Glutamate-induced Oxidative Stress Research Articles

Protective Effect of Peanut Sprout Extract on Glutamate‐induced Neuronal Toxicity and Kainic acid‐induced Memory Loss

Glutamate, one of major excitatory neurotransmitter in central nervous system, has been known to induce oxidative stress such as reactive oxygen species (ROS) by overstimulation of neuron. In this context, we investigated the neuroprotective effect of peanut sprout extract (PSE) against glutamate/kainic acid‐induced oxidative stress in the mouse hippocampal HT22 cells and ICR mice. PSE significantly increased the cell viability and decreased the ROS generation under glutamate‐induced oxidative stress in HT22 cells. PSE induced heme oxygenase‐1(HO‐1) known as a phase 2 enzyme and, its protective effect was abolished by HO‐1 and PI3K inhibitors. Furthermore, to examine the effect of PSE on learning and memory abilities in mice, kainic acid was treated to induce learning and memory loss, an analogue of glutamate. Passive avoidance and Y‐maze tests were performed to examine abilities of learning memory and spatial cognition respectively. The results showed that PSE had protective effect on kainic acid‐induced memory loss in mice, suggesting that PSE protects neuronal cells from glutamate/kainic acid‐induced oxidative stress through induction of HO‐1.Grant Funding Source: ASN

Abstract TP107: Stats Regulate Transcription Of 12/15-lox In Mouse Cortical Neurons Under Glutamate-induced Oxidative Stress

Background and Purpose— Oxidative stress during reperfusion following cerebral ischemic stroke causes neuronal death and various forms of cerebrovascular injury. 12/15-lipoxygenase (12/15-LOX) is a major contributor to these processes; however, the regulation of 12/15-LOX expression in the ischemic brain is still unknown. In this study, we investigated the transcriptional regulation of 12/15-LOX in primary cortical neurons subjected to oxidative stress. Methods— Immature cortical neurons were treated with glutamate. Western blots were performed for analysis of 12/15-LOX protein level and phosphorylation of STAT1 and 6 in response to glutamate stimuli. Primary mouse cerebral cortical neurons were transfected with small interfering RNA probes targeted to STAT1 or 6 and were used for real time RT-PCR for analysis of changes in 12/15-LOX mRNA level, and cell viability assay following glutamate treatment. Results— In preliminary screening of putative transcriptional factors on the promoters of mouse and human 12/15-LOX genes, we detected multiple putative binding motifs for STATs in the promoters of 12/15-LOX. Following glutamate treatment of primary cortical neurons, phosphorylation of STAT1 (Y701) and STAT6 (Y641), as well as 12/15-LOX protein levels, were significantly increased. Importantly, we found significant increases in mRNA levels of 12/15-LOX in mouse cerebral cortices subjected to ischemia reperfusion, which was paralleled in glutamate-treated primary cortical neurons. Gene silencing of STAT1 or 6 by siRNA transfection in primary cortical neurons reduced the levels of both 12/15-LOX mRNA and protein under glutamate-induced oxidative stress. Moreover, inhibition of STAT1 or 6 by siRNA transfection protected primary cortical neurons against glutamate-induced oxidative stress. Conclusions— This study increases our understanding of the transcriptional regulation of the 12/15-LOX gene in cerebral ischemic injury and oxidative stress, which will be important to guard against neuronal cell death. Identifying regulators of 12/15-LOX transcription may suggest new molecular targets for stroke therapy.

Protective effects of onion-derived quercetin on glutamate-mediated hippocampal neuronal cell death

Background:Neurodegenerative diseases are characterized by progressive neuron degeneration in specific functional systems of the central or peripheral nervous system. This study investigated the protective effects of quercetin isolated from onion on neuronal cells and its protective mechanisms against glutamate-induced apoptosis in HT22 cells.Materials and Methods:HT22 cells were cultured to study the neuroprotective mechanism of quercetin against glutamate-mediated oxidative stress. The intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (ΔΨm) were measured. The protein expression of calpain, spectrin, Bcl-2, Bax, Bid, cytochrome c, and mitogen-activated protein kinases (MAPKs) was evaluated by Western blotting.Results:Quercetin had a protective effect by reducing both intracellular ROS overproduction and glutamate-mediated Ca2+ influx. These effects were due to the downregulation of several apoptosis-related biochemical markers. Calpain expression was reduced and spectrin cleavage was inhibited by quercetin in glutamate-exposed HT22 cells. Disruption of the mitochondrial membrane potential (ΔΨm), activation of the pro-apoptotic proteins Bid and Bax, and cytochrome c release in response to glutamate-induced oxidative stress were reduced. Quercetin also suppressed phosphorylation of MAPKs.Conclusion:This is the first report on the detailed mechanisms of the protective effect of quercetin on HT22 cells. Onion extract and quercetin may be useful for preventing or treating neurodegenerative disorders.

