miR-711 upregulation induces neuronal cell death after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of mortality and disability. MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at post-transcriptional level and may be key modulators of neuronal apoptosis, yet their role in secondary injury after TBI remains largely unexplored. Changes in miRs after controlled cortical impact (CCI) in mice were examined during the first 72 h using miR arrays and qPCR. One selected miR (711) was examined with regard to its regulation and relation to cell death; effects of miR-711 modulation were evaluated after CCI and using in vitro cell death models of primary cortical neurons. Levels of miR-711 were increased in the cortex early after TBI and in vitro models through rapid upregulation of miR-711 transcription (pri-miR-711) rather than catabolism. Increases coincided with downregulation of the pro-survival protein Akt, a predicted target of miR-711, with sequential activation of forkhead box O3 (FoxO3)a/glycogen synthase kinase 3 (GSK3)α/ß, pro-apoptotic BH3-only molecules PUMA (Bcl2-binding component 3) and Bim (Bcl2-like 11 (apoptosis facilitator)), and mitochondrial release of cytochrome c and AIF. miR-711 and Akt (mRNA) co-immunoprecipitated with the RNA-induced silencing complex (RISC). A miR-711 hairpin inhibitor attenuated the apoptotic mechanisms and decreased neuronal death in an Akt-dependent manner. Conversely, a miR-711 mimic enhanced neuronal apoptosis. Central administration of the miR-711 hairpin inhibitor after TBI increased Akt expression and attenuated apoptotic pathways. Treatment reduced cortical lesion volume, neuronal cell loss in cortex and hippocampus, and long-term neurological dysfunction. miR-711 changes contribute to neuronal cell death after TBI, in part by inhibiting Akt, and may serve as a novel therapeutic target.

Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death.

Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.

Selective caspase activation may contribute to neurological dysfunction after experimental spinal cord trauma.

Caspases are implicated in apoptotic cell death after spinal cord injury (SCI), but the relative contribution of these proteases to the secondary injury process has been only partially described. We examined the activation of caspases 1, 2, 3, 6, 8, and 9 from 1 hr to 7 days after moderate contusion injury induced by a weight-drop method in the rat. Tissue homogenates from a 1-cm segment of cord that contained the site of impact were processed by fluorometric enzymatic activity assays and/or immunoblotting methods. Caspases 3, 8, and 9 were activated from 1 to 72 hr after injury, whereas caspases 1, 2, and 6 were not. Double-label immunohistochemistry utilizing antibodies for CNS cell-type-specific markers and active subunits of caspases 3, 8, or 9 showed that, at 4 and 72 hr after injury, these caspases were primarily activated in neurons and oligodendrocytes, rather than in astrocytes. Active caspase subunits were present in neurons within the necrotic lesion core at 4 hr after injury and in cells more than several segments away at 4 or 72 hr after injury. Intrathecal injection of the pan-caspase inhibitor Boc-Asp (OMe)-fluoromethylketone (Boc-d-fmk) at 15 min after injury improved locomotor function 21 and 28 days later. Treatment with the selective caspase 3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk) improved function at 21 days after injury. These data suggest that caspases 3, 8, and 9 may be differentially activated in white and gray matter after spinal cord trauma and that such activation may contribute to subsequent neurological dysfunction.

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways.

Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.

Consistent and reproducible slice selection in rodent brain using a novel stereotaxic device for MRI.

Typically small animal radiological images are obtained after placing the animal in the center of the imaging device using beds or platforms, and then adjusting the position after obtaining a scout image. Such a process does not permit the reproducible visualization of the same anatomical plane with repeated examinations. We have developed a device that allows stereotaxic placement of an animal in precisely the same position for repeated examinations. The instrument incorporates a full range of physiological monitoring and life support systems including temperature control, anesthesia delivery and respiratory monitoring. Using magnetic resonance imaging (MRI), the accuracy and reliability of this device is demonstrated in a rat traumatic brain injury (TBI) model.

Administration of either anti-intercellular adhesion molecule-1 or a nonspecific control antibody improves recovery after traumatic brain injury in the rat.

Intercellular adhesion molecule-1 (ICAM-1) is an endothelial protein that facilitates invasion of leukocytes into the CNS in response to injury or inflammation. ICAM-1 expression correlates with the severity of clinical head injuries, but its importance in secondary injury events is not fully understood. Therefore, we evaluated ICAM-1 expression and the effect of anti-ICAM-1 treatment on motor recovery and neutrophil invasion after traumatic brain injury induced via the lateral fluid-percussion method in the rat. ICAM-1 was expressed in large and small blood vessels within the injured cortex at 10 and 24 h after injury. Repeated administration of anti-ICAM-1 antibody (clone 1A29) at 1, 10, and again at 24 h after injury significantly improved performance in two of three motor tests, compared to saline controls. Equal doses of nonspecific control antibody (IgG) also significantly improved motor test scores, compared to saline controls. Cortical myeloperoxidase activity, an indicator of neutrophil invasion, was significantly reduced 26 h after injury in animals treated with anti-ICAM-1. Animals treated with IgG showed a trend toward reduction that did not reach significance. These data suggest that ICAM-1 may be involved in neutrophil invasion and neurological dysfunction after TBI, but also implicate a role for a nonspecific antibody effect in improved functional outcome.

Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia.

Activation of group I metabotropic glutamate receptors (mGluR) has been implicated in the pathophysiology of acute central nervous system injury. However, the relative roles of the two group I subtypes, mGluR1 or mGluR5, in such injury has not been well examined. We compared the effects of treatment with the newly developed, selective mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) and the selective mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) in a rat intraluminal filament model of temporary middle cerebral artery occlusion (MCAo). Rats were administered MPEP or CHPG i.c.v. beginning 15 or 135 min after induction of ischemia for 2 h. Infarct size was measured after either 22 or 70 h of reperfusion, and neurological function was quantified at 2, 24, 48 and 72 h. Treatment with MPEP or CHPG at 15 min reduced 24 h infarct volume by 61 and 44%, respectively. The neuroprotective effects were dose dependent. Delaying MPEP treatment until 135 min eliminated the neuroprotective effects. In other studies, using early MPEP treatment (15 min) at optimal doses, infarct volume was reduced by 44% at 72 h and this was correlated with significant neurological recovery. These data suggest that both MPEP and CHPG are neuroprotective when administered after focal cerebral ischemia. In separate, recent studies we found that although MPEP does act as an mGluR5 antagonist and blocks agonist induced phosphoinositide hydrolysis, it also serves as a non-competitive NMDA antagonist; in contrast, other results indicate that CHPG mediated neuroprotection may reflect anti-apoptotic activity. Therefore, both types of compounds may prove to have therapeutic potential for the treatment of stroke.

Differential expression of apoptotic protease-activating factor-1 and caspase-3 genes and susceptibility to apoptosis during brain development and after traumatic brain injury.

Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute brain injury. Recent studies have provided evidence that neuronal susceptibility to apoptosis induced by traumatic or ischemic injury decreases during brain development. However, the molecular mechanisms responsible for this age-dependent phenomenon remain unclear. Here we demonstrate that, during brain maturation, the potential of the intrinsic apoptotic pathway is progressively reduced and that such repression is associated with downregulation of apoptotic protease-activating factor-1 (Apaf-1) and caspase-3 gene expression. A similar decline in apoptotic susceptibility associated with downregulation of Apaf-1 expression as a function of developmental age was also found in cultured primary rat cortical neurons. Injury-induced cytochrome c-specific cleavage of caspase-9 followed by activation of caspase-3 in mature brain correlated with marked increases in Apaf-1 and caspase-3 mRNA and protein expression. These results suggest that differential expression of Apaf-1 and caspase-3 genes may underlie regulation of apoptotic susceptibility during brain development, as well as after acute injury to mature brain, through the intrinsic pathway of caspase activation.

Small shifts in craniotomy position in the lateral fluid percussion injury model are associated with differential lesion development.

Previous studies have shown that location and direction of injury may affect outcome in experimental models of traumatic brain injury. Significant variability in outcome data has also been noted in studies using the lateral fluid percussion brain injury model (FPI) in rats. In recent studies from our laboratory, we observed considerable variability in localization and severity of tissue damage as a function of small changes in craniotomy position. To further address this issue, we examined the relationship between craniotomy position and brain lesion size/location in rats subjected to moderate FPI (2.28 +/- 0.18 atmospheres). With placement of a 5-mm craniotomy adjacent to the sagittal suture, there was both ipsilateral and contralateral damage as detected at 3 weeks posttrauma using T2-weighted magnetic resonance imaging (MRI). The MRI lesions were generally restricted to the hippocampus and subcortical layers. Shifting of the craniotomy site laterally was associated with increased ipsilateral tissue damage and a greater cortical component that correlated with distance from the sagittal suture. In contrast, the contralateral MRI lesion did not change significantly in size or location unless the center of the craniotomy was placed more than 3.5 mm from the sagittal suture, under which condition contralateral damage could no longer be detected. Ipsilateral tissue damage as determined from the MRI scans was linearly correlated to motor outcome but not with cognitive outcome as assessed by the Morris Water Maze. We conclude that craniotomy position is critical in determining extent and location of tissue injury produced during the lateral FPI model in rats. Addressing such potential variability is essential for studies that address either injury mechanisms or therapeutic treatments.

Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury.

BACKGROUND: Apoptosis plays an important pathophysiologic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA fragmentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after experimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. MATERIALS AND METHODS: We used mice deficient in DFF45 and wild-type controls. Oligonucleosomal DNA fragmentation induced by TBI was analyzed using in vivo and in vitro assays. Expression and integrity of DFF45 and DFF40 proteins was assessed by Western analysis. Other outcome measurements included neurologic scoring, learning/memory tests, lesion volume measurements (MRI), and assessment of cell viability in vitro among others. RESULTS: We compared the effects of controlled cortical impact (CCI) trauma in DFF45 knockout mice and wild-type controls. Analysis of TBI-induced DNA fragmentation in brain cortex from wild-type and DFF45 knockout mice indicates that, although somewhat delayed, oligonucleosomal cleavage of DNA occurs after TBI in DFF45 knockout mice. DFF45 knockouts showed no significant differences in behavioral outcomes or lesion volumes after TBI as compared to wild-type controls. Using an in vitro reconstitution system, we also demonstrated that cleavage of DFF45 by caspase-3 is not sufficient for DNA fragmentation induced by protein extracts from rat brain cortex. We found that endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg2+ and Ca2+, but is not inhibited by Zn2+. Primary neuronal cultures from DFF45 knockouts failed to show DNA laddering in response to staurosporine, but did show prominent, albeit delayed, DNA fragmentation following treatment with etoposide. In contrast, primary neurons from wild-type animals demonstrated marked DNA fragmentation following treatment with staurosporine or etoposide. CONCLUSIONS: The results of this study suggest that, in addition to DFF45/40, other endonucleases may be essential for chromatin degradation during neuronal apoptosis in adult brain after TBI.

Multiple caspases are involved in beta-amyloid-induced neuronal apoptosis.

beta-amyloid peptide (Abeta) has been implicated in the pathogenesis of Alzheimer disease and has been reported to induce apoptotic death in cell culture. Cysteine proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. Multiple caspases have been implicated in Abeta toxicity; these reports are conflicting. We show that treatment of cerebellar granule cells (CGC) with Abeta25-35 causes apoptosis associated with increased activity of caspases-2, -3 and -6. Selective inhibition of each of these three caspases provides significant protection against Abeta-mediated apoptosis. In contrast, no change in caspase-1 activity was seen after Abeta25-35 application, nor was inhibition of caspase-1 neuroprotective. Similar to CGC, cortical neuronal cultures treated with Abeta25-35 demonstrate increased caspase-3 activity but not caspase-1 activity. Furthermore, significant neuroprotection is elicited by selective inhibition of caspase-3 in cortical neurons administered Abeta25-35, whereas selective caspase-1 inhibition has no effect. Taken together, these findings indicate that multiple executioner caspases may be involved in neuronal apoptosis induced by Abeta.

Exacerbation of neuronal cell death by activation of group I metabotropic glutamate receptors: role of NMDA receptors and arachidonic acid release.

Both ionotropic and metabotropic glutamate receptors have been implicated in the pathogenesis of neuronal injury. Activation of group I metabotropic glutamate receptors (mGluR) exacerbates neuronal cell death, whereas inhibition is neuroprotective. However, the mechanisms involved remain unknown. Activation of group I mGluR modulates multiple signal transduction pathways including stimulation of phosphoinositide hydrolysis, potentiation of NMDA receptor activity, and release of arachidonic acid. Here we demonstrate that whereas activation of group I mGluR by (S)-3,5-dihydroxyphenylglycine (DHPG) potentiates NMDA-induced currents and intracellular calcium increases in rat cortical neuronal cultures, partial effects of group I mGluR activation or inhibition on neuronal injury induced by oxygen-glucose deprivation remain despite NMDA receptor blockade. DHPG stimulation also increases basal arachidonic acid release from rat neuronal-glial cultures and potentiates injury-induced arachidonic acid release in these cultures. Thus, activation of group I mGluR may exacerbate neuronal injury through multiple mechanisms, which include positive modulation of NMDA receptors and enhanced release of arachidonic acid.

Effect of serine protease inhibitors on posttraumatic brain injury and neuronal apoptosis.

N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.

Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in vitro and improvesoOutcome after brain trauma.

The effects of selective blockade of group I metabotropic glutamate receptor subtype 1 (mGluR1) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. The selective mGluR1 antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), and (S)-(+)-alpha-amino-4-carboxy-2-methylbezeneacetic acid (LY367385) provided significant neuroprotection in rat cortical neuronal cultures subjected to mechanical injury, in both pretreatment or posttreatment paradigms. Administration of the antagonists also attenuated glutamate-induced neuronal cell death in the cultures. Coapplication of these antagonists with the N-methyl-d-aspartate (NMDA) receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had additive neuroprotective effects in glutamate injured cultures. Intracerebroventricular administration of AIDA to rats markedly improved recovery from motor dysfunction after lateral fluid percussion induced traumatic brain injury (TBI). Treatment with mGluR1 antagonists also significantly reduced lesion volumes in rats after TBI, as evaluated by MRI. It appears that these compounds mediate their neuroprotective effect through an mGluR1 antagonist action, as demonstrated by inhibition of agonist induced phosphoinositide hydrolysis in our in vitro system. Moreover, AIDA, CPCCOEt, and LY367385, at concentrations shown to be neuroprotective, had no significant effects on the steady state NMDA evoked whole cell current. Taken together, these data suggest that modulation of mGluR1 activity may have substantial therapeutic potential in brain injury.

mGluR5 antagonists 2-methyl-6-(phenylethynyl)-pyridine and (E)-2-methyl-6-(2-phenylethenyl)-pyridine reduce traumatic neuronal injury in vitro and in vivo by antagonizing N-methyl-D-aspartate receptors.

The effect of selective group I metabotropic glutamate receptor subtype 5 (mGluR5) antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB-1893) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. Treatment with MPEP and SIB-1893 showed significant neuroprotective effects in rat cortical neuronal cultures subjected to mechanical injury. Application of the antagonists also attenuated glutamate- and N-methyl-D-aspartate (NMDA)-induced neuronal cell death in vitro. Intracerebroventricular administration of MPEP to rats markedly improved motor recovery and reduced deficits of spatial learning after lateral fluid percussion-induced traumatic brain injury. Lesion volumes as assessed by magnetic resonance imaging were also substantially reduced by MPEP treatment. Although we show that MPEP acts as a potent mGluR5 antagonist in our culture system, where it completely blocks agonist-induced phosphoinositide hydrolysis, electrophysiological and pharmacological studies indicate that MPEP and SIB-1893 also inhibit NMDA receptor activity at higher concentrations that are neuroprotective. Taken together, these data suggest that MPEP and SIB-1893 may have therapeutic potential in brain injury, although the mechanisms of neuroprotective action for these drugs may reflect their ability to modulate NMDA receptor activity.

Traumatic brain injury: developmental differences in glutamate receptor response and the impact on treatment.

Perinatal brain injury following trauma, hypoxia, and/or ischemia represents a substantial cause of pediatric disabilities including mental retardation. Such injuries lead to neuronal cell death through either necrosis or apoptosis. Numerous in vivo and in vitro studies implicate ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors in the modulation of such cell death. Expression of glutamate receptors changes as a function of developmental age, with substantial implications for understanding mechanisms of post-injury cell death and its potential treatment. Recent findings suggest that the developing brain is more susceptible to apoptosis after injury and that such caspase mediated cell death may be exacerbated by treatment with N-methyl-D-aspartate receptor antagonists. Moreover, group I metabotropic glutamate receptors appear to have opposite effects on necrotic and apoptotic cell death. Understanding the relative roles of glutamate receptors in post-traumatic or post-ischemic cell death as a function of developmental age may lead to novel targeted approaches to the treatment of pediatric brain injury.

Caspase-dependent apoptotic pathways in CNS injury.

Recent studies have suggested a role for neuronal apoptosis in cell loss following acute CNS injury as well as in chronic neurodegeneration. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to contribute significantly to the morphological and biochemical manifestations of apoptotic cell death. Caspases are translated as inactive zymogens and become active after specific cleavage. Of the 14 identified caspases, caspase-3 appears to be the major effector of neuronal apoptosis induced by a variety of stimuli. A role for caspase-3 in injury-induced neuronal cell death has been established using semispecific peptide caspase inhibitors. This article reviews the current literature relating to pathways regulating caspase activation in apoptosis associated with acute and chronic neurodegeneration, and suggests that identification of critical upstream caspase regulatory mechanisms may permit more effective treatment of such disorders.

Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism.

1. The metabotropic glutamate receptors (mGluRs) are a family of G-protein linked receptors that can be divided into three groups (group I, II and III). A number of studies have implicated group I mGluR activation in acute neuronal injury, but until recently it was not possible to pharmacologically differentiate the roles of the two individual subunits (mGluR1 and mGluR5) in this group. 2. We investigated the role of mGluR5 in acute NMDA and glutamate mediated neurodegeneration in cultured rat cortical cells using the mGluR5 antagonists MPEP and SIB-1893, and found that they provide significant protection at concentrations of 20 or 200 microM. 3. These compounds act as effective mGluR5 antagonists in our cell culture system, as indicated by the ability of SIB-1893 to prevent phosphoinositol hydrolysis induced by the specific mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). 4. However, they also significantly reduce NMDA evoked current recorded from whole cells voltage clamped at -60 mV, and significantly decrease the duration of opening of NMDA channels recorded in the outside out patch configuration. 5. This suggests that although MPEP and SIB-1893 are effective mGluR5 antagonists, they also act as noncompetitive NMDA receptor antagonists. Therefore, the neuroprotective effects of these compounds are most likely mediated through their NMDA receptor antagonist action, and caution should be exercised when drawing conclusions about the roles of mGluR5 based on their use.

Caspase pathways, neuronal apoptosis, and CNS injury.

Caspases are a family of mammalian proteases related to the ced-3 gene of Caenorhabditis elegans. They mediate many of the morphological and biochemical features of apoptosis, including structural dismantling of cell bodies and nuclei, fragmentation of genomic DNA, destruction of regulatory proteins, and propagation of other pro-apoptotic molecules. Based on their substrate specificities and DNA sequence homologies, the 14 currently identified caspases may be divided into three groups: apoptotic initiators, apoptotic executioners, and inflammatory mediators. Caspases are activated through two principal pathways, known as the "extrinsic pathway," which is initiated by cell surface death receptor ligation, and the intrinsic pathway, which arises from mitochondria. Endogenous inhibitors, such as the inhibitors of apoptosis (IAP) family, modulate caspase activity at various points within these pathways. Upon activation, caspases appear to play an important role in sequelae of traumatic brain injury, spinal cord injury, and cerebral ischemia. In addition, they may also play a role in mediating cell death in chronic neurodegenerative conditions such as Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. This article reviews the current literature on the role of caspases in acute and chronic CNS injury, and provides evidence for the potential therapeutic use of caspase inhibitors in the setting of these conditions.