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Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast-induced traumatic brain injury. Specifically, we employ a bioengineered 3D brain-like human tissue culture system composed of collagen-infused silk protein donut-like hydrogels embedded with human IPSC-derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D-printed surrogate skull-like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast-induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF-H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury, composed of neurons, microglia, and astrocytes, and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for the development of therapies for TBI patients.

Total body radiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated the impact of radiation-induced iron release on the spleen of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed ~20-60-fold increased Fe3+ in the spleen 7-14 days post-irradiation, also associated with altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not prevent iron deposition in the spleen, and did not significantly modulate most iron-binding proteins. Spleen volumes were markedly decreased 7-14 days, correlating with high Fe3+. At these time points, caspase-3 was activated and we identified four markers of ferroptosis, iron-dependent programmed cell death. Interestingly, p21/Waf1, a marker of accelerated senescence, was not upregulated in vivo. Macrophage inflammation is an important effect of TBI. We investigated the effects of radiation and Fe3+ on the cultured J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, within 24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron binding proteins and triggers ferroptosis.

Angiotensin II (Ang II)-mediated activation of its type I receptor (AT1R) in the central nervous system promotes glial proliferation, local inflammation, and a decrease of cerebral blood flow. Angiotensin-(1-7) (Ang-(1-7))-an Ang II derivative peptide-signals through the Mas receptor (MasR) in opposition to Ang II/AT1R, promoting anti-inflammatory, vasodilatory, and neuroprotective effects. As our laboratory has previously demonstrated beneficial effects of AT1R inhibition following controlled cortical impact (CCI) in mice, we asked whether activation of Ang-(1-7)/MasR signaling would also be beneficial in this model. Adult male C57BL/6 mice were injured by CCI. Ang-(1-7) or vehicle was administered subcutaneously (S.Q.) at 1 mg/kg/day at 1 or 6 h post-injury, until animals were sacrificed at 3 or 29 days post-injury (dpi). Ang-(1-7) attenuated motor deficits at 3 dpi and improved performance in the Morris Water Maze at 28 dpi. Brain histology or magnetic resonance imaging (MRI) indicated that Ang-(1-7)-treated mice had smaller lesion volumes at 3, 10, 24, and 29 dpi. Pre-treatment with A779, a MasR antagonist, prevented Ang-(1-7) from reducing lesion volume at 3 dpi, suggesting that the benefits of Ang-(1-7) were MasR-dependent. Immunohistochemistry revealed that Ang-(1-7) reduced microgliosis at 3 and 29 dpi, and astrogliosis at 29 dpi. Ang-(1-7) decreased neuronal and capillary loss at 29 dpi. In summary, S.Q. administration of Ang-(1-7) after injury had anti-inflammatory, neuroprotective, and cerebrovascular-protective actions leading to improved functional and pathological recovery in a mouse model of traumatic brain injury (TBI). These data show for the first time that Ang-(1-7) has potential therapeutic use for TBI.

Total body irradiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated radiation-induced iron in the spleens of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed increased splenic Fe3+ and altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not affect iron deposition in the spleen or modulate iron-binding proteins. Caspase-3 was activated after ~7–14 days, indicating apoptosis had occurred. We also identified markers of iron-dependent apoptosis known as ferroptosis. The p21/Waf1 accelerated senescence marker was not upregulated. Macrophage inflammation is an effect of TBI. We investigated the effects of radiation and Fe3+ on the J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, after ~24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron-binding proteins and triggers apoptosis and possible ferroptosis.

The increasing potential for radiation exposure from nuclear accidents or terrorist activities has intensified the need to develop pharmacologic countermeasures against injury from total body irradiation (TBI). Many initial experiments to develop and test these countermeasures utilize murine irradiation models. Yet, the route of drug administration can alter the response to irradiation injury. Studies have demonstrated that cutaneous injuries can exacerbate damage from radiation, and thus surgical implantation of osmotic pumps for drug delivery could adversely affect the survival of mice following TBI. However, daily handling and injections to administer drugs could also have negative consequences. This study compared the effects of subcutaneous needlesticks with surgical implantation of osmotic pumps on morbidity and mortality in a murine model of hematopoietic acute radiation syndrome (H-ARS). C57BL/6 mice were sham irradiated or exposed to a single dose of 7.7 Gy60Co TBI. Mice were implanted with osmotic pumps containing sterile saline seven days prior to irradiation or received needlesticks for 14 days following irradiation or received no treatment. All irradiated groups exhibited weight loss. Fewer mice with osmotic pumps survived to 30 days post irradiation (37.5%) than mice receiving needlesticks or no treatment (70% and 80%, respectively), although this difference was not statistically significant. However, mice implanted with the pump lost significantly more weight than mice that received needlesticks or no treatment. These data suggest that surgical implantation of a drug-delivery device can adversely affect the outcome in a murine model of H-ARS.