활성산소 소거활성에 따른 꾸지뽕잎 추출물의 신경세포 보호 효과

In an attempt to identify the neuroprotective effect of Cudrania tricuspidata (CT) leaves against ROS (reactive oxygen species)-induced oxidative stress in neuronal cells, the extracts from CT leaves were investigated using PC12 cells and N18-RE-105 cells. The methanolic and ethanolic extracts from CT were denoted as CTM (Cudrania tricuspidata Leaves methanolic extracts) and CTE (Cudrania tricuspidata Leaves ethanolic extracts), respectively. The neuroprotective effects of the extracts were measured by DCF-DA assay, MTT reduction assay, and LDH release assay. The PC12 cells exposed to <TEX>$H_2O_2$</TEX>-induced oxidative stress and the N18-RE-105 cells exposed to glutamate-induced oxidative stress were treated with various concentrations of CTM and CTE. The results, CTM treatments resulted in the induction of a dose-dependent protective effect in PC12 cells and N18-RE-105 cells. Interestingly, CTE also showed neuroprotective effect in PC12 cells and N18-RE-105 cells. Therefore, these results suggest that CTM and CTE could be a new potential candidate as neuroprotective agents against ROS-induced oxidative stress in neuronal cells.

Effects of Resveratrol and trans-3,5,4'-Trimethoxystilbene on Glutamate-Induced Cytotoxicity, Heme Oxygenase-1, and Sirtuin 1 in HT22 Neuronal Cells

Resveratrol (trans-3,5,4’-trihydroxystilbene) has received considerable attention recently for the potential neuroprotective effects in neurodegenerative disorders where heme oxygenase-1 (HO-1) and sirtuin 1 (SIRT1) represent promising therapeutic targets. Resveratrol has been known to increase HO-1 expression and SIRT1 activity. In this study, the effects of resveratrol and trans-3,5,4’-trimethoxystilbene (TMS), a resveratrol derivative, on cytotoxicity caused by glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation have been investigated by using murine hippocampal HT22 cells, which have been widely used as an in vitro model for investigating glutamate-induced neurotoxicity. Resveratrol protected HT22 neuronal cells from glutamateinduced cytotoxicity and increased HO-1 expression as well as SIRT1 activity in a concentration-dependent manner. Cytoprotec-tion afforded by resveratrol was partially reversed by the specific inhibition of HO-1 expression by HO-1 small interfering RNA and the nonspecific blockage of HO-1 activity by tin protoporphyrin IX, but not by SIRT1 inhibitors. Surprisingly, TMS, a resveratrol derivative with methoxyl groups in lieu of the hydroxyl groups, and trans-stilbene, a non-hydroxylated analog, failed to protect HT22 cells from glutamate-induced cytotoxicity and to increase HO-1 expression and SIRT1 activity. Taken together, our findings suggest that the cytoprotective effect of resveratrol was at least in part associated with HO-1 expression but not with SIRT1 activation and, importantly, that the presence of hydroxyl groups on the benzene rings of resveratrol appears to be necessary for cytoprotection against glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation in HT22 neuronal cells.

