Mitochondrial dynamics following global cerebral ischemia

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.     

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm)

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH_m). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.     

Mitochondria-derived reactive oxygen species mediate caspase-dependent and -independent neuronal deaths

Mitochondrial dysfunction and oxidative stress are implicated in many neurodegenerative diseases. Mitochondria-targeted drugs that effectively decrease oxidative stress, protect mitochondrial energetics, and prevent neuronal loss may therefore lend therapeutic benefit to these currently incurable diseases. To investigate the efficacy of such drugs, we examined the effects of mitochondria-targeted antioxidants MitoQ₁₀ and MitoE₂ on neuronal death induced by neurotrophin deficiency. Our results indicate that MitoQ₁₀ blocked apoptosis by preventing increased mitochondria-derived reactive oxygen species (ROS) and subsequent cytochrome c release, caspase activation, and mitochondrial damage in nerve growth factor (NGF)-deprived sympathetic neurons, while MitoE₂ was largely ineffective. In this paradigm, the most proximal point of divergence was the ability of MitoQ₁₀ to scavenge mitochondrial superoxide (O₂⁻). MitoQ₁₀ also prevented caspase-independent neuronal death in these cells demonstrating that the mitochondrial redox state significantly influences both apoptotic and nonapoptotic pathways leading to neuronal death. We suggest that mitochondria-targeted antioxidants may provide tools for delineating the role and significance of mitochondrial ROS in neuronal death and provide a new therapeutic approach for neurodegenerative conditions involving trophic factor deficits and multiple modes of cell death.     

4-hydroxynonenal increases neuronal susceptibility to oxidative stress

Increased levels of reactive oxygen species occur in neurodegenerative disorders and may promote neuron death. The lipid peroxidation product 4-hydroxynonenal (HNE) is increased in neurons following oxidative stress and promotes neuron death in vitro and in vivo. The present study examined the possibility that HNE can increase neuron vulnerability to oxidative stress. Application of low concentrations of HNE (50-500 nM) increased neuron death induced by beta-amyloid or glutamate when added within 3 hr of injury. In addition, treatment with HNE exacerbated mitochondrial reactive oxygen species formation and loss of mitochondrial membrane potential in response to beta-amyloid and glutamate. The ability to exacerbate oxidative stress, mitochondrial dysfunction, and neuron death appears to be specific to HNE, because application of other lipid peroxidation products had no effect. These data indicate a role for low levels of HNE in promoting reactive oxygen species accumulation and neuron degeneration by altering mitochondrial homeostasis. In addition, the present study indicates a possible mechanism for reactive oxygen species and lipid peroxidation toxicity in neurodegenerative conditions.     

Bid mediates fission,membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death

Mitochondria are highly dynamic organelles that undergo permanent fusion and fission, a process that is important for mitochondrial function and cellular survival. Emerging evidence suggests that oxidative stress disturbs mitochondrial morphology dynamics, resulting in detrimental mitochondrial fragmentation. In particular, such fatal mitochondrial fission has been detected in neurons exposed to oxidative stress, suggesting mitochondrial dynamics as a key feature in intrinsic death pathways. However, the regulation of mitochondrial fission in neurons exposed to lethal stress is largely unknown. Here, we used a model of glutamate toxicity in HT-22 cells for investigating mitochondrial fission and fusion in neurons exposed to oxidative stress. In these immortalized hippocampal neurons, glutamate induces glutathione depletion and increased formation of reactive oxygen species (ROS). Glutamate toxicity resulted in mitochondrial fragmentation and peri-nuclear accumulation of the organelles. Further, mitochondrial fission was associated with loss of mitochondrial outer membrane potential (MOMP). The Bid-inhibitor BI-6c9 prevented MOMP and mitochondrial fission, and protected the cells from cell death. In conclusion, oxidative stress induced by glutamate causes mitochondrial translocation of Bid thereby inducing mitochondrial fission and associated mitochondrial cell death pathways. Inhibiting regulators of pathological mitochondrial fragmentation is proposed as an efficient strategy of neuroprotection.     

