Phosphoproteomic analysis of neuronal cell death by glutamate-induced oxidative stress

Oxidative stress is one of the major causes of neuronal cell death in disorders such as perinatal hypoxia and ischemia. Protein phosphorylation is the most significant PTM of proteins and plays an important role in stress-induced signal transduction. Thus, the analysis of alternative protein phosphorylation states which occur during oxidative stress-induced cell death could provide valuable information regarding cell death. In this study, a reference phosphoproteome map of the mouse hippocampal cell line HT22 was constructed based on 125 spots that were identified by MALDI-TOF or LC-ESI-Q-TOF-MS analysis. In addition, proteins of HT22 cells at various stages of oxidative stress-induced cell death were separated by 2-DE and alterations in phosphoproteins were detected by Pro-Q Diamond staining. A total of 17 spots showing significant quantitative changes and seven newly appearing spots were identified after glutamate treatment. Splicing factor 2, peroxiredoxin 2, S100 calcium binding protein A11, and purine nucleoside phosphorylase were identified as up- or down-regulated proteins. CDC25A, caspase-8, and cyp51 protein appeared during oxidative stress-induced cell death. The data in this study from phosphoproteomic analysis provide a valuable resource for the understanding of HT22 cell death mechanisms mediated by oxidative stress.     

Isoliquiritigenin isolated from Glycyrrhiza uralensis protects neuronal cells against glutamate-induced mitochondrial dysfunction

Glutamate-mediated excitotoxicity, which is associated with reactive oxygen species (ROS), is hypothesized to be a major contributor to pathological cell death in the mammalian central nervous system, and to be involved in many acute and chronic brain diseases. Here, we showed that isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis (Gu), one of the most frequently prescribed oriental herbal medicines, protected HT22 hippocampal neuronal cells from glutamate-induced oxidative stress. In addition, we clarified the molecular mechanisms by which it protects against glutamate-induced neuronal cell death. ISL reversed glutamate-induced ROS production and mitochondrial depolarization, as well as glutamate-induced changes in expression of the apoptotic regulators Bcl-2 and Bax. Pretreatment of HT22 cells with ISL suppresses the release of apoptosis-inducing factor from mitochondria into the cytosol. Taken together, our results suggest that ISL may protect against mitochondrial dysfunction by limiting glutamate-induced oxidative stress. In conclusion, our results demonstrated that ISL isolated from Gu has protective effects against glutamate-induced mitochondrial damage and hippocampal neuronal cell death. We expect ISL to be useful in the development of drugs to prevent or treat neurodegenerative diseases.     

HSP27 but not HSP70 has a potent protective effect against alpha-synuclein-induced cell death in mammalian neuronal cells

alpha-Synuclein is a neuronally expressed protein which is mutated in familial Parkinson's disease. We have previously shown that disease-associated mutants of alpha-synuclein cause enhanced neuronal cell death in response to a variety of stimuli, whereas wild-type alpha-synuclein has a protective effect against some stimuli, whilst enhancing the death response to others. We demonstrate, for the first time, that over-expression of the heat shock protein HSP27 has a potent protective anti-apoptotic effect against the damaging effects of wild-type and particularly of mutant alpha-synuclein. In contrast, HSP70 has some protective effect against the damaging effect of the wild-type protein, but has no effect against the mutant proteins, whilst HSP56 has no protective effect in this system. Our results indicate that disease-associated mutants of alpha-synuclein enhance its death-inducing properties and lead to increased apoptosis, which can be mitigated by either the use of specific caspase inhibitors or HSP27 over-expression. This potent protective effect of HSP27 against the mutant and wild-type proteins may be of potential therapeutic importance.     