Chronic inflammation mediated by persistent microglial activation is associated with the pathogenesis of neurodegenerative diseases. The mechanisms underlying chronic microglial activation are poorly understood. We have previously shown that anti-inflammatory TGF-β signaling is inhibited in LPS-treated microglia. In this study, we assessed whether different disease-related microglial activators could downregulate TGF-β induction of gene expression. We examined the effects of amyloid β (Aβ) (1-42)- or heat-killed Listeria monocytogenes (HKLM) on the TGF-β-regulated gene expression in primary rat microglia. We found that Aβ (1-42) oligomers and HKLM, in addition to LPS, suppressed TGF-β-mediated induction of gene expression in part through reducing expression of TβR1 mRNA encoding the TGF-β receptor 1 in primary microglia. Aβ (1-42) and LPS also prevented induction of TGF-β-induced genes in primary microglia. Additionally, Aβ (1-42) rescued primary microglia from TGF-β-mediated cell death without increasing cell proliferation. Blockage of NFκB signaling, but not the ERK or IRF3 pathways, inhibited Aβ (1-42)- and LPS-mediated reduction of TβR1 mRNA. Finally, LPS and Aβ (1-42) induced transient upregulation of mRNAs encoding SnoN and Bambi, inhibitors of TGF-β signaling. Our data indicate that one mechanism through which activators may prolong microglial stimulation is through direct inhibition of anti-inflammatory signaling. A more detailed understanding of the interaction between inflammatory and anti-inflammatory pathways may reveal potential targets for ameliorating chronic inflammation and hence speed the development of therapeutics to address neurodegenerative diseases.

Traumatic brain injury alters the signaling environment of the adult neurogenic niche and may activate unique proliferative cell populations that contribute to the post-injury neurogenic response. Runx1 is not normally expressed by adult neural stem or progenitor cells (NSPCs) but is induced in a subpopulation of putative NSPCs after brain injury in adult mice. In order to investigate the role of Runx1 in NSPCs, we established neurosphere cultures of adult mouse subventricular zone NSPCs. We show that Runx1 is basally expressed in neurosphere culture. Removal of the mitogen bFGF or addition of 1% FBS decreased Runx1 expression. Inhibition of endogenous Runx1 activity with either Ro5-3335 or shRNA-mediated Runx1 knockdown inhibited NSPC proliferation without affecting differentiation. Lentiviral mediated over-expression of Runx1 in neurospheres caused a significant change in cell morphology without reducing proliferation. Runx1-overexpressing neurospheres changed from floating spheres to adherent colonies or individual unipolar or bipolar cells. Flow cytometry analysis indicated that Runx1 over-expression produced a significant increase in expression of the neuronal marker TuJ1 and a minor increase in the astrocytic marker S100β. Thus, Runx1 expression drove adult NSPC differentiation, predominantly toward a neuronal lineage. These data suggest that Runx1 could be manipulated after injury to promote neuronal differentiation to facilitate repair of the CNS.

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.

See Moon (doi:10.1093/awv239) for a scientific commentary on this article.Traumatic brain injury frequently leads to long-term cognitive problems and physical disability yet remains without effective therapeutics. Traumatic brain injury results in neuronal injury and death, acute and prolonged inflammation and decreased blood flow. Drugs that block angiotensin II type 1 receptors (AT1R, encoded by AGTR1) (ARBs or sartans) are strongly neuroprotective, neurorestorative and anti-inflammatory. To test whether these drugs may be effective in treating traumatic brain injury, we selected two sartans, candesartan and telmisartan, of proven therapeutic efficacy in animal models of brain inflammation, neurodegenerative disorders and stroke. Using a validated mouse model of controlled cortical impact injury, we determined effective doses for candesartan and telmisartan, their therapeutic window, mechanisms of action and effect on cognition and motor performance. Both candesartan and telmisartan ameliorated controlled cortical impact-induced injury with a therapeutic window up to 6 h at doses that did not affect blood pressure. Both drugs decreased lesion volume, neuronal injury and apoptosis, astrogliosis, microglial activation, pro-inflammatory signalling, and protected cerebral blood flow, when determined 1 to 3 days post-injury. Controlled cortical impact-induced cognitive impairment was ameliorated 30 days after injury only by candesartan. The neurorestorative effects of candesartan and telmisartan were reduced by concomitant administration of the peroxisome proliferator-activated receptor gamma (PPARγ, encoded by PPARG) antagonist T0070907, showing the importance of PPARγ activation for the neurorestorative effect of these sartans. AT1R knockout mice were less vulnerable to controlled cortical impact-induced injury suggesting that the sartan's blockade of the AT1R also contributes to their efficacy. This study strongly suggests that sartans with dual AT1R blocking and PPARγ activating properties have therapeutic potential for traumatic brain injury.

