Department of Defense grant: USAMRMC X81XWH-05-1-0001. We thank David Molnar and Dr. Juan Carlos de Rivero Vaccari for technical assistance.
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