Prevention Of Neuronal Cell Death Research Articles

The Syvn1 inhibits neuronal cell ferroptosis by activating Stat3/Gpx4 axis in rat with spinal cord injury.

Spinal cord injury (SCI) leads to secondary neuronal death, which severely impedes recovery of motor function. Therefore, prevention of neuronal cell death after SCI is an important strategy. Ferroptosis, a new form of cell death discovered in recent years, has been shown to be involved in the regulation of SCI. However, the role and potential mechanisms of ferroptosis in secondary SCI are not fully understood. In this study, we report that the E3 ubiquitin ligase Syvn1 suppresses ferroptosis and promotes functional recovery from SCI in vitro and in vivo. Mechanistically, screened with bioinformatics, immunoprecipitation, and mass spectrometry, we identified Stat3, a transcription factor that induces the expression of the ferroptosis inhibitor Gpx4, as a substrate of Syvn1. Furthermore, we identified neurons as the primary cellular source of Syvn1 signalling. Moreover, we determined the binding domains of Syvn1 and Stat3 in HEK 293 T cells using full-length proteins and a series of truncated Flag-tagged and Myc-tagged fragments. Furthermore, we created the cell and animal models with silencing or overexpression of Syvn1 and Stat3 and found that Syvn1 inhibits neuronal ferroptosis by stabilizing Stat3, which subsequently activates the ferroptosis regulator Gpx4 in SCI. In summary, the Syvn1-mediated Stat3/Gpx4 signalling axis attenuates neuronal ferroptosis, reduces neuronal death, and promotes SCI repair. Therefore, our findings provide potential new targets and intervention strategies for the treatment of SCI.

Investigating the Relationship Between Neuronal Cell Death and Early DNA Methylation After Ischemic Injury.

Cerebral ischemia induces neuronal cell death and causes various kinds of brain dysfunction. Therefore, prevention of neuronal cell death is most essential for protection of the brain. On the other hand, it has been reported that epigenetics including DNA methylation plays a pivotal role in pathogenesis of some diseases such as cancer. Accumulating evidences indicate that aberrant DNA methylation is related to cell death. However, DNA methylation after cerebral ischemia has not been fully understood yet. The aim of this present study was to investigate the relationships between DNA methylation and neuronal cell death after cerebral ischemia. We examined DNA methylation under the ischemic condition by using transient middle cerebral artery occlusion and reperfusion (MCAO/R) model rats and N-methyl-D-aspartate (NMDA)–treated cortical neurons in primary culture. In this study, we demonstrated that DNA methylation increased in these neurons 24 h after MCAO/R and that DNA methylation, possibly through activation of DNA methyltransferases (DNMT) 3a, increased in such neurons immediately after NMDA treatment. Furthermore, NMDA-treated neurons were protected by treatment with a DNMT inhibitor that were accompanied by inhibition of DNA methylation. Our results showed that DNA methylation would be an initiation factor of neuronal cell death and that inhibition of such methylation could become an effective therapeutic strategy for stroke.

Citric Acid Protects Dopaminergic Cells against Rotenone-Induced Neurodegeneration

We investigated the effect of citric acid on rotenone-induced oxidative stress and neuro- and hepato-toxicity. Swiss mice received subcutaneous injections of rotenone at a dose of 1.5 mg/kg once every other day for two weeks either alone or in combination with citric acid at 200 or 400 mg/kg given orally. The control group was treated with the vehicle dimethyl sulfoxide. The brain and liver levels of the lipid peroxidation product malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (TAC), and paraoxonase-1 (PON-1) activity were measured and as well as brain striatal concentrations of nuclear factor-kappa B (NF-kB) and tyrosine hydroxylase. Histopathology of the brain and liver tissue was also performed. Results indicated that MDA and NO content were increased whereas TAC and PON-1 decreased in the brain and liver tissue following rotenone injection. There was also increased NF-kB and decreased tyrosine hydroxylase concentrations in the brain of rotenone-treated mice. Rotenone-treated mice showed decreased striatal cell size and pyknotic nuclei. There were also interstitial hemorrhage and focal inflammatory cells infiltration in cerebral cortex, and degenerated neurons in cortex and hippocampus. The liver exhibited inflammatory cell infiltration and fibrosis. The administration of citric acid protected against rotenone-induced histopathological changes, decreased MDA, NO, and increased TAC and PON-1 activity in the brain and liver. It also reduced NF-kB and increased tyrosine hydroxylase in the brain of rotenone-treated mice. These data indicate that citric acid prevents rotenone-induced neuronal and liver damage via a decrease in oxidative stress. These findings suggest that supplementation with citric acid could be of value in the prevention of neuronal cell death in Parkinson’s disease.