α/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia. The hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined. We found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model. WWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain.

The role of type IIA receptor protein tyrosine phosphatases (RPTPs), which includes LAR, RPTPσ and RPTPδ, in the nervous system is becoming increasingly recognized. Evidence supports a significant role for these RPTPs during the development of the nervous system as well as after injury, and mutations in RPTPs are associated with human disease. However, a major open question is the nature of the ligands that interact with type IIA RPTPs in the adult brain. Candidates include several different proteins as well as the glycosaminoglycan chains of proteoglycans. In order to investigate this problem, we used a receptor affinity probe assay with RPTPσ-AP fusion proteins on sections of adult mouse brain and to cultured neurons. Our results demonstrate that the major binding sites for RPTPσ in adult mouse brain are on neurons and are not proteoglycan GAG chains, as RPTPσ binding overlaps with the neuronal marker NeuN and was not significantly altered by treatments which eliminate chondroitin sulfate, heparan sulfate, or both. We also demonstrate no overlap of binding of RPTPσ with perineuronal nets, and a unique modulation of RPTPσ binding to brain by divalent cations. Our data therefore point to neuronal proteins, rather than CSPGs, as being the ligands for RPTPσ in the adult, uninjured brain.

Accumulating evidence indicates that activated microglia contribute to the neuropathology involved in many neurodegenerative diseases and after traumatic injury to the CNS. The cytokine transforming growth factor-beta 1 (TGF-β1), a potent deactivator of microglia, should have the potential to reduce microglial-mediated neurodegeneration. It is therefore perplexing that high levels of TGF-β1 are found in conditions where microglia are chronically activated. We hypothesized that TGF-β1 signaling is suppressed in activated microglia. We therefore activated primary rat microglia with lipopolysaccharide (LPS) and determined the expression of proteins important to TGF-β1 signaling. We found that LPS treatment decreased the expression of the TGF-β receptors, TβR1 and TβR2, and reduced protein levels of Smad2, a key mediator of TGF-β signaling. LPS treatment also antagonized the ability of TGF-β to suppress expression of pro-inflammatory cytokines and to induce microglial cell death. LPS treatment similarly inhibited the ability of the TGF-β related cytokine, Activin-A, to down-regulate expression of pro-inflammatory cytokines and to induce microglial cell death. Together, these data suggest that microglial activators may oppose the actions of TGF-β1, ensuring continued microglial activation and survival that eventually may contribute to the neurodegeneration prevalent in chronic neuroinflammatory conditions.

Transforming growth factor-β (TGF-β) signaling is involved in pathological processes following brain injury. TGF-β signaling through Smad3 contributes significantly to the immune response and glial scar formation after brain injury. However, TGF-β is also neuroprotective, suggesting that Smad3 signaling may also be involved in neuroprotection after injury. We found expression of the TGF-β type II receptor (TβRII) and Smad3 protein to be strongly and rapidly induced in neurons in the ipsilateral cortex and CA1 region of the hippocampus after stab wound injury. In contrast, astrocytic expression of TβRII and Smad3 was induced more slowly. Comparison of the response of wild-type and Smad3 null mice to cortical stab wound injury showed a more pronounced loss of neuronal viability in Smad3 null mice. Neuronal density was more strongly reduced in Smad3 null mice than in wild-type mice at 1 and 3days post lesion in both the ipsilateral cortex and hippocampal CA1 region. Fluoro-Jade B, TUNEL staining, and cleaved caspase-3 staining also demonstrated increased neuronal degeneration at early time points after injury in the ipsilateral hemisphere in Smad3 null mice. Taken together, our results suggest that TGF-β cytokine family signaling through Smad3 protects neurons in the damaged cortex and hippocampus at early time points after injury.