Mitochondrial superoxide dismutase SOD2, but not cytosolic SOD1, plays a critical role in protection against glutamate-induced oxidative stress and cell death in HT22 neuronal cells

Oxidative cell death is an important contributing factor in neurodegenerative diseases. Using HT22 mouse hippocampal neuronal cells as a model, we sought to demonstrate that mitochondria are crucial early targets of glutamate-induced oxidative cell death. We show that when HT22 cells were transfected with shRNA for knockdown of the mitochondrial superoxide dismutase (SOD2), these cells became more susceptible to glutamate-induced oxidative cell death. The increased susceptibility was accompanied by increased accumulation of mitochondrial superoxide and loss of normal mitochondrial morphology and function at early time points after glutamate exposure. However, overexpression of SOD2 in these cells reduced the mitochondrial superoxide level, protected mitochondrial morphology and functions, and provided resistance against glutamate-induced oxidative cytotoxicity. The change in the sensitivity of these SOD2-altered HT22 cells was neurotoxicant-specific, because the cytotoxicity of hydrogen peroxide was not altered in these cells. In addition, selective knockdown of the cytosolic SOD1 in cultured HT22 cells did not appreciably alter their susceptibility to either glutamate or hydrogen peroxide. These findings show that the mitochondrial SOD2 plays a critical role in protecting neuronal cells from glutamate-induced oxidative stress and cytotoxicity. These data also indicate that mitochondria are important early targets of glutamate-induced oxidative neurotoxicity.     

Cyclin-dependent kinase 5 (CDK5) and neuronal cell death

Many neurological disorders like Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis (ALS) or stroke have in common a definite loss of CNS neurons due to apoptotic or necrotic neuronal cell death. Previous studies suggested that proapoptotic stimuli may trigger an abortive and, therefore, eventually fatal cell cycle reentry in postmitotic neurons. Neuroprotective effects of small molecule inhibitors of cyclin-dependent kinases (CDKs), which are key regulators of cell cycle progression, support the cell cycle theory of neuronal apoptosis. However, growing evidence suggests that deregulated CDK5, which is not involved in cell cycle control, rather than cell cycle relevant members of the CDK family, promotes neuronal cell death. Here we summarize the current knowledge about the involvement of CDK5 in neuronal cell death and discuss possible up- or downstream partners of CDK5. Moreover, we discuss potential therapeutic options that might arise from the identification of CDK5 as an important upstream element of neuronal cell death cascades.     

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death ) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.     

Impaired mitochondrial function protects against free radical-mediated cell death

Free radical damage can have fatal consequences. Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS). Many agents also generate ROS. Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated. Respiratory-deficient cells lacking mitochondrial DNA (rho(0)) were up to 100-fold more resistant than isogenic rho(+) cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents. Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became "petites." The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains. These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains. Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function. The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.Subject terms: Apoptosis, Cell death in the nervous system, Neurodegeneration     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of α-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol®) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents α-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.     

Microglia and neuronal cell death

Microglia, the brain's innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.     

Resistance of mitochondrial DNA-depleted cells against cell death: role of mitochondrial superoxide dismutase

We have shown that mitochondrial DNA-depleted (rho(0)) SK-Hep1 hepatoma cells are resistant to apoptosis, contrary to previous papers reporting normal apoptotic susceptibility of rho(0) cells. We studied the changes of gene expression in SK-Hep1 rho(0) cells. DNA chip analysis showed that MnSOD expression was profoundly increased in rho(0) cells. O(2)(.) contents increased during rho(0) cell derivation but became normalized after establishment of rho(0) phenotypes, suggesting that MnSOD induction is an adaptive process to increased O(2)(.). rho(0) cells were resistant to menadione, paraquat, or doxorubicin, and O(2)(.) contents after treatment with them were lower in rho(0) cells compared with parental cells because of MnSOD overexpression. Expression levels and activity of glutathione peroxidases were also increased in rho(0) cells, rendering them resistant to exogenous H(2)O(2). rho(0) cells were resistant to p53, and intracellular ROS contents after p53 expression were lower compared with parental cells. Other types of rho(0) cells also showed increased MnSOD expression and resistance against ROS. Heme oxygenase-1 expression was increased in rho(0) cells, and a heme oxygenase-1 inhibitor decreased the induction of MnSOD in rho(0) cells and their resistance against ROS donors. These results indicate that rho(0) cells are resistant to cell death contrary to previous reports and suggest that an adaptive increase in the expression of antioxidant enzymes renders cancer cells or aged cells with frequent mitochondrial DNA mutations to resist against oxidative stress, host anti-cancer surveillance, or chemotherapeutic agents, conferring survival advantage on them.     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of alpha-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents alpha-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.     

Activation of autophagy during glutamate-induced HT22 cell death

Recent evidence suggests that autophagy plays a role in oxidative injury-induced cell death. Here we examined whether glutamate-mediated oxidative toxicity induces autophagy in murine hippocampal HT22 cells and if autophagy induction affects the molecular events associated with cell death. Markers for autophagy induction including LC3 conversion, suppression of mTOR pathway, and GFP-LC3 dot formation were enhanced by glutamate treatment. By contrast, autophagy inhibition blocked glutamate-induced LC3 conversion and consequently reduced cell death. Activation of ERK1/2, a hallmark of glutamate-induced cytotoxicity, was also decreased by autophagy inhibition. Interestingly, autophagy inhibition also affected the expression of chaperones including Hsp60 and Hsp70, which are differentially regulated during HT22 cell death. Conversely, knock-down of Hsp60 greatly decreased LC3 conversion. Together these results suggest that glutamate-induced cytotoxicity involves autophagic cell death and chaperones may play a role in this process.     

Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death

Excessive activation of neuronal glutamate receptors has been implicated in the pathophysiology of stroke, epilepsy, and traumatic brain injury. Previously, it has been demonstrated that excitotoxic glutamate exposure results in the induction of an extended neuronal depolarization (END), as well as protracted elevations in free intracellular calcium ([Ca(2+)](i)). Both END and the prolonged [Ca(2+)](i) elevations were shown to correlate with subsequent neuronal death. In the current study, we used whole-cell current-clamp electrophysiology and fura-ff Ca(2+) imaging to determine the electrophysiological basis of END. We found that removal of extracellular Ca(2+) but not Na(+) in the post-glutamate period resulted in complete reversal of END, allowing neurons to rapidly repolarize to their initial resting membrane potential (RMP). In addition, removal of extracellular Ca(2+) was sufficient to eliminate the protracted [Ca(2+)](i) elevations induced by excitotoxic glutamate exposure. To investigate the mechanism through which extracellular Ca(2+) was effecting these changes, pharmacological antagonists of well-characterized routes of Ca(2+) entry were tested for their effects on END. Antagonists of glutamate receptors and voltage-gated Ca(2+) channels (VGCCs) had no significant effect on the membrane potential of neurons in END. Likewise, inhibitors of the Na(+)/Ca(2+) exchange (NCX) were ineffective. In contrast, addition of 500 microM ZnCl(2) or 100 microM GdCl(3) to control extracellular medium (containing normal levels of extracellular Ca(2+)) in the post-glutamate period resulted in rapid and complete reversal of END. Addition of 1mM CdCl(2) to control medium had only modest effects on END. These data provide the first direct evidence that END induced by excitotoxic glutamate exposure is caused by an influx of extracellular Ca(2+) and demonstrate that the previously irreversible condition of END can be reversed by removing extracellular Ca(2+). In addition, understanding the electrophysiological basis of this novel Ca(2+)-induced extended depolarization may provide an insight into the pathophysiology of stroke, traumatic brain injury, and other forms of neuronal injury.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

The role of Bax in glutamate-induced nerve cell death

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.     

Lipocalin-type prostaglandin D synthase protects against oxidative stress-induced neuronal cell death

L-PGDS [lipocalin-type PGD (prostaglandin D) synthase] is a dual-functional protein, acting as a PGD2-producing enzyme and a lipid transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. In the present study we demonstrate the protective effect of L-PGDS on H2O2-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H2O2-treated neuronal cells, and the L-PGDS level was highly associated with H2O2-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H2O2-mediated cell death. A cell viability assay revealed that L-PGDS protected against H2O2-induced cell death in a concentration-dependent manner. Furthermore, the titration of free thiols in H2O2-treated L-PGDS revealed that H2O2 reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight)-MS spectrum of H2O2-treated L-PGDS showed a 32 Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable with those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.     

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.