Prophylactic Zinc and Therapeutic Selenium Administration Increases the Antioxidant Enzyme Activity in the Rat Temporoparietal Cortex and Improves Memory after a Transient Hypoxia‐Ischemia

In the cerebral hypoxia-ischemia rat model, the prophylactic administration of zinc can cause either cytotoxicity or preconditioning effect, whereas the therapeutic administration of selenium decreases the ischemic damage. Herein, we aimed to explore whether supplementation of low doses of prophylactic zinc and therapeutic selenium could protect from a transient hypoxic-ischemic event. We administrated zinc (0.2 mg/kg of body weight; ip) daily for 14 days before a 10 min common carotid artery occlusion (CCAO). After CCAO, we administrated sodium selenite (6 μg/kg of body weight; ip) daily for 7 days. In the temporoparietal cerebral cortex, we determined nitrites by the Griess method and lipid peroxidation by the Gerard-Monnier assay. qPCR was used to measure mRNA of nitric oxide synthases, antioxidant enzymes, chemokines, and their receptors. We measured the enzymatic activity of SOD and GPx and protein levels of chemokines and their receptors by ELISA. We evaluated long-term memory using the Morris-Water maze test. Our results showed that prophylactic administration of zinc caused a preconditioning effect, decreasing nitrosative/oxidative stress and increasing GPx and SOD expression and activity, as well as eNOS expression. The therapeutic administration of selenium maintained this preconditioning effect up to the late phase of hypoxia-ischemia. Ccl2, Ccr2, Cxcl12, and Cxcr4 were upregulated, and long-term memory was improved. Pyknotic cells were decreased suggesting prevention of neuronal cell death. Our results show that the prophylactic zinc and therapeutic selenium administration induces effective neuroprotection in the early and late phases after CCAO.

After Treatment with Methylene Blue is Effective against Delayed Encephalopathy after Acute Carbon Monoxide Poisoning.

Delayed encephalopathy after acute carbon monoxide (CO) poisoning (DEACMP) is the most severe and clinically intractable complication that occurs following acute CO poisoning. Unfortunately, the mechanism of DEACMP is still vague. Growing evidence indicates that delayed cerebral damage after CO poisoning is related to oxidative stress, abnormal neuro-inflammation, apoptosis and immune-mediated injury. Our recent report indicated that methylene blue (MB) may be a promising therapeutic agent in the prevention of neuronal cell death and cognitive deficits after transient global cerebral ischaemia (GCI). In this study, we aimed to investigate the potential of MB therapy to ameliorate the signs and symptoms of DEACMP. Rats were exposed to 1000 ppm CO for 40 min. in the first step; CO was then increased to 3000 ppm, which was maintained for another 20 min. The rats were implanted with 7-day release Alzet osmotic mini-pumps subcutaneously under the back skin, which provided MB at a dose of 0.5 mg/kg/day 1 hr after CO exposure. The results showed that MB significantly suppressed oxidative damage and expression of pro-inflammatory factors, including tumour necrosis factor-α and interleukin (IL)-1β. MB treatment also suitably modulated mitochondrial fission and fusion, which is helpful in the preservation of mitochondrial function. Furthermore, MB dramatically attenuated apoptosis and neuronal death. Lastly, behavioural studies revealed that MB treatment preserved spatial learning and memory in the Barnes maze test. Our findings indicated that MB may have protective effects against DEACMP.

Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury

ObjectivePreterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. Methods3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. ResultsMSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions InterpretationMSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Memantine Treatment for Prevention of Neuronal Cell Death in Traumatic Brain Injury

Memantine Treatment for Prevention of Neuronal Cell Death in Traumatic Brain Injury

Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus

The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1–3R. Throughout the last 20years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GAL1R and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL1R selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats.The peptide ligands were evaluated in vitro for their binding preference in a competitive 125I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL1R in the prevention of neuronal cell death.

Cytokine signaling by grafted neuroectodermal stem cells rescues motoneurons destined to die

Following an injury to their axons close to the cell body, adult motoneurons generally die. This type of injury, typically caused by avulsion of the spinal ventral root, initiates the activation of astrocytes and microglial cells and the extracellular space becomes loaded with excessive amounts of excitotoxic glutamate. We have provided evidence that, following ventral root avulsion and reimplantation, murine embryonic neuroectodermal stem cells (NE-GFP-4C) grafted into the rat spinal cord rescue the vast majority of the motoneurons that would otherwise die, and enable them to reinnervate peripheral targets. Stem cell grafts produced the modulatory cytokines IL-1-alpha, IL-6, IL-10, TNF-alpha and MIP-1-alpha, but not neurotrophic factors. The neurons and astrocytes in the ventral horn of grafted animals also produced IL-6 and MIP-1-alpha, indicating a strong interaction between the graft and the host tissue. The infusion of function-blocking antibodies against all cytokines into the grafted cords completely abolished their motoneuron-rescuing effect, while neutralization of only IL-10 suggested its strong effectivity as concerns motoneuron survival and a milder effect on reinnervation.It is suggested that, apart from the anti-inflammatory function of IL-10, the pro-inflammatory cytokines produced exert a strong modulatory function in the CNS, promoting the prevention of neuronal cell death.

Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein

Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1α), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1α and the associated ubiquitin–proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1α stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106–126). Lactoferrin prevented PrP (106–126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1α stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106–126)-induced neurotoxicity via the up-regulation of HIF-1α stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease.

Erythropoietin modulates the immune-inflammatory response of a SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS)

Temporal patterns of inflammatory cytokine levels reflect the immune-inflammatory role in pathogenic mechanisms of SOD1 animal model of Amyotrophic Lateral Sclerosis (ALS) and these cytokines have important roles in both toxic and protective functions depending on the stage of disease progression in ALS patients. Erythropoietin (EPO) has various neuroprotective effects, including the reduction of inflammation, the enhancement of survival signals, and the prevention of neuronal cell death. This study was undertaken to evaluate the temporal pattern of inflammatory cytokine levels induced by EPO treatment in the SOD1G93A mice model of ALS. We treated mice with 5 IU of EPO per gram of animal weight once every other week after the mice were 60 days old, and pro/anti-inflammatory cytokines were analyzed at 30, 60, 90, and 120 days of age. In untreated controls, pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, CCL2 (MCP-1), CCL5 (RANTES), CXCL10 (IP-10), and IL-17A) were gradually increased with aging. In contrast, increment of anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) showed the highest level at 90 days of age and their levels were remarkably faded until 120 days of age. EPO treatment, however, showed significantly decreased level of pro-inflammatory cytokines. And, up-regulated levels of anti-inflammatory cytokines with EPO were highly maintained until 120 days. In addition, the treatment of EPO delayed symptom onset, prolonged time of rotarod failure, and showed more preserved number of motoneurons. These findings suggest that EPO may be a potential therapeutic candidate having ability to modulate immune-inflammation in ALS

BAFF Controls Neural Cell Survival through BAFF Receptor

Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell–activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