Neuroprotective Effects of Dehydroglyasperin C through Activation of Heme Oxygenase-1 in Mouse Hippocampal Cells

Licorice, the root of the Glycyrrhiza species ( Glycyrrhiza uralensis Fisher), is known to have antioxidant, anti-inflammatory, antiviral, and antitumor properties. The objective of this study is to explore the neuroprotective effect of dehydroglyasperin C (DGC) against glutamate-induced oxidative stress in mouse hippocampal HT22 cells. DGC significantly reduced cytotoxicity and reactive oxygen species (ROS) generation induced by glutamate in HT22 cells, whereas DGC did not restore glutathione depletion caused by glutamate. In addition, it was further investigated whether DGC affected the expression of heme oxygenase (HO)-1, one of the major cellular antioxidant defense systems, and it was found that DGC dose-dependently increased HO-1 expression. DGC-mediated cytoprotection of HT22 neuronal cells from glutamate insult was abrogated by either HO-1 inhibitor (Tin protoporphyrin, SnPP) or AKT inhibitor (LY294002). In conclusion, the present results demonstrate for the first time that DGC protects neuronal cells against glutamate-induced oxidative injury through the induction of HO-1 expression, which is, in turn, activated maybe through Nrf2-Keap1 and PI3K/AKT signaling pathways.

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.

DGC protects neuronal cells against glutamate‐induced oxidative injury through the induction of HO‐1 expression

Oxidative stress has been related to the development of chronic neurodegenerative diseases and acute neurological injury. Licorice, the root of the Glycyrrhiza species (Glycyrrhiza uralensis Fisher), is known to have antioxidant, anti‐inflammatory, anti‐viral, and anti‐tumor properties. The object of this study is to explore the neuroprotective effects of dehydroglyasperin C (DGC) against glutamate‐induced oxidative stress in the mouse hippocampal HT22 cells. DGC significantly reduced cytotoxicity and reactive oxygen species (ROS) generation induced by glutamate in HT22 cells, whereas DGC did not restore glutathione depletion caused by glutamate. In addition, we investigated further whether DGC affected the expression of heme oxygenase (HO)‐1, one of major cellular antioxidant defense system, and found that DGC dose‐dependently increased HO‐1 expression. DGC‐mediated cytoprotection of HT22 neuronal cells from glutamate insult was abrogated by either Tin protophorphyrin (SnPP), an inhibitor of HO‐1, or AKT inhibitor (LY294002). In conclusion, the present results demonstrate for the first time that DGC protects neuronal cells against glutamate‐induced oxidative injury through the induction of HO‐1 expression which is, in turn, activated by PI3k/AKT signaling pathway.

New Flavonoid Glycosides from Pyrrosia petiolosa and their Neuroprotective Activities Against Glutamate-Induced Oxidative Stress in HT22 Hippocampal Cells

New Flavonoid Glycosides from Pyrrosia petiolosa and their Neuroprotective Activities Against Glutamate-Induced Oxidative Stress in HT22 Hippocampal Cells

The combination of organoselenium compounds and guanosine prevents glutamate-induced oxidative stress in different regions of rat brains

This study was designed to investigate the protective effects of the combination of guanosine and 2 organoselenium compounds (ebselen and diphenyl diselenide) against glutamate-induced oxidative stress in different regions of rat brains. Glutamate caused an increase in reactive oxygen species (ROS) generation and a decrease in [3H]-glutamate uptake in striatal, cortical, and hippocampal slices. Guanosine, ebselen, and diphenyl diselenide prevented glutamate-induced ROS production in striatal, cortical and hippocampal slices. The combination of guanosine with organoselenium compounds was more effective against glutamate-induced ROS production than the individual compounds alone. Guanosine prevented [3H]-glutamate uptake inhibition in striatal, cortical, and hippocampal slices. Thus, protection against the harmful effects of glutamate is possibly due to the combination of the antioxidant properties of organoselenium compounds and the stimulatory effect of guanosine on glutamate uptake. In conclusion, the combination of antioxidants and glutamatergic system modulators could be considered a potential therapy against the prooxidant effects of glutamate.

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 alteration of components in the fermented Hwangryunhaedok-tang and its neuroprotective activity.