Background Bone morphogenetic protein-2 (BMP-2) is a pleiotropic, secreted molecule with diverse effects. The potent ability of BMP-2 to stimulate bone growth prompted its widespread clinical use for arthrodesis (spine fusion). However, elevated post-operative pain in patients treated with BMP-2 has been increasingly reported. Determining whether BMP-2 induces pain directly or whether it induces neuroinflammation, which could lower the threshold for pain, is important for developing therapeutic interventions. We therefore modeled the clinical use of BMP-2 for posterior lumbar fusion by implanting absorbable collagen sponges soaked with either recombinant human BMP-2 (rhBMP-2) or vehicle above the L4–L5 transverse processes of rat spine. Results Using microarray analysis we found that implantation of rhBMP-2-soaked absorbable collagen sponges resulted in altered expression of numerous pro-inflammatory genes in the adjacent dorsal root ganglia (DRG) showing that implantation of rhBMP-2/absorbable collagen sponges triggers potent neuroinflammatory responses in the DRG-2. Interestingly, direct BMP-2 treatment of DRG explants resulted in changes in gene expression that were not specifically pro-inflammatory. Rats implanted with rhBMP-2 in absorbable collagen sponges also exhibited a transient change in thermal and mechanical sensitivity indicating that rhBMP-2 applied to the lumbar spine could increase pain sensitivity. Immunohistochemical analysis indicated macrophage infiltration in the DRG and spinal nerve in rats implanted with rhBMP-2/absorbable collagen sponges or absorbable collagen sponges alone, but not in rats that underwent surgery without implantation of the absorbable collagen sponges suggesting that the sponges contributed to the biological response. Indeed, analysis of DRGs taken from rats implanted with absorbable collagen sponges without rhBMP-2 showed a significant change in gene expression distinct from DRGs from rats undergoing surgery only. Conclusions Our data indicate that implantation of rhBMP-2/absorbable collagen sponges on the lumbar spine triggers potent neuroinflammatory responses in the DRG. Importantly, however, these BMP-2 effects may be partially mediated through a response to the absorbable collagen sponges.

Background α/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia. Methods The hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined. Results We found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model. Conclusions WWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0783-4) contains supplementary material, which is available to authorized users.

Traumatic brain injury (TBI) results in complex pathological reactions, the initial lesion worsened by secondary inflammation and edema. Angiotensin II (Ang II) is produced in the brain and Ang II receptor type 1 (AT₁R) overstimulation produces vasoconstriction and inflammation. Ang II receptor blockers (ARBs) are neuroprotective in models of stroke but little is known of their effect when administered in TBI models. We therefore performed controlled cortical impact (CCI) injury on mice to investigate whether the ARB candesartan would mitigate any effects of TBI. We administered candesartan or vehicle to mice 5 h before CCI injury. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%, decreased the number of dying neurons, lessened the number of activated microglial cells, protected cerebral blood flow (CBF), and reduced the expression of the cytokine TGFβ1 while increasing expression of TGFβ3. Candesartan-treated mice also showed better motor skills on the rotarod 3 days after injury, and improved performance in the Morris water maze 4 weeks after injury. These results indicate that candesartan is neuroprotective, reducing neuronal injury, decreasing lesion volume and microglial activation, protecting CBF and improving functional behavior in a mouse model of TBI. Co-treatment with a peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist significantly reduced some of the beneficial effects of candesartan after CCI, suggesting that PPARγ activation may contribute to part or to all of the neuroprotective effect of candesartan. Overall, our data suggest that ARBs with dual AT₁R-blocking and PPARγ activation properties may have therapeutic value in treating TBI.

Background: Treatment of trauma-related spinal instability with use of recombinant human bone morphogenetic protein-2 (rhBMP-2) may appear as a viable option, but little is known of the direct effects of rhBMP-2 on the injured spinal cord. In the current study, we investigated the acute and long-term effects of using rhBMP-2 in the posterolateral spine at the level of a spinal cord injury in rats. Methods: Fifty-two rats underwent a T10 dorsal hemisection and were assigned to one of two groups: the vehicle control group (twenty-four rats) or the rhBMP-2 group (twenty-four rats). Within each group, animals were further subdivided according to the follow-up period: one week and six weeks after the lesion. For the acute phase, an additional group of four rats received recombinant human albumin, to account for the cross-species inflammatory response. Postoperatively, locomotor function was assessed on a weekly basis with use of an open field scale and digital footprint analysis. After the animals were killed, they were perfused and the spinal cords analyzed for inflammatory markers, gliosis, and extracellular matrix proteins with use of immunohistochemistry. Results: At one week, there was a significant increase in reactive astrocyte, macrophage-microglia, and fibroblast immunoreactivity around the lesion in the rhBMP-2-treated rats relative to controls. Additionally, there was increased staining for chondroitin sulfate proteoglycans. Similar intergroup morphologic differences persisted at six weeks. Functionally, in the acute phase, rhBMP-2-treated animals demonstrated more open field and fine motor control deficits relative to the controls. By six weeks, both groups had equivalent functional scores, but those treated with rhBMP-2 retained significantly greater paw angle changes than the control animals. Conclusions: Our findings indicate that in a rat model, rhBMP-2 use in the vicinity of a penetrating spinal cord injury triggers detrimental changes in the morphology of the spinal cord lesion and alters functional recovery. Clinical Relevance: Our findings suggest that rhBMP-2 used in its current form may impede neurologic recovery in a subset of patients with an open dural spinal cord injury, as we observed detrimental effects on histological and behavioral testing in our rat model.

The use and “off-label” indications for recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal arthrodesis have been significantly expanded over the last decade. New surgical approaches and pathologies treated often place the exogenous protein near the spinal cord or peripheral nerves, yet little data exist to the potential interaction between rhBMP-2 and the nervous system. The current review was undertaken to provide a basic science perspective on the wide-ranging effects that rhBMP-2, a potent growth factor, has on the injured spinal cord and the local dorsal root ganglia (DRG). Results from the early animal studies on neural safety of rhBMP-2 were compared with the more recent in vivo work characterizing protein impact on the injured spinal cord. Potential mechanism of the rhBMP-2–induced radiculitis after lumbar arthrodesis is also discussed. The original pre-FDA approval animal study did not uncover any interaction between rhBMP-2 and the spinal cord or the nerve rootlets comprising the cauda equina. Recent in vivo work indicated, however, that in a penetrating injury model, rhBMP-2 triggers direct signaling in all spinal cord cells. In the rat, this interaction was deleterious to spontaneous recovery by exacerbating the inflammatory response to injury, increasing the glial scar, and making it more inhibitory to axonal regeneration. With respect to posterolateral lumbar arthrodesis in a noninjury model, rhBMP-2 use contributed to a transient postoperative mechanical hyperalgesia. Potential mechanism of this allodynia is through an observed inflammatory response within and around the local DRG. In summary, contrary to the original beliefs in the clinical community, rhBMP-2 does elicit a profound signaling response within the spinal cord and the peripheral ganglia. Recent preclinical studies indicate that rhBMP-2, if provided direct access to the spinal cord parenchyma or the DRG, can trigger significant inflammation and morphologic changes within these tissues that could be deleterious to neurologic recovery.

The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and its indications for spinal fusion continue to be expanded with recent reports citing spinal trauma application. However, there are no data establishing the effects of rhBMP-2 on the injured spinal cord.The purpose of this study was to evaluate the extent of bone morphogenetic protein (BMP)-specific intrathecal signaling after application to the spine at various time points after a spinal cord injury (SCI).This is an in vivo rat study using a combination of the dorsal hemisection SCI and the posterolateral arthrodesis animal models.Sixty-five female Sprague-Dawley rats underwent either a T9-T10 dorsal hemisection SCI (n=52) or laminectomy only (n=13). Spinal cord injury animals were further subdivided into four follow-up groups (n=13/group): 30 minutes, 24 hours, 7 days, and 21 days, at which time one of two secondary surgeries were performed: Eight rats per time point received either 43 microg of rhBMP-2 per side or sterile water control over T9-T11 on absorbable collagen sponges (ACSs). Animals were perfused after 24 hours, and spinal cords were immunohistochemically analyzed. Sections of the lesion were stained with BMP-specific pSmad 1, 5, 8 antibody and costained with cell-specific markers. pSmad-positive cells were then counted around the lesion. The remaining five rats (n=5/time point) had luciferase (blood spinal cord barrier [BSCB] permeability marker) injected through the jugular vein. Subsequently, spinal cords were collected and luciferase activity was quantified around the lesion and in the cervical samples (controls) using a luminometer.After injury, a significant increase in the number of pSmad-positive cells was observed when rhBMP-2 was implanted at the 30-minute, 24-hour, and 7-day time points (p.05). Costaining revealed BMP-specific signaling activation in neurons, glial cells, macrophages, and fibroblasts. Spinal cord permeability to luciferase was significantly increased at 30 minutes, 24 hours, and 7 days post lesion (p.05). A significant linear regression was established between the extent of BSCB permeability and pSmad signaling (r(2)=0.66, p=.000).Our results indicate that rhBMP-2 use around a spinal cord lesion elicits a robust signaling response within the spinal cord parenchyma. All CNS cell types and the invading fibroblasts are activated to the extent dependent on the integrity of the meningeal and BSCB barriers. Therefore, in the presence of a SCI and/or dural tear, rhBMP-2 diffuses intrathecally and activates a signaling cascade in all major CNS cell types, which may increase glial scarring and impact neurologic recovery.

Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated chondroitin sulfate GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings show that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.

We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid-like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five- and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury-induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H-7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF-treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury.

After peripheral nerve axotomy, vasoactive intestinal peptide (VIP) gene expression is upregulated in neurons, whereas ciliary neurotrophic factor (CNTF) accumulates extracellularly at the lesion site. Although CNTF-induced VIP gene expression has been reported in cultured sympathetic neurons and neuroblastoma cells, it still remains to be determined if CNTF and VIP play interrelated roles in nerve injury. The corneal endothelium, like sympathetic neurons, derives from the neural crest. Previously, we demonstrated that a sublethal-level of oxidative stress induces CNTF release from corneal endothelial (CE) cells in situ. Here, we show that human CE cells express the 53 kDa ligand-binding alpha subunit of the CNTF receptor (CNTFRalpha). We further demonstrate that CNTF induces VIP immunoreactivity in human donor corneas. To determine if the increase in VIP immunoreactivity was reflected by an increase in gene expression, donor human corneas were bisected and treated with CNTF or vehicle, and analyzed by real-time RT-qPCR. Two experiments using different sets of bisected corneas indicated that CNTF induced increases in VIP mRNA levels of 6.5+/-2.2-fold (N=7 corneas) and 2.3+/-0.6-fold (N=10 corneas) (mean+/-S.E.M.), respectively. Whereas VIP is produced as a CE autocrine factor against oxidative stress, the present study suggested that oxidative stress-released CNTF plays a role in protecting CE cells against oxidative stress injury by upregulating VIP expression.

Following central nervous system injury, adult mammalian neurons do not regenerate through regions of scar formation. This regenerative failure is due in part to the inhibitory environment of the glial scar at the lesion site. Following injury, transforming growth factor beta (TGF-beta) is strongly induced and is important to many aspects of the response to injury, including deposition of extracellular matrix (ECM) in the glial scar. However, the pathways through which TGF-beta signals to mediate these effects are not known. In order to examine the contribution of the TGF-beta-induced transcription factor, Smad3, to formation of the glial scar after traumatic brain injury, we utilized mice that do not express Smad3. We report that Smad3 null mice heal stab wounds to the cerebral cortex more rapidly than do wild-type mice. In Smad3 null mice many aspects of glial scar formation and the immune response to injury were altered. Fewer neutrophils, macrophages/microglia, NG2-positive cells and GFAP-positive cells were detected immediately around the lesion in Smad3 null mice. Expression of fibronectin and laminin was also reduced. Injury-induced cell proliferation was significantly lower in Smad3 null mice around the lesion. There was no overall difference between wild-type and Smad3 null mice in immunoreactivity for TGF-beta(1) after injury. Thus, our experiments suggest that TGF-beta signaling through Smad3 contributes significantly to the immune response and scar formation after cortical stab wound injury, delaying recovery through multiple mechanisms.

Traumatic brain injury (TBI) results in a loss of brain tissue at the moment of impact in the cerebral cortex. Subsequent secondary injury involves the release of molecular signals with dramatic consequences for the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The mechanisms behind the progression of tissue loss remain under investigation. In this study, we analyzed the spatial-temporal profile of blood flow, apoptotic and astrocytic-vascular events in the cortical regions around the impact site at time points ranging from 5 hours to 2 months after TBI. We performed a mild-moderate controlled cortical impact injury in young adult mice and analyzed the glial and vascular response to injury. We observed a dramatic decrease in perilesional cerebral blood flow (CBF) immediately following the cortical impact that lasted until days later. CBF finally returned to baseline levels by 30 days post-injury (dpi). The initial impact also resulted in an immediate loss of tissue and cavity formation that gradually increased in size until 3 dpi. An increase in dying cells localized in the pericontusional region and a robust astrogliosis were also observed at 3 dpi. A strong vasculature interaction with astrocytes was established at 7 dpi. Glial scar formation began at 7 dpi and seemed to be compact by 60 dpi. Altogether, these results suggest that TBI results in a progression from acute neurodegeneration that precedes astrocytic activation, reformation of the neurovascular unit to glial scar formation. Understanding the multiple processes occurring after TBI is critical to the ability to develop neuroprotective therapeutics to ameliorate the short and long-term consequences of brain injury.