Loss of Otx2 in the Adult Retina Disrupts Retinal Pigment Epithelium Function, Causing Photoreceptor Degeneration

Photoreceptors are specialized neurons of the retina that receive nursing from the adjacent retinal pigment epithelium (RPE). Frequent in the elderly, photoreceptor loss can originate from primary dysfunction of either cell type. Despite intense interest in the etiology of these diseases, early molecular actors of late-onset photoreceptor degeneration remain elusive, mostly because of the lack of dedicated models. Conditional Otx2 ablation in the adult mouse retina elicits photoreceptor degeneration, providing a new model of late-onset neuronal disease. Here, we use this model to identify the earliest events after Otx2 ablation. Electroretinography and gene expression analyses suggest a nonautonomous, RPE-dependent origin for photoreceptor degeneration. This is confirmed by RPE-specific ablation of Otx2, which results in similar photoreceptor degeneration. In contrast, constitutive Otx2 expression in RPE cells prevents degeneration of photoreceptors in Otx2-ablated retinas. We use chromatin immunoprecipitation followed by massive sequencing (ChIP-seq) analysis to identify the molecular network controlled in vivo by Otx2 in RPE cells. We uncover four RPE-specific functions coordinated by Otx2 that underpin the cognate photoreceptor degeneration. Many direct Otx2 target genes are associated with human retinopathies, emphasizing the significance of the model. Importantly, we report a secondary genetic response after Otx2 ablation, which largely precedes apoptosis of photoreceptors, involving inflammation and stress genes. These findings thus provide novel general markers for clinical detection and prevention of neuronal cell death.

Nicotinamide attenuates the injury-induced decrease of hippocalcin in ischemic brain injury

Nicotinamide is an important cofactor in the prevention of brain damage during focal cerebral ischemia. Hippocalcin is a calcium buffer protein that modulates intracellular calcium concentration and attenuates apoptosis. In this study, we investigated whether nicotinamide modulates hippocalcin expression during cerebral ischemia. Male Sprague–Dawley rats were treated with vehicle or nicotinamide (500mg/kg) 2h after the onset of middle cerebral artery occlusion (MCAO) and cerebral cortex tissues were collected 24h after MCAO. Nicotinamide treatment decreased infarct volume in the cerebral cortex of MCAO-operated animals. Our proteomic approach revealed a decrease in hippocalcin expression in vehicle-treated animals during MCAO, which was attenuated by nicotinamide treatment. We used RT-PCR and Western blot analyses to demonstrate that nicotinamide clearly restored the injury-induced decrease in hippocalcin expression. Glutamate toxicity also decreased hippocalcin levels in cultured hippocampal cells, while nicotinamide treatment prevented the glutamate exposure-induced decrease in hippocalcin levels. These results suggest that nicotinamide modulates hippocalcin expression in cerebral ischemic injury and consequently contributes to the prevention of neuronal cell death.

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca(2+) channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca(2+) channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca(2+)-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.

Effects of Bax gene deletion on social behaviors and neural response to olfactory cues in mice

Bax is a pro-death protein that plays a crucial role in developmental neuronal cell death. Bax(-/-) mice exhibit increased neuron number and lack several neural sex differences. Here we examined the effects of Bax gene deletion on social behaviors (olfactory preference, social recognition, social approach and aggression) and the neural processing of olfactory cues. Bax deletion eliminated the normal sex difference in olfactory preference behavior. In the social recognition test, both genotypes discriminated a novel conspecific, but wild-type males and Bax(-/-) animals of both sexes spent much more time than wild-type females investigating stimulus animals. Similarly, Bax(-/-) mice were more sociable than wild-type mice in a social approach test. Bax deletion had no effect on aggression in a resident/intruder paradigm where males, regardless of genotype, exhibited a shorter latency to attack. Thus, the prevention of neuronal cell death by Bax gene deletion results in greater sociability as well as the elimination of sex differences in some social behaviors. To examine olfactory processing of socially relevant cues, we counted c-Fos-immunoreactive (Fos-ir) cells in several nodes of the accessory olfactory pathway after exposure to male-soiled or control bedding. In both genotypes, exposure to male-soiled bedding increased Fos-ir cells in the posterodorsal medial amygdala, principal nucleus of the bed nucleus of the stria terminalis and medial preoptic nucleus (MPN), and the response in the MPN was greater in females than in males. However, a reduction in Fos-ir cells was seen in the anteroventral periventricular nucleus of Bax(-/-) mice.

Prevention of neuronal cell death by anticonvulsants in experimental epilepsy (extended abstract)

In human temporal lobe epilepsy, neuronal cell damage is found in the hippocampus (1) as well as in the amygdaloid complex (2) and entorhinal cortex (3). Data from animal studies show that recurrent seizures selectively injure certain populations of neurons in the temporal lobe structures (4). Whether or not recurrent seizures cause neuronal damage in human epilepsy is still under investigation. A new challenge for anti-epilepsy medication is not only to suppress clinical seizures but also to prevent any neuronal damage caused by the seizures.

Recombinant human erythropoietin suppresses symptom onset and progression of G93A‐SOD1 mouse model of ALS by preventing motor neuron death and inflammation

Multifactorial pathogenic mechanisms, including inflammation, attenuated survival signals and enhanced death signals, are involved in amyotrophic lateral sclerosis (ALS). Erythropoietin (EPO) has recently been highlighted as a cytokine with various potent neuroprotective effects, including reduction of inflammation, enhancement of survival signals and prevention of neuronal cell death. This study was undertaken to evaluate the effect of recombinant human EPO (rhEPO) on ALS model mice. We treated 96 ALS model mice with vehicle only, or 1, 2.5 or 5 imu of rhEPO/g of mouse once every other week after they were 60 days old. The treatment significantly prolonged symptom onset and life span, preserved more motor neurons, enhanced survival signals, and attenuated inflammatory signals in a dose-dependent manner. These data suggest that treatment with rhEPO represents a potential therapeutic strategy for ALS.

Biomarkers for apoptosis in Alzheimer's disease

Alzheimer's disease (AD) affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. Today there is no cure for this devastating and always lethal disease and therefore it is of great interest for patients, relatives and societies to find new drugs that can hinder the disease process. During the progression of AD a substantial amount of neurons degenerate in the brain. The mechanisms of cell death involved in AD have not been fully elucidated. However, there are several reports showing that neurons die partly by apoptosis in the AD brain. Drugs blocking apoptosis could therefore be potentially useful for early prevention of neuronal cell death. Biomarkers for apoptosis should be important tools in the evaluation of drug effects and in the diagnostics of AD. Here we review the current knowledge in the field and discuss potential biomarkers for apoptosis in AD.

Protective Effect of DY-9760e, a Calmodulin Antagonist, against Neuronal Cell Death

An excessive elevation of intracellular Ca(2+) levels is known to play a key role in the pathological events following cerebral ischemia. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a potent calmodulin antagonist that attenuates brain damage in focal ischemia models. In the present study, we investigated the effect of DY-9760e on neuronal cell death induced by a variety of cell-toxic stimuli that increase intracellular Ca(2+). Cell death was induced by the exposure of primary cultured neurons to excitotoxic agents such as glutamate and N-methyl-D-aspartate, membrane-depolarizing agents such as veratridine and high KCl, or thapsigargin an endoplasmic reticulum Ca(2+)-ATPase inhibitor. Treatment with DY-9760e resulted in a dose-dependent prevention of neuronal cell death elicited by excitotoxicity, voltage-gated channel opening, and inhibition of endoplasmic reticulum Ca(2+)-ATPase. These results indicate that DY-9760e can rescue neurons from various types of cell-toxic stimuli, which may contribute to attenuation of brain injury after cerebral ischemia.