Background:Hwangryunhaedok-tang is a traditional herbal prescription that has sedative activity, hypotensive and anti-bacterial effects.Objective:In this study, we investigated the alteration of contents of components in Hwangryunhaedok-tang, antioxidant activity and neuroprotective activity by fermentation with Lactobacillus acidophilus KFRI 128.Materials and Methods:Contents of three marker compounds (geniposide, berberine and palmatine) and unknown compounds in the Hwangryunhaedok-tang (HR) and the fermented Hwangryunhaedok-tang (FHR) were measured and compared using the established high-performance liqued chromatograph coupled with a photodiode (HPLC-DAD) method. The antioxidant activity of HR and FHR were determined by DPPH free radical and hydrogen peroxide (H2O2) scavenging assay. Also, the neuroprotective activities of HR and FHR against glutamate-induced oxidative stress in a mouse hippocampal cell line (HT22) were evaluated by MTT assay.Results:The contents of geniposide and palmatine were decreased but the content of berberine was increased in the FHR. And the contents of unknown compounds (1), (2), (3), (4) and (5) in the HR were altered by fermentation. Electron donating activity (EDA, %) value of FHR was higher than HR for DPPH radical scavenging activity and H2O2 scavenging activity, respectively. In the MTT assay, FHR showed more potent neuroprotective activity than HR by 513.90%.Conclusion:The FHR using microorganism could convert compounds in HR and enhance the antioxidant and neuroprotective activity.

Neuroprotective Diarylheptanoids from the Leaves and Twigs ofJuglans sinensisagainst Glutamate-Induced Toxicity in HT22 Cells

A new diarylheptanoid, juglanin C(1), was isolated from the 80 % methanolic extract of the leaves and twigs of JUGLANS SINENSIS with three known diarylheptanoids, juglanin A(2), juglanin B (3), and (5R)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1(4-hydroxyphenyl)-3-heptanone (4), using bioactivity-guided fractionation and chromatographic techniques. Among the isolated diarylheptanoids, compounds 1 and 2 significantly showed neuroprotective activities against glutamate-induced toxicity in HT22 cells. These two diarylheptanoids significantly reduced the overproduction of cellular peroxide in glutamate-injured HT22 cells. Moreover, these two diarylheptanoids significantly maintained antioxidative defense systems, including glutathione, glutathione reductase, and glutathione peroxidase, under glutamate-induced oxidative stress in HT22 cells.

Effect of Neuroprotective Flavonoids ofAgrimonia eupatoriaon Glutamate-Induced Oxidative Injury to HT22 Hippocampal Cells

A methanolic extract of Agrimonia eupatoria (Rosaceae) significantly attenuated glutamate-induced oxidative stress in HT22 hippocampal cells. A new flavonoid, characterized as kaempferol 3-O-beta-D-(2''-O-acetyl-6''-(E)-p-coumaroyl)-glucopyranoside (2''-acetyl-tiliroside (1), was isolated from the methanolic extract of A. eupatoria stems together with nine known flavonoids. Compounds 4, 7, 8 and 9 all showed a neuroprotective effect on glutamate-induced toxicity in HT22 cells.

Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons

Glutamate toxicity involves increases in intracellular calcium levels and enhanced formation of reactive oxygen species (ROS) causing neuronal dysfunction and death in acute and chronic neurodegenerative disorders. The molecular mechanisms mediating glutamate-induced ROS formation are, however, still poorly defined. Using a model system that lacks glutamate-operated calcium channels, we demonstrate that glutamate-induced acceleration of ROS levels occurs in two steps and is initiated by lipoxygenases (LOXs) and then significantly accelerated through Bid-dependent mitochondrial damage. The Bid-mediated secondary boost of ROS formation downstream of LOX activity further involves mitochondrial fragmentation and release of mitochondrial apoptosis-inducing factor (AIF) to the nucleus. These data imply that the activation of Bid is an essential step in amplifying glutamate-induced formation of lipid peroxides to irreversible mitochondrial damage associated with further enhanced free radical formation and AIF-dependent execution of cell death.

Decursinol and decursin protect primary cultured rat cortical cells from glutamate-induced neurotoxicity