Neuroprotective Effect of Baicalein on Hydrogen Peroxide-Mediated Oxidative Stress and Mitochondrial Dysfunction in PC12 Cells

Oxidative stress plays an important role in many neurodegenerative disorders. In this study, the effect of baicalein, a natural flavonoid isolated from the root of Scutellaria baicalensis G., on hydrogen peroxide (H₂O₂)-induced cytotoxicity in PC12 cells were investigated. Exposure of PC12 cells to 0.15 mM H₂O₂ for 20 min induced a significant decrease in cell viability accompanied by increased oxidative stress, mitochondrial dysfunction, downregulation of Bcl-2, upregulation of Bax, and cell apoptosis. Pretreatment of PC12 cells with baicalein inhibited H₂O₂-induced cell viability loss, intracellular reactive oxygen species generation, and lipid peroxidation in a dose-dependent manner. Meanwhile, baicalein potentially inhibited H₂O₂-induced cell apoptosis characterized with the DNA fragment. And the mitochondrial pathway involving the mitochondrial dysfunction associated with cell apoptosis including membrane potential loss, the release of cytochrome c, the downregulation of Bcl-2, upregulation of Bax induced by H₂O₂ were also abrogated in the presence of baicalein. Taken together, these results suggest that baicalein can block H₂O₂-induced apoptosis by prevention of oxidative stress as well as regulation of Bcl-2 family members and suppression of mitochondria dysfunction, which might be beneficial for the treatment of oxidative stress in aging and age-associated neurodegenerative diseases.     

Protective Effect of Melatonin on Methamphetamine-Induced Apoptosis in Glioma Cell Line

Methamphetamine (METH) is a highly addictive drug causing neurodegenerative diseases. METH has been known to be neurotoxic by inducing oxidative stress, free radical, and pro-inflammatory cytokines. Previous studies have shown that METH could induce neuron and glial cell death, especially inducing glial cell-mediated neurotoxicity that plays a critical role in stress-induced central nervous system damage. Therefore, the aim of the present study is to explore the mechanisms of METH-induced cell death in the glial cell. METH-induced glial cells death is mediated via mitochondrial damage pathway. METH activates the upregulation of the Bax, cytochrome c, cleavage caspase 9 and 3 proteins, and downregulation of Bcl-X_L protein in cascade. Pretreatment with melatonin, a neurohormone secreted by the pineal gland, effectively reduced glial cell death. Moreover, melatonin increased the Bcl-X_L/Bax ratio but reduced the level of cytochrome c, cleavage caspase 9 and 3 proteins. Therefore, these results demonstrated that melatonin could reduce the cytotoxic effect of METH by decreasing the mitochondrial death pathway activation in glial cells. This outcome suggests that melatonin might be beneficial as the neuroprotection in neurodegenerative diseases caused by METH or other pathogens.     

Elevation of mitochondrial glutathione by gamma-glutamylcysteine ethyl ester protects mitochondria against peroxynitrite-induced oxidative stress

Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders by directing neurons to cell death. Protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying onset or progression of these diseases. We have investigated the ability of gamma-glutamylcysteine ethyl ester (GCEE) to upregulate mitochondrial glutathione (GSH) in vivo or in vitro and protect against subsequent in vitro peroxynitrite (ONOO-) damage. Mitochondria pretreated in vitro with GCEE were protected against oxidative damage induced by peroxynitrite, as assessed by mitochondrial swelling, changes in mitochondrial membrane potential, 3-nitrotyrosine formation, protein carbonyl formation, and cytochrome c release. Loss of mitochondrial function in neuronal cell cultures by the oxidants 2,2,'Azobis(2-amidino-propane)dihydrochloride (AAPH) and ONOO- was ameliorated by treatment with GCEE. In vivo studies showed that mitochondria isolated from animals injected intraperitoneally with GCEE were protected partially against oxidative modifications induced by ONOO-. Taken together, these results suggest that GCEE may be effective in increasing mitochondrial GSH and may be prove to have therapeutic relevance in neurodegenerative disorders associated with oxidative stress and mitochondrial dysfunction.     

