Rapid phosphorylation of histone H2A.X following ionotropic glutamate receptor activation.

Excessive activation of ionotropic glutamate receptors increases oxidative stress, contributing to the neuronal death observed following neurological insults such as ischemia and seizures. Post-translational histone modifications may be key mediators in the detection and repair of damage resulting from oxidative stress, including DNA damage, and may thus affect neuronal survival in the aftermath of insults characterized by excessive glutamate release. In non-neuronal cells, phosphorylation of histone variant H2A.X (termed gamma-H2AX) occurs rapidly following DNA double-strand breaks. We investigated gamma-H2AX formation in rat cortical neurons (days in vitro 14) following activation of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors using fluorescent immunohistochemical techniques. Moreover, we evaluated the co-localization of gamma-H2AX 'foci' with Mre11, a double-strand break repair protein, to provide further evidence for the activation of this DNA damage response pathway. Here we show that minimally cytotoxic stimulation of ionotropic glutamate receptors was sufficient to evoke gamma-H2AX in neurons, and that NMDA-induced gamma-H2AX foci formation was attenuated by pretreatment with the antioxidant, Vitamin E, and the intracellular calcium chelator, BAPTA-AM. Moreover, a subset of gamma-H2AX foci co-localized with Mre11, indicating that at least a portion of gamma-H2AX foci is damage dependent. The extent of gamma-H2AX induction following glutamate receptor activation corresponded to the increases we observed following conventional DNA damaging agents [i.e. non-lethal doses of gamma-radiation (1 Gy) and hydrogen peroxide (10 microm)]. These data suggest that insults not necessarily resulting in neuronal death induce the DNA damage-evoked chromatin modification, gamma-H2AX, and implicate a role for histone alterations in determining neuronal vulnerability following neurological insults.

Neuroprotective effects of selective group II mGluR activation in brain trauma and traumatic neuronal injury.

The effects of group II mGluR activation by selective agonist (-)-2-oxa-4-aminobicyclo[3.1. 0]hexane-4,6-dicarboxylate (LY379268) were examined in a mouse model of controlled cortical impact (CCI)-induced brain injury and in primary neuronal/glial and neuronal cultures subjected to mechanical trauma. Systemic administration of LY379268 to mice at 30 min after CCI significantly improved both motor and cognitive recovery as compared with vehicle-treated control animals. LY379268 also significantly reduced cell death induced by mechanical injury in rat neuronal/glial and neuronal cultures, as measured by lactate dehydrogenase (LDH) release assay. The neuroprotective effect of LY379268 in vitro was abolished by co-administration of the mGluR2/3 antagonist (s)-alpha-ethylglutamic acid (EGLU); however, co-application of selective mGluR3 antagonist beta-N-acetyl-aspartyl-glutamate (NAAG) had no significant influence in the same system. Together, these findings demonstrate the neuroprotective activity of group II mGluR activation and underscore the role of the mGluR2 subtype for this effect.

Expression of two temporally distinct microglia-related gene clusters after spinal cord injury.

The dual role of microglia in cytotoxicity and neuroprotection is believed to depend on the specific, temporal expression of microglial-related genes. To better clarify this issue, we used high-density oligonucleotide microarrays to examine microglial gene expression after spinal cord injury (SCI) in rats. We compared expression changes at the lesion site, as well as in rostral and caudal regions after mild, moderate, or severe SCI. Using microglial-associated anchor genes, we identified two clusters with different temporal profiles. The first, induced by 4 h postinjury to peak between 4 and 24 h, included interleukin-1beta, interleukin-6, osteopontin, and calgranulin, among others. The second was induced 24 h after SCI, and peaked between 72 h and 7 days; it included C1qB, Galectin-3, and p22(phox). These two clusters showed similar expression profiles regardless of injury severity, albeit with slight decreases in expression in mild or severe injury vs. moderate injury. Expression was also decreased rostral and caudal to the lesion site. We validated the expression of selected cluster members at the mRNA and protein levels. In addition, we demonstrated that stimulation of purified microglia in culture induces expression of C1qB, Galectin-3, and p22(phox). Finally, inhibition of p22(phox) activity within microglial cultures significantly suppressed proliferation in response to stimulation, confirming that this gene is involved in microglial activation. Because microglial-related factors have been implicated both in secondary injury and recovery, identification of temporally distinct clusters of genes related to microglial activation may suggest distinct roles for these groups of factors.

Novel neuroprotective tripeptides and dipeptides.

It has long been recognized that thyrotropin-releasing hormone (TRH) and certain TRH analogues are neuroprotective in a variety of animal models of CNS trauma. In addition to these neuroprotective actions, TRH and most TRH analogues have other physiological actions that may not be desirable for treatment of acute injury, such as analeptic, autonomic, and endocrine effects. We have developed a series of dual-substituted TRH analogues that have strong neuroprotective actions, but are largely devoid of these other physiological actions. In addition, we have developed a family of cyclized dipeptides (diketopiperazines), structurally somewhat related to a metabolic product of TRH, that appear even more effective as neuroprotective agents in vitro and in vivo, and may have nootropic properties. Here, we review these novel tripeptide and dipeptide compounds.

Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.

Neuroprotective effects of novel small peptides in vitro and after brain injury.

Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and beta-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.

Ceramide induces neuronal apoptosis through mitogen-activated protein kinases and causes release of multiple mitochondrial proteins.

Ceramide accumulates in neurons during various disorders associated with acute or chronic neurodegeneration. In these studies, we investigated the mechanisms of ceramide-induced apoptosis in primary cortical neurons using exogenous C(2) ceramide as well as inducing endogenous ceramide accumulation using inhibitors of glucosylceramide synthetase. Ceramide induced the translocation of certain, but not all, pro-apoptotic mitochondrial proteins: cytochrome c, Omi, SMAC, and AIF were released from the mitochondria, whereas Endonuclease G was not. Ceramide also selectively altered the phosphorylation state of members of the MAPK superfamily, causing dephosphorylation of ERK1/2 and hyperphosphorylation of p38 MAP kinases, but not affecting the phosphorylation of JNK or ERK5. Inhibitors of the p38 MAP kinase pathway (SB-202190 or SB-203580) and an inhibitor of the ERK1/2 pathway (U0126) reduced ceramide-induced neuronal death. These p38 and ERK1/2 inhibitors appear to block ceramide-activated apoptotic signaling upstream of the mitochondria, as they attenuated mitochondrial release of cytochrome c, Omi, AIF, and SMAC, as well as reducing ceramide-induced caspase-3 activation.

Neuroprotective activity of the mGluR5 antagonists MPEP and MTEP against acute excitotoxicity differs and does not reflect actions at mGluR5 receptors.

1 Neuroprotection has been reported after either activation or blockade of the group I metabotropic glutamate receptor subtype 5 (mGluR5). However, some recent evidence suggests that protection provided by mGluR5 antagonists may reflect their ability to inhibit N-methyl-D-aspartate (NMDA) receptor activity. 2 Here, in both rat and mouse cortical neurons, we compare the neuroprotective actions of two mGluR5 antagonists: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), which has been commonly used and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), a more recently developed compound believed to have greater mGluR5 selectivity. We have previously shown that MPEP directly reduces single-channel NMDA receptor open time at the same concentrations (20 microM or greater) that show neuroprotection, whereas MPEP antagonizes mGluR5 agonist ((RS)-2-chloro-5-hydroxyphenylglycine (CHPG))-induced changes in inositol phosphates (IP) at concentrations as low as 0.2 microM. 3 In the present studies, MTEP significantly inhibited CHPG-mediated IP hydrolysis at concentrations as low as 0.02 microM. In contrast to MPEP, which significantly reduced glutamate- or NMDA-mediated cell death in primary rat neuronal cultures at a concentration of 20 microM, small neuroprotective effects were observed with MTEP only at a concentration of 200 microM. Neither MPEP- nor MTEP-mediated mGluR5 inhibition had any effect on etoposide-induced apoptotic cell death. In rat cortical neurons, the neuroprotective effects of MTEP at very high concentrations, like those of MPEP, reflect ability to directly reduce NMDA receptor peak and steady-state currents. 4 We also compared the effects of MPEP and MTEP in primary cortical neuronal cultures from parental and mGluR5 knockout mice. Both agents were neuroprotective, at high concentrations in normal as well as in the knockout cultures. In contrast to rat cortical neurons, neither MPEP nor MTEP appears to directly alter NMDA receptor activity. 5 Combined, these studies support the conclusion that MTEP has greater mGluR5 selectivity than MPEP, and that neuroprotection provided by either antagonist in neuronal cultures does not reflect inhibition of mGluR5 receptors.

Identification of novel neuroprotective agents using pharmacophore modeling.

In addition to its endocrine function, for which it was named, thyrotropin-releasing hormone (TRH) has substantial neuroprotective actions as well as other physiological effects. We have developed a number of modified TRH analogues as well as cyclic dipeptides structurally related to a major metabolic product of TRH, which have enhanced neuroprotective activity but none of the other major physiological effects of TRH. The extensive structure-activity data developed with these compounds were used to develop a pharmacophore model. Subsequently, a web-based pharmacophore searching program was used to query several large three-dimensional databases. Of the 219 compounds identified whose structures met the pharmacophore model, 15 were chosen for study in a classical model of neuronal cell death in vitro; five of these, 2-6, showed neuroprotective activity. Thus, pharmacophore modeling developed from neuroprotective small peptides can be used to identify novel lead compounds as neuroprotective agents.

MGLuR5 activation reduces beta-amyloid-induced cell death in primary neuronal cultures and attenuates translocation of cytochrome c and apoptosis-inducing factor.