We previously reported six neuroprotective decursinol derivatives, coumarins from Angelica gigas (Umbelliferae) roots. To elucidate the action patterns of decursinol derivatives, we investigated the neuroprotective effects of decursinol and decursin, which showed highly significant activity and were major constituents of A. gigas, using primary cultures of rat cortical cells in-vitro. At concentrations of 0.1-10.0 microM, both decursinol and decursin exerted a significant neuroprotective activity pretreatment and throughout treatment. In addition, decursin had a neuroprotective impact in the post-treatment paradigm implying that decursin might possess different action mechanisms from that of decursinol in the protection of neurons against glutamate injury. Both decursinol and decursin effectively reduced the glutamate-induced increased intracellular calcium ([Ca(2+)](i)) in cortical cells, suggesting that these two coumarins may exert neuroprotection by reducing calcium influx by overactivation of glutamate receptors. This suggestion was supported by the result that decursinol and decursin protected neurons against kainic acid (KA)-induced neurotoxicity better than against that induced by N-methyl-D-aspartate (NMDA). Moreover, both decursinol and decursin significantly prevented glutamate-induced decreases in glutathione, a cellular antioxidant, and glutathione peroxidase activity. In addition, both compounds efficiently reduced the overproduction of cellular peroxide in glutamate-injured cortical cells. These results suggested that both decursinol and decursin protected primary cultured rat cortical cells against glutamate-induced oxidative stress by both reducing calcium influx and acting on the cellular antioxidative defence system. Moreover, decursin is considered to probably have a different action mechanism from that of decursinol in protecting cortical cells against glutamate injury.

Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage

To investigate the mechanisms of excitotoxic effects of glutamate on human neuroblastoma SH-SY5Y cells. SH-SY5Y cell viability was measured by MTT assay. Other damaged profile was detected by lactate dehydrogenase (LDH) release and by 4', 6-diamidino-2-phenylindole (DAPI) staining. The cytosolic calcium concentration was tested by calcium influx assay. The glutamate-induced oxidative stress was analyzed by cytosolic glutathione assay, superoxide dismutase (SOD) assay and extracellular malondialdehyde (MDA) assay. Glutamate treatment caused damage in SH-SY5Y cells, including the decrease of cell viability, the increase of LDH release and the alterations of morphological structures. Furthermore, the concentration of cytoplasmic calcium in SH-SY5Y cells was not changed within 20 min following glutamate treatment, while cytosolic calcium concentration significantly increased within 24 h after glutamate treatment, which could not be inhibited by MK801, an antagonist of NMDA receptors, or by LY341495, an antagonist of metabotropic glutamate receptors. On the other hand, oxidative damage was observed in SH-SY5Y cells treated with glutamate, including decreases in glutathione content and SOD activity, and elevation of MDA level, all of which could be alleviated by an antioxidant Tanshinone IIA (Tan IIA, a major active ingredient from a Chinese plant Salvia Miltiorrhiza Bge). Glutamate exerts toxicity in human neuroblastoma SH-SY5Y cells possibly through oxidative damage, not through calcium homeostasis destruction mediated by NMDA receptors.

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.

ESP‐102, a combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, protects against glutamate‐induced toxicity in primary cultures of rat cortical cells

It was reported previously that ESP-102, a combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, significantly improved scopolamine-induced memory impairment in mice and protected primary cultured rat cortical cells against glutamate-induced toxicity. To corroborate this effect, the action patterns of ESP-102 were elucidated using the same in vitro system. ESP-102 decreased the cellular calcium concentration increased by glutamate, and inhibited the subsequent overproduction of cellular nitric oxide and reactive oxygen species to the level of control cells. It also preserved cellular activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase reduced in the glutamate-injured neuronal cells. While a loss of mitochondrial membrane potential was observed in glutamate treated cells, the mitochondrial membrane potential was maintained by ESP-102. These results support that the actual mechanism of neuroprotective activity of ESP-102 against glutamate-induced oxidative stress might be its antioxidative activity.

12/15‐Lipoxygenase targets neuronal mitochondria under oxidative stress

12/15-Lipoxygenase (12/15-LOX) is an important mediator of brain injury following experimental stroke in rodents. It contributes to neuronal death, but the underlying mechanism remains unclear. We demonstrate here that in neuronal HT22 cells subjected to glutamate-induced oxidative stress, 12/15-LOX damages mitochondria, and this represents the committed step that condemns the cell to die. Importantly these events, including breakdown of the mitochondrial membrane potential, the production of reactive oxygen species, and cytochrome c release, can all be replicated by incubation of 12/15-LOX with mitochondria in vitro, without the need to add other cytosolic factors. Proteasome activity is required downstream of mitochondrial damage to complete the cell death cascade, but proteasome inhibition is only partially protective. These findings position 12/15-LOX as the central executioner in an oxidative stress-related neuronal death program.