An error was introduced in the preparation of this article for publication. The word "At2" is incorrect in the article title. The correct title is "Commercially Available Angiotensin II AT2 Receptor Antibodies Are Nonspecific". The correct citation is: Hafko R, Villapol S, Nostramo R, Symes A, Sabban EL, et al. (2013) Commercially Available Angiotensin II AT2 Receptor Antibodies Are Nonspecific. PLoS ONE 8(7): e69234. doi:10.1371/journal.pone.0069234

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin’s action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr 986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr 986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin’s weight-reducing effects in obese individuals.

The vasoactive intestinal peptide cytokine response element (VIP CyRE) is responsible for mediating the transcriptional induction of the VIP gene to the neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). In investigating the sequence and function of the CyRE, we found a region of DNA with homology to the distal NFAT site in the IL-2 promoter. In this paper we characterize this sequence and show that the VIP NFAT site recognizes T cell NFAT with similar affinity to the previously characterized IL-2 NFAT site. However, despite its location in the middle of the CyRE, we find no CNTF/LIF induced binding to it. Instead we show that in NBFL neuroblastoma cells, the calcium ionophore A23187 induces a protein to bind to the VIP NFAT site. This A23187-mediated induction of nuclear protein binding to an NFAT oligonucleotide is dependent on extracellular calcium but not dependent on de novo protein synthesis. Thus, this protein has the characteristics of an NFAT-like protein and is recognized by an NFAT3-specific antiserum suggesting that it is indeed an NFAT protein. The location of the NFAT site in the VIP CyRE suggests that this may be one mechanism through which different signaling pathways engage in cross talk to alter VIP gene transcription.

The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors.

Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1–3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6–8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9–11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.

Neuropoietic cytokines, including ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), have survival effects on cells of the peripheral and central nervous systems (PNS and CNS). CNTF and LIF also produce differentiation in some cells of the PNS. We have shown previously that CNTF activates the signal transducers and activators of transcription (STAT) family of transcription factors, and that this signaling pathway may be one of several employed by CNTF to regulate the expression of the vasoactive intestinal peptide (VIP) gene in cells of the PNS (Symes et al.: Proc Natl Acad Sci USA 90:572-576, 1993; Symes et al.: Mol Endocrinol 8:1750-1763, 1994). To investigate the mechanisms of action of CNTF in the CNS, we have analyzed the activation of STAT proteins in a septal-derived cell line, SN48, and in primary CNS cultures. CNTF treatment of SN48 cells produces a sustained activation of Stat3. CNTF treatment of SN48 cells also activated transcription mediated by the VIP cytokine responsive element (CyRE) which contains a STAT binding site. Mutation of the STAT site in the CyRE attenuated transcriptional activation by CNTF, indicating the importance of STAT proteins to CNTF-dependent transcriptional activation of SN48 cells. In cultures of embryonic rat septum and other brain areas, in addition to Stat3, CNTF also activates Stat1. As in cells of the PNS and non-neuronal cells, the Janus kinase (Jak)-STAT pathway is activated in CNS cells by cytokines. The SN48 cell line may be valuable in further characterization of regulation of the Jak-STAT pathway by neuropoietic cytokines.

