Protective effect of casuarinin against glutamate-induced apoptosis in HT22 cells through inhibition of oxidative stress-mediated MAPK phosphorylation

Glutamate is the major excitatory neurotransmitter in the central nervous system and is involved in oxidative stress during neurodegeneration. In the present study, casuarinin prevented glutamate-induced HT22 murine hippocampal neuronal cell death by inhibiting intracellular reactive oxygen species (ROS) production. Moreover, casuarinin reduced chromatin condensation and annexin-V-positive cell production induced by glutamate. We also confirmed the underlying protective mechanism of casuarinin against glutamate-induced neurotoxicity. Glutamate markedly increased the phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38, which are crucial in oxidative stress-mediated neuronal cell death. Conversely, treatment with casuarinin diminished the phosphorylation of ERK1/2 and P38. In conclusion, the results of this study suggest that casuarinin, obtained from natural products, acts as potent neuroprotective agent by suppressing glutamate-mediated apoptosis through the inhibition of ROS production and activation of the mitogen activated protein kinase (MAPK) pathway. Thus, casuarinin can be a potential therapeutic agent in the treatment of neurodegenerative diseases.     

Sanguiin H-11 from Sanguisorbae radix protects HT22 murine hippocampal cells against glutamate-induced death

Excessive glutamate level induces neuronal death in acute brain injuries and chronic neurodegenerative diseases. Natural compounds from medicinal and food plants have been attracting interest as a treatment for neurological disorders. Sanguiin H-11 (SH-11), a hydrolysable ellagitannin, inhibits neutrophil movement and nitric oxide -production. However, its neuroprotective effect has not been studied. Therefore, the present study examined the protective effect of SH-11 from Sanguisorbae radix and its mechanism against glutamate-induced death in HT22 cells. Our results showed that SH-11 possessed a strong antioxidant activity and prevented glutamate-induced death in HT22 cells. As a strong antioxidant, SH-11 significantly reduced glutamate-induced increases in intracellular reactive oxygen species accumulation and calcium ion influx. Western blotting analysis showed that glutamate-induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinases 1/2, c-Jun N-terminal kinase, and p38, was significantly decreased by SH-11. Furthermore, SH-11 significantly decreased the number of annexin V-positive HT22 cells, which is indicating apoptotic cell death. In conclusion, our results suggested that SH-11 exerted a potent neuroprotective activity against glutamate-mediated apoptotic cell death by inhibiting oxidative stress-mediated MAPK activation.     

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.     

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.     

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.     

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl¹-NH-Aryl² scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H₂O₂ induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H₂O₂ in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.     

Redox active calcium ion channels and cell death

Calcium plays a key role in both apoptotic and necrotic cell death. Emptying of intracellular calcium stores and/or alteration in intracellular calcium levels can modulate cell death in almost all cell types. These calcium fluxes are determined by the activity of membrane channels normally under tight control. The channels may be ligand activated or voltage dependent as well as being under the control of affector molecules such as calmodulin. It has become increasingly apparent that many calcium channels are affected by reactive oxygen or reactive nitrogen species; ROS/RNS. This may be part of the normal signaling pathways in the cell or by the action of exogenously generated ROS or RNS often by toxins. This review covers the recent literature on the activity of these redox active channels as related to cell death.     

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.     

Glutamate-induced cell death in neuronal HT22 cells is attenuated by extracts from St. John''s wort (Hypericum perforatum L.)

Glutamate-induced cell death of hippocampal HT22 cells is a model system for neuronal disorders due to depletion of glutathione levels and increase of intracellular reactive oxygen species. Standardized extracts of Hypericum perforatum (HPE) contain flavonoids known for antioxidative properties. In the above model, cytoprotective effects at a concentration of 0.05% HPE by attenuation of calcium fluxes and cellular energy statuses are reported.     

Antagonizing TGF-beta induced liver fibrosis by a retinoic acid derivative through regulation of ROS and calcium influx

Transforming growth factor-beta1 (TGF-β1) mediates the regulation of extracellular matrix via reactive oxygen species (ROS) and calcium influx, both are activators of hepatic stellate cells (HSC) which play a critical role in hepatic fibrogenesis. Hence one can use ROS assay as the main screening tool for molecules that might antagonize the process of liver fibrosis. A retinoic acid derivative isolated from the mycelium of Phellinus linteus that down-regulates ROS generation and calcium influx in HSC-T6 cells was thus obtained in our screening process. The retinoic acid derivative also reverses an early liver fibrosis, as assayed by liver contents of hydroxyproline, α-smooth muscle actin (α-SMA), and collagen 1A2, in an early liver fibrosis model we established previously where an inducible expression vector containing a TGF-β gene was hydrodynamically transferred into a testing animal. Retinoic acid derivative thus acts both in vitro and in vivo to prevent liver fibrosis at an early phase.     

Rice OsSIPK and its orthologs: A “central master switch” for stress responses

Mitogen-activated protein kinase (MAPK) plays a central role in controlling a vast array of plant biochemical and physiological processes. It is regulated by a characteristic phosphorelay system in which a series of three kinases phosphorylate and activate each other. Over the past years, several plants MAPKs have been identified and characterized. Of these, rice OsSIPK (Salicylic acid (SA)-Induced Protein Kinase) and its orthologs in other plants are of particular interest. A large body of evidence demonstrates the involvement of SIPKs in fine-tuned regulation of the plant responses to ozone, wounding, SA, and jasmonic acid (JA). Interestingly, their function appears to be conserved across reference plants, such as rice, tobacco, and Arabidopsis. In this minireview, we discuss the recent progress on rice OsSIPK and its orthologs as a “central master switch” for mediating plant responses against ozone, wounding, and JA as examples.     

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.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.