Activation of metabotropic glutamate receptor 5 (mGluR5) has been shown to reduce caspase-dependent apoptosis in primary neuronal cultures induced by staurosporine and etoposide. beta-Amyloid (Abeta)-induced neurotoxicity in culture appears to be in part caspase mediated. In the present studies the effects of treatment with an mGluR5 agonist or antagonist on Abeta-induced neuronal apoptosis were examined in rat cortical neuronal cultures. Pretreatment with the selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) markedly reduced the number of apoptotic cells after exposure to Abeta (25-35), as well as associated LDH release. Blockade of mGluR5 by the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP) attenuated these effects of CHPG. A similar neuroprotective effect of mGluR5 activation by CHPG was observed in cultures treated with full-length Abeta peptide (1-42). CHPG attenuated Abeta (25-35)-induced cytochrome c release and decreased levels of active caspase-3 protein. CHPG also reduced translocation of apoptosis-inducing factor (AIF) induced by Abeta (25-35). Thus, mGluR5 activation limits the release of mitochondrial proteins associated with induction of both caspase-dependent and -independent apoptosis.

The "dark side" of endocannabinoids: a neurotoxic role for anandamide.

Endocannabinoids, including 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell death in several cell lines. Here we show that anandamide causes neuronal cell death in vitro and exacerbates cell loss caused by stretch-induced axonal injury or trophic withdrawal in rat primary neuronal cultures. Administered intracerebroventricularly, AEA causes sustained cerebral edema, as reflected by diffusion-weighted magnetic resonance imaging, regional cell loss, and impairment in long-term cognitive function. These effects are mediated, in part, through VR1 as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in pro-inflammatory/microglial-related responses. Thus, anandamide produces neurotoxic effects both in vitro and in vivo through multiple mechanisms independent of the CB1 receptor.

Novel small peptides with neuroprotective and nootropic properties.

The tripeptide thyrotropin-releasing hormone (TRH) and/or related analogues have shown neuroprotective activity across multiple animal trauma models as well as in a small clinical trial of spinal cord injury. The metabolic product of TRH (cyclo-his-pro) retains physiological activity. We have developed a number of novel cyclic dipeptides that are structurally similar to cyclo-his-pro, and have examined their neuroprotective activity across multiple in vitro models of neuronal injury and after traumatic brain injury (TBI) in rodents. Four such compounds were found to reduce cell death after trophic withdrawal or traumatic injury in primary neuronal cultures; two also protected against glutamate or beta-amyloid neurotoxicity. All compounds significantly improved motor and cognitive recovery after controlled cortical impact injury in mice, and markedly reduced lesion volumes as shown by high field magnetic resonance imaging. Further, compound 35b, which is being developed for clinical trials, also showed considerable neuroprotection after fluid percussion induced TBI in rats, and improved cognitive function after daily administration in chronically brain injured rats. At a mechanistic level, the drugs attenuate both apoptotic and necrotic cell death in primary neuronal cultures, markedly reduce intracellular calcium accumulation after injury, and limit changes in mitochondrial membrane potential and associated cytochrome c release. In addition, microarray studies show that 35b reduces transcriptional changes after injury for a number of genes (and proteins) that may be associated with secondary injury, including cell cycle genes, aquaporins and cathepsins. It also upregulates brain-derived neurotrophic factor (BDNF), heat shock proteins (HSP) and hypoxia inducible factor (HIF). Thus, these novel dipeptides have multipotential actions that make them candidates for the treatment of both acute and chronic neurodegeneration.

Ceramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3beta, and induction of the mitochondrial-dependent intrinsic caspase pathway.

Neuronal apoptosis has been implicated as an important mechanism of cell death in acute and chronic neurodegenerative disorders. Ceramide is a product of sphingolipid metabolism which induces neuronal apoptosis in culture, and ceramide levels increase in neurons during various conditions associated with cell death. In this study we investigate the mechanism of ceramide-induced apoptosis in primary cortical neuronal cells. We show that ceramide treatment initiates a cascade of biochemical alterations associated with cell death: earliest signal transduction changes involve Akt dephosphorylation and inactivation followed by dephosphorylation of proapoptotic regulators such as BAD (proapoptotic Bcl-2 family member), Forkhead family transcription factors, glycogen synthase kinase 3-beta, mitochondrial depolarization and permeabilization, release of cytochrome c into the cytosol, and caspase-3 activation. Bongkrekic acid, an agent that inhibits mitochondrial depolarization, significantly reduces ceramide-induced cell death and correlated caspase-3 activation. Together, these data demonstrate the importance of the mitochondrial-dependent intrinsic pathway of caspase activation for ceramide-induced neuronal apoptosis.

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway.

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.

Ceramide-induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis.

Ceramide is a sphingolipid that has been implicated both in apoptosis and protection from cell death. We show that in both rat cerebellar granule cells and cortical neuronal cultures application of C(2)-ceramide causes cell death in a dose- and time-dependent manner. Similar effects were observed with the exogenous application of bacterial sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the plasma membrane and leads to endogenous ceramide accumulation. Furthermore, endogenous ceramide levels were increased during apoptosis induced by nutrient deprivation or etoposide treatment. These findings suggest that upregulation of ceramide levels, which may be generated through activation of sphingomyelinase, contributes to neuronal apoptosis.