Cyanide preconditioning protects brain endothelial and NT2 neuron-like cells against glucotoxicity: role of mitochondrial reactive oxygen species and HIF-1α

The current study was undertaken to address the role of mitochondrial reactive oxygen species (ROS), and hypoxia inducible factor-1 alpha (HIF-1α) signaling pathway in the protection against high glucose levels in brain endothelial and NT2 neuron-like cells. Rat brain endothelial cells (RBE4) treated with non-toxic concentrations of cyanide (≤ 1 μM; 1 h) exhibited an increase in ROS levels, particularly hydrogen peroxide (H₂O₂). Cyanide also induced a modest mitochondrial depolarization, an increase in oxygen consumption and a structural (smaller mitochondria) and spatial (perinuclear region) reorganization of mitochondrial network. The stabilization and nuclear activation of HIF-1α in the presence of cyanide were also observed, which resulted in an increase in vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and erythropoietin (EPO) protein levels reflecting an adaptive response. Importantly, preconditioning induced by cyanide protected brain endothelial cells against high glucose-mediated damage by the prevention of apoptotic cell death. In mitochondrial DNA-depleted NT2 (NT2 ρ0) cells, cyanide (0.1 μM) was unable to stimulate ROS production and, consequently, protect against glucotoxicity. Conversely, in NT2 cells, the parental cells with functional mitochondria, cyanide significantly increased ROS levels protecting against high glucose-induced neuronal cell loss and activation of caspase-3. The free radical scavenger N-acetyl-L-cysteine and the specific HIF-1α inhibitor 2-methoxyestradiol completely abolished the protective effects of cyanide preconditioning. Altogether our results demonstrate that mitochondrial preconditioning induced by cyanide triggers a protective response mediated by mitochondrial ROS and HIF-1α activation and signaling, which render brain endothelial and neuronal cells resistant against glucotoxicity.     

Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines

There is an increasing recognition that mitochondrial dysfunction is associated with autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction and how mitochondrial abnormalities might interact with other physiological disturbances such as oxidative stress. Reserve capacity is a measure of the ability of the mitochondria to respond to physiological stress. In this study, we demonstrate, for the first time, that lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) have an abnormal mitochondrial reserve capacity before and after exposure to reactive oxygen species (ROS). Ten (44%) of 22 AD LCLs exhibited abnormally high reserve capacity at baseline and a sharp depletion of reserve capacity when challenged with ROS. This depletion of reserve capacity was found to be directly related to an atypical simultaneous increase in both proton-leak respiration and adenosine triphosphate-linked respiration in response to increased ROS in this AD LCL subgroup. In this AD LCL subgroup, 48-hour pretreatment with N-acetylcysteine, a glutathione precursor, prevented these abnormalities and improved glutathione metabolism, suggesting a role for altered glutathione metabolism associated with this type of mitochondrial dysfunction. The results of this study suggest that a significant subgroup of AD children may have alterations in mitochondrial function, which could render them more vulnerable to a pro-oxidant microenvironment as well as intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxins. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors.     

Iptakalim ameliorates MPP+‐induