BAX contributes to apoptotic-like death following neonatal hypoxia-ischemia: evidence for distinct apoptosis pathways.

BACKGROUND: Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied. MATERIALS AND METHODS: Bax +/+, +/-, and -/- mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation. RESULTS: We found that bax -/- mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax -/- mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax -/- mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/- and bax -/- mice. CONCLUSIONS: These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.     

Lack of X-linked inhibitor of apoptosis protein leads to increased apoptosis and tissue loss following neonatal brain injury

Neurological deficits caused by H-I (hypoxia-ischaemia) to the perinatal brain are often severely debilitating and lead to motor impairment, intellectual disability and seizures. Perinatal brain injury is distinct from adult brain injury in that the developing brain is undergoing the normal process of neuronal elimination by apoptotic cell death and thus the apoptotic machinery is more easily engaged and activated in response to injury. Thus cell death in response to neonatal H-I brain injury is partially due to mitochondrial dysfunction and activation of the apoptosome and caspase 3. An important regulator of the apoptotic response following mitochondrial dysfunction is XIAP (X-linked inhibitor of apoptosis protein). XIAP inhibits apoptosis at the level of caspase 9 and caspase 3 activation, and lack of XIAP in vitro has been shown to lead to increased apoptotic cell death. In the present study we show that mice lacking the gene encoding the XIAP protein have an exacerbated response to neonatal H-I injury as measured by tissue loss at 7 days following the injury. In addition, when the XIAP-deficient mice were studied at 24 h post-H-I we found that the increase in injury correlates with an increased apoptotic response in the XIAP-deficient mice and also with brain imaging changes in T2-weighted magnetic resonance imaging and apparent diffusion coefficient that correspond to the location of apoptotic cell death. These results identify a critical role of XIAP in regulating neuronal apoptosis in vivo and demonstrate the enhanced vulnerability of neurons to injury in the absence of XIAP in the developing brain.     

Selective,reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model.     

Polyphenol amentoflavone affords neuroprotection against neonatal hypoxic-ischemic brain damage via multiple mechanisms

Flavonoids are naturally occurring polyphenolic compounds that have many biological properties, including antioxidative, anti-inflammatory and neuroprotective effects. Here, we report that amentoflavone significantly reduced cell death induced by staurosporine, etoposide and sodium nitroprusside in neuroblastoma SH-SY5Y cells. In post-natal day 7 rats, hypoxic-ischemic (H-I) brain damage induced by unilateral carotid ligation and hypoxia resulted in distinct features of neuronal cell death including apoptosis and necrosis. In this model, a systemic administration of amentoflavone (30 mg/kg) markedly reduced the H-I-induced brain tissue loss with a wide therapeutic time window up to 6 h after the onset of hypoxia. Amentoflavone blocked the activation of caspase 3, characteristic of apoptosis, and the proteolytic cleavage of its substrates following H-I injury. Amentoflavone also reduced the excitotoxic/necrotic cell death after H-I injury in vivo and after oxygen/glucose deprivation in mouse mixed cultures in vitro. Treatment of mouse microglial cells with amentoflavone resulted in a significant decrease in the lipopolysaccharide-induced production of nitric oxide and induction of inducible nitric oxide synthase and cyclo-oxygenase-2. Furthermore, amentoflavone decreased the inflammatory activation of microglia after H-I injury when assessed by the microglial-specific marker OX-42. These data demonstrate for the first time that amentoflavone strongly protects the neonatal brain from H-I injury by blocking multiple cellular events leading to brain damage.     

Caspase-3 mediated neuronal death after traumatic brain injury in rats

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.     

Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases

Neurotrophins support neuronal survival and differentiation via Trk receptors, yet can also induce cell death via the p75 receptor. In these studies, we investigated signaling mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases. Although p75 is structurally a member of the Fas/TNFR1 receptor family, caspase-8 was not required for p75-mediated death, unlike other members of this receptor family. In contrast, p75-mediated neuronal death was associated with mitochondrial loss of cytochrome c and required Apaf-1 and caspase-9, -6, and -3. In particular, caspase-6 plays a central role in mediating neurotrophin-induced death, illuminating a novel role for this caspase. Inhibition of DIABLO/Smac, which blocks inhibitor of apoptosis proteins, protected cells from death, whereas simultaneous inhibition of both DIABLO/Smac and MIAP3 allowed trophin-induced death to proceed. In vivo, pilocarpine-induced seizures, previously shown to up-regulate p75 expression and increase neurotrophin production, caused activation of caspase-6 and -3 and cleavage of poly(ADP-ribose) polymerase in p75-expressing hippocampal neurons. In p75(-/-) mice, no activated caspase-3 was detected, and there was a marked reduction in the number of dying neurons after pilocarpine treatment compared with wild type mice. Neurotrophin-induced p75-mediated death is likely to play an important role in mediating neuronal loss consequent to brain injury.     

Autophagic neuron death in neonatal brain ischemia/hypoxia

Hypoxia/ischemia (H/I) brain injury at birth is an important cause of cerebral palsy, mental retardation, and epilepsy. The H/I insult also causes energy failure, oxidative stress, and unbalanced ion fluxes, leading to high induction of autopahgy in brain neurons. Since the mice unable to execute autophagy (due to brain-specific deletion of Atg7 or Atg5) die by massive loss of cerebral and cerebellar neurons with accumulation of ubiquitin aggregates, induction of neuronal autophagy after H/I injury is generally considered neuroprotective by maintaining cellular homeostasis. However, our recent results show that hippocampal pyramidal neurons undergoing caspase-dependent or -independent death following neonatal H/I injury possess abundant LC3-positive granules, and such H/I neuronal death is largely prevented by Atg7 deficiency. In the present review we discuss the roles of autophagy and other forms of programmed cell death in the neonatal H/I brain insult.     

Autophagic neuron death in neonatal brain ischemia/hypoxia

Hypoxia/ischemia (H/I) brain injury at birth is an important cause of cerebral palsy, mental retardation, and epilepsy. The H/I insult also causes energy failure, oxidative stress, and unbalanced ion fluxes, leading to high induction of autopahgy in brain neurons. Since the mice unable to execute autophagy (due to brain-specific deletion of Atg7 or Atg5) die by massive loss of cerebral and cerebellar neurons with accumulation of ubiquitin aggregates, induction of neuronal autophagy after H/I injury is generally considered neuroprotective by maintaining cellular homeostasis. However, our recent results show that hippocampal pyramidal neurons undergoing caspase-dependent or -independent death following neonatal H/I injury possess abundant LC3-positive granules, and such H/I neuronal death is largely prevented by Atg7 deficiency. In the present review we discuss the roles of autophagy and other forms of programmed cell death in the neonatal H/I brain insult.     

Targeted gene inactivation of calpain-1 suppresses cortical degeneration due to traumatic brain injury and neuronal apoptosis induced by oxidative stress

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role.     

Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.     

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na(+), K(+) -ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na(+), K(+) -ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain.     

Role of DNA-dependent protein kinase in neuronal survival

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.     

Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency

Blockade of ionotropic glutamate receptors induces neuronal cell apoptosis. We investigated if mitochondria-mediated death signals would contribute to neuronal apoptosis following administration of glutamate antagonists. The administration of MK-801 and CNQX (MK-801/CNQX), the selective antagonists of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, produced widespread neuronal death in neonatal rat brain and cortical cell cultures. MK-801/CNQX-induced neuronal apoptosis was prevented by zVAD-fmk, a broad inhibitor of caspases, but insensitive to inhibitors of calpain or cathepsin D. Activation of caspase-3 was observed within 6-12 h and sustained over 36 h after exposure to MK-801/CNQX, which cleaved PHF-1 tau, the substrate for caspase-3. Activation of caspase-3 was blocked by high K+ and mimicked by BAPTA-AM, a selective Ca2+ chelator. Reducing extracellular Ca2+, but not Na+, activated caspase-3, suggesting an essential role of Ca2+ deficiency in MK-801/CNQX-induced activation of caspases. Cortical neurons treated with MK-801/CNQX triggered activation of caspase-9, release of cytochrome c from mitochondria, and translocation of Bax into mitochondria. The present study suggests that blockade of ionotropic glutamate receptors causes caspase-3-mediated neuronal apoptosis due to Ca2+ deficiency that is coupled to the sequential mitochondrial death pathway.     

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu−/−) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu−/− and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu−/− mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu−/− slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d -2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons.     

Temporal profile and cell subtype distribution of activated caspase-3 following experimental traumatic brain injury

This study investigated the temporal expression and cell subtype distribution of activated caspase-3 following cortical impact-induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase-3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase-3 activation on proteolysis of the cytoskeletal protein alpha-spectrin. Increased protein levels of p18 and the caspase-3-specific 120-kDa breakdown product to alpha-spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase-3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase-3-positive cells revealed that the number of caspase-3-positive neurons exceeded the number of caspase-3-positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18-immunopositive cells exhibiting apoptotic-like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or alpha-spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase-3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase-3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.     

Mitochondrial Impairment in the Developing Brain After Hypoxia–Ischemia

The pattern of cell death in the immature brain differs from that seen in the adult CNS. During normal development, more than half of the neurons are removed through apoptosis, and mediators like caspase-3 are highly upregulated. The contribution of apoptotic mechanisms in cell death appears also to be substantial in the developing brain, with a marked activation of downstream caspases and signs of DNA fragmentation. Mitochondria are important regulators of cell death through their role in energy metabolism and calcium homeostasis, and their ability to release apoptogenic proteins and to produce reactive oxygen species. We find that secondary brain injury is preceded by impairment of mitochondrial respiration, signs of membrane permeability transition, intramitochondrial accumulation of calcium, changes in the Bcl-2 family proteins, release of proapoptotic proteins (cytochrome C, apoptosis inducing factor) and downstream activation of caspase-9 and caspase-3 after hypoxia-ischemia. These data support the involvement of mitochondria-related mechanisms in perinatal brain injury.     

Macrophage colony stimulating factor prevents NMDA-induced neuronal death in hippocampal organotypic cultures

Macrophage colony stimulating factor (M-CSF) and its receptor are up-regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M-CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase-3 activation in organotypic hippocampal cultures, whereas treatment with M-CSF protected hippocampal neurons from NMDA-induced apoptosis. Caspase-3 activation was inhibited by M-CSF treatment to the same degree as with the caspase inhibitor Z-VAD-FMK. These results suggest that M-CSF has neuroprotective properties through inhibition of caspase-3 that could promote neuronal survival after excitotoxic insult. The role of M-CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.     

Clusterin levels increase during neuronal development

The expression of clusterin has been shown to be elevated in several models of experimentally induced programmed cell death and in association with a number of neurodegenerative conditions. In order to test whether this protein is expressed in neurons during development, the expression of clusterin was examined in the developing nervous system, using immunohistochemistry and mRNA analysis. Clusterin expression was observed in the earliest neurons of the cortical plate on embryonic day (E) 12. Thereafter, the intensity of clusterin staining continued to increase in an age-dependent manner, with the greatest intensity of staining being found in the postnatal mature brain. Virtually all neurons were clusterin-positive and there was no evidence for the appearance of clusterin-positive cells specifically during epochs of programmed neuronal death in the embryo. This study suggests that clusterin has a role in neuronal maturation and it is unlikely to be associated exclusively with neuronal cell death. © 1993 John Wiley & Sons, Inc.     

Wild-type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.