Metabolic Alterations and the Protective Effect of Punicalagin Against Glutamate-Induced Oxidative Toxicity in HT22 Cells

Oxidative stress is involved in many neurological diseases, including Alzheimer’s disease. Punicalagin (PC) is a hydrolysable polyphenol derived from Punica granatum and a potent antioxidant. In this study, the neuroprotective effect of PC on glutamate-induced oxidative stress was evaluated in the mouse hippocampal cell line, HT22. PC treatment protected HT22 cells from glutamate-induced cell death in a concentration-dependent manner, potentially attenuated glutamate-induced intracellular reactive oxygen species (ROS) and restored the mitochondrial membrane depolarization. Metabolic alterations after glutamate-induced oxidative stress and the protective effect of PC were evaluated with HPLC and GC-MS profiling methods with multivariate statistical analyses. Alterations in ten metabolites were identified, including amino acids, aspartic acid, asparagine, threonine, anserine, cysteine, tryptophan, lysine, as well as fatty acids palmitic acid, stearic acid, and palmitoleic acid. Metabolic pathway analysis revealed the involvement of multiple affected pathways, such as cysteine and methionine metabolism, tryptophan metabolism, alanine, aspartate, and glutamate and fatty acid oxidation. These results clearly demonstrate that PC is a promising therapeutic agent for oxidative stress-associated diseases.     

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm)

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.     

α-Synuclein Protects Neurons from Apoptosis Downstream of Free-Radical Production Through Modulation of the MAPK Signalling Pathway

α-Synuclein is a pre-synaptic chaperone and its accumulation contributes to differential cell loss in Parkinson’s disease. Cytoplasmic expression of α-synuclein can directly modulate apoptotic pathways and contribute to cell survival, whereas induced over-expression of the protein causes oxidative stress through mitochondrial and cytosolic free-radical production. This study aimed to clarify the contribution of endogenous α-synuclein to oxidative stress and its association with cell death. Primary cortical neurons were derived from α-synuclein knock-out (Snca-/-) and wild-type (C57BL/6; WT) mice and treated with in vitro models of oxidative-stress, complex I inhibition and excitotoxicity. Mitochondrial free radical production was determined in isolated mitochondria derived from each mouse strain. Snca-/- derived cortical cultures were more susceptible (P < 0.05) to oxidative-stress, but not excitotoxicity. This result was determined by significant increases in cell death (Propidium-Iodide staining) after 6 h treatment in Snca-/- (45 % ± 2.7 SEM), relative to WT (33 % ± 3.9 SEM) cultures. α-Synuclein also confers significant (P < 0.05) resistance to low-dose (5 nM) rotenone toxicity, with a twofold reduction in cell death in WT, compared with Snca-/- cortical neurons. The expression of α-synuclein had no effect on cortical glutathione levels, or the production of reactive oxygen intermediates in isolated mitochondria. These data indicate that endogenous levels of α-synuclein confer resistance to oxidative stress downstream of free radical production and scavenging. The current data suggest that α-synuclein prevents cytochrome c release and apoptosis through inhibition of the MAPK signalling pathway.     

Induction of cytochrome c-mediated apoptosis by amyloid beta 25-35 requires functional mitochondria

Accumulating data suggest a central role for mitochondria and oxidative stress in neurodegenerative apoptosis. We previously demonstrated that amyloid-beta peptide 25-35 (Abeta 25-35) toxicity in cultured cells is mediated by its effects on functioning mitochondria. In this study, we further explored the hypothesis that Abeta 25-35 might induce apoptotic cell death by altering mitochondrial physiology. Mitochondria in Ntera2 (NT2 rho+) human teratocarcinoma cells exposed to either staurosporine (STS) or Abeta 25-35 were found to release cytochrome c, with subsequent activation of caspases 9 and 3. However, NT2 cells depleted of mitochondrial DNA (rho0 cells), which maintain a normal mitochondrial membrane potential (Deltapsi(m)) despite the absence of a functional electron transport chain (ETC), demonstrated cytochrome c release and caspase activation only with STS. We further observed increased reactive oxygen species (ROS) production and decreased reduced glutathione (GSH) levels in rho+ and rho0 cells treated with STS, but only in rho+ cells treated with Abeta 25-35. We conclude that under in vitro conditions, Abeta can induce oxidative stress and apoptosis only when a functional mitochondrial ETC is present.     