Chondroitin sulfate proteoglycans (CSPGs) play a pivotal role in many neuronal growth mechanisms including axon guidance and the modulation of repair processes following injury to the spinal cord or brain. Many actions of CSPGs in the central nervous system (CNS) are governed by the specific sulfation pattern on the glycosaminoglycan (GAG) chains attached to CSPG core proteins. To elucidate the role of CSPGs and sulfated GAG chains following traumatic brain injury (TBI), controlled cortical impact injury of mild to moderate severity was performed over the left sensory motor cortex in mice. Using immunoblotting and immunostaining, we found that TBI resulted in an increase in the CSPGs neurocan and NG2 expression in a tight band surrounding the injury core, which overlapped with the presence of 4-sulfated CS GAGs but not with 6-sulfated GAGs. This increase was observed as early as 7 days post injury (dpi), and persisted for up to 28 dpi. Labeling with markers against microglia/macrophages, NG2+ cells, fibroblasts, and astrocytes showed that these cells were all localized in the area, suggesting multiple origins of chondroitin-4-sulfate increase. TBI also caused a decrease in the expression of aggrecan and phosphacan in the pericontusional cortex with a concomitant reduction in the number of perineuronal nets. In summary, we describe a dual response in CSPGs whereby they may be actively involved in complex repair processes following TBI. J. Comp. Neurol. 520:3295–3313, 2012. © 2012 Wiley Periodicals, Inc.

Differentiation factors have been identified that influence the phenotype of sympathetic neurons by altering expression of classical neurotransmitters and neuropeptides. Investigation of the molecular mechanisms through which such factors act would be facilitated by the availability of a neuronal cell line that responds to these factors in a fashion similar to sympathetic neurons. We have identified a human neuroblastoma cell line, NBFL, that responds to the differentiation factor ciliary neurotrophic factor (CNTF) by coordinately inducing multiple neuropeptide genes as do sympathetic neurons. Treatment of NBFL cells with CNTF increases vasoactive intestinal polypeptide (VIP), somatostatin, and calcitonin gene-related peptide (CGRP) mRNAs but does not change other neurotransmitter properties. The induction of VIP mRNA by CNTF in NBFL cells is dose dependent, rapid, sustained, and independent of new protein synthesis. Genomic 5' flanking sequences located within a 1.59-kilobase region of the human VIP gene and distinct from the previously defined cAMP-responsive element subserve transcriptional activation by CNTF. Further examination of NBFL cells should permit the elucidation of the molecular mechanisms by which CNTF and other differentiation factors coordinately activate neuropeptide gene transcription to influence neuronal differentiation. Similar mechanisms may mediate the effect of CNTF on neuronal survival.

Objective To investigate transforming growth factor β (TGFβ) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Methods Real-time reverse transcription–polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFβ-mediated CTGF promoter activity. Results CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFβ treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFβ-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFβ was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFβ expression in the degenerated state. Conclusion TGFβ, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.

The calcitonin/α-CORP (CT/CGRP) gene is ectopically expressed in a wide variety of neoplasia. We have investigated the molecular mechanisms responsible for this ectopic expression in the human cell line BEN, which is derived from a poorly differentiated squamous cell lung carcinoma. We show that a trans-acting factor which represses expression of the CT/CGRP gene in HeLa cells is absent or inactive in BEN cells, and have localised the repressor binding site to a 53 bp fragment 1500 bp upstream of the transcription start site.

Transforming growth factor-beta signaling through Smad3 inhibits cell proliferation in many cell types. As cell proliferation in the brain is an integral part of neurogenesis, we sought to determine the role of Smad3 in adult neurogenesis through examining processes and structures important to neurogenesis in adult Smad3 null mice. We find that there are fewer proliferating cells in neurogenic regions of adult Smad3 null mouse brains and reduced migration of neuronal precursor cells from the subventricular zone to the olfactory bulb. Alterations in astrocyte number and distribution within the rostral migratory stream of Smad3 null mice give rise to a smaller and more disorganized structure that may impact on neuronal precursor cell migration. However, the proportion of proliferating cells that become neurons is similar in wild type and Smad3 null mice. Our results suggest that signaling through Smad3 is needed to maintain the rate of cell division of neuronal precursors in the adult brain and hence the amount of neurogenesis, without altering neuronal cell fate.

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or “leak” current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCϵ is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.

Calcium signaling is a critical component of astrocyte activation but little information is available regarding the identity and function of transcriptional targets of calcium signaling in these cells. As a first step in elucidating the mechanisms that astrocytes use to regulate transcription in response to raised intracellular calcium concentrations, we have investigated expression and activation of the calcium activated transcription factors of the NFAT family. We report here that NFAT4 is expressed in U373 astrocytoma cells and in primary cultures of astrocytes. Treatment of U373 cells or primary astrocytes with the calcium ionophore A23187, or the neurotransmitter glutamate, leads to NFAT nuclear translocation and increased DNA binding to a NFAT consensus site through a cyclosporin A-sensitive process. These data suggest that NFAT4 functions as a calcium-responsive transcription factor in astrocytes.