Hippocampal Neurons Exposed to the Environmental Contaminants Methylmercury and Polychlorinated Biphenyls Undergo Cell Death via Parallel Activation of Calpains and Lysosomal Proteases

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants commonly found as contaminants in the same food sources. Even though their neurotoxic effects are established, the mechanisms of action are not fully understood. In the present study, we have used the mouse hippocampal neuronal cell line HT22 to investigate the mechanisms of neuronal death induced by MeHg, PCB 153, and PCB 126, alone or in combination. All chemicals induced cell death with morphological changes compatible with either apoptosis or necrosis. Mitochondrial functions were impaired as shown by the significant decrease in mitochondrial Ca²⁺ uptake capacity and ATP levels. MeHg, but not the PCBs, induced loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. Also, pre-treatment with the antioxidant MnTBAP was protective only against cell death induced by MeHg. While caspase activation was absent, the Ca²⁺-dependent proteases calpains were activated after exposure to MeHg or the selected PCBs. Furthermore, lysosomal disruption was observed in the exposed cells. Accordingly, pre-treatment with the calpain specific inhibitor PD150606 and/or the cathepsin D inhibitor Pepstatin protected against the cytotoxicity of MeHg and PCBs, and the protection was significantly enhanced when the two inhibitors were combined. Simultaneous exposures to lower doses of MeHg and PCBs suggested mostly antagonistic interactions. Taken together, these data indicate that MeHg and PCBs induce caspase-independent cell death via parallel activation of calpains and lysosomal proteases, and that in this model oxidative stress does not play a major role in PCB toxicity.     

Lamotrigine inhibition of rotenone- or 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.     

Nanomolar Naloxone Attenuates Neurotoxicity Induced by Oxidative Stress and Survival Motor Neuron Protein Deficiency

Oxidative stress and survival motor neuron (Smn) protein deficiency are the major causes of motor neuronal death. Naloxone exhibits neuroprotection against ischemic stroke and anti-inflammation. In this study, we determined whether nanomolar naloxone provides neuroprotection under oxidative stress (H₂O₂) and Smn deficiency in a motor neuron-like cell line, NSC34. In H₂O₂-treated NSC34 cells, naloxone (1–10 nM) increased cell survival and mitochondria membrane potential. In addition, naloxone decreased NADPH oxidase (NOX) 2 activation, reactive oxygen species production and oxygen consumption rate. Moreover, naloxone increased anti-apoptotic Bcl-2 expression, attenuated apoptotic protein (Bax, cytochrome c, and caspase) expression and decreased apoptotic death. Furthermore, naloxone also increased Smn mRNA and protein expression. In Smn knockdown NSC34 cells, Smn deficiency significantly increased H₂O₂ cytotoxicity. Naloxone exhibited neuroprotection at higher concentrations in Smn knockdown NSC34 cells than in control cells. In conclusion, naloxone attenuated neurotoxicity induced by H₂O₂ and Smn deficiency. Our findings also revealed the involvement of Smn protein in naloxone protection and oxidative stress-related neurotoxicity.     

Mitochondrial dysfunction induced by oxidative stress in the brains of hamsters infected with the 263 K scrapie agent

Scrapie, one of the prion diseases, is a transmissible neurodegenerative disease of sheep and other animals. Clinical symptoms of prion diseases are characterized by a long latent period, followed by progressive ataxia, tremor, and death. To study the induction of neurodegeneration during scrapie infection, we have analyzed the activities of various antioxidant enzymes and mitochondrial enzymes in cerebral cortex, brain stem, and cerebellum of scrapie-infected hamsters. The activity of mitochondrial Mn-superoxide dismutase (SOD) was decreased, while the activities of cytosolic Cu/Zn-SOD and catalase were not altered in infected brains. The activities of glutathione peroxidase and glutathione reductase were increased in scrapie-infected hamsters. The decreased activity of Mn-SOD might result in increasing oxidative stress in the mitochondria of infected brain; this concept is supported by our findings of a high level of lipid peroxidation, and low levels of ATPase and cytochrome c oxidase activity in the infected cerebral mitochondria. In addition, structural abnormalities of mitochondria have been observed in the neurons of hippocampus and cerebral cortex of infected brain. These results suggest that mitochondrial dysfunction caused by oxidative stress gives rise to neurodegeneration in prion disease. Received: 7 October 1997 / Revised, accepted: 26 February 1998     

Metabotropic glutamate receptor regulation of neuronal cell death

The metabotropic glutamate receptors (mGluRs) are a family of glutamate-sensitive receptors that regulate neuronal function separately from the ionotropic glutamate receptors. By coupling to guanosine nucleotide-binding proteins (G proteins), mGluRs are able to regulate neuronal injury and survival, likely through a series of downstream protein kinase and cysteine protease signaling pathways that affect mitochondrial regulated programmed cell death (PCD). The physiological relevance of this system is supported by evidence that mGluRs are associated with cell survival in several central nervous system neurodegenerative diseases. Evidence is presented that mGluRs are also able to prevent PCD in the peripheral nervous system, and that this may provide a novel mechanism for treatment of diabetic neuropathy. In dorsal root ganglion (DRG) neurons, a high glucose load increases generation of reactive oxygen species (ROS), destabilizes the inner mitochondrial membrane potential (Deltapsi(M)), induces cytochrome c release from the mitochondrial intermembrane space, and induces downstream activation of caspases. In high-glucose conditions, the group II metabotropic glutamate agonist N-acetylaspartylglutamate (NAAG) blocks caspase activation and is completely reversed by the mGluR3 antagonist (S)-alpha-ethylglutamic acid (EGLU). Furthermore, the direct mGluR3 agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) prevents induction of ROS. Together these findings are consistent with an emerging concept that mGluRs may protect against cellular injury by regulating oxidative stress in the neuron. More complete understanding of the complex PCD regulatory pathways mediated by mGluRs will provide new therapeutic approaches for the treatment of a wide variety of neurodegenerative diseases.     

Dynamin-related protein 1 mediates mitochondria-dependent apoptosis in chlorpyrifos-treated SH-SY5Y cells

Recent studies have demonstrated that dynamin-related protein 1 (Drp1), a mitochondrial fission protein, mediates mitochondria-dependent apoptosis through mitochondrial division. However, little is known about the mechanism by which Drp1 modulates apoptosis in response to chlorpyrifos (CPF)-induced toxicity. In this study, we determined that CPF-induced mitochondrial apoptosis is mediated by Drp1 translocation in SH-SY5Y human neuroblastoma cells. Our results showed that CPF treatment induced intrinsic apoptosis by activating caspase-9, caspase-3, and cytochrome c release in SH-SY5Y cells. Cytosolic Drp1 translocated to the mitochondria in CPF-treated cells and was phosphorylated at Ser616. Treating cells with CPF induced the generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs). Inhibiting this ROS generation and MAPK activation abolished CPF-induced expression of phospho-Drp1. Furthermore, Drp1 was required for p53 to translocate to the mitochondria under CPF-induced oxidative stress. Treating cells with mitochondrial-division inhibitor-1 (mdivi-1), which blocks Drp1 translocation, increased the viability of CPF-treated cells by abrogating Drp1 translocation and caspase-3 activation. Specifically, pretreating cells with mdivi-1 inhibited Bax translocation to the mitochondria by blocking p53 signaling. Taken together, these data reveal a novel mechanism by which Drp1 activates mitochondrial-dependent apoptosis and indicate that inhibiting Dpr1 function can protect against CPF-induced cytotoxicity. We propose that inhibiting Drp1 is a possible therapeutic approach for pesticide-induced toxicity when hyperactivated Drp1 contributes to pathology.     

Paraquat-induced apoptotic cell death in cerebellar granule cells

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP⁺ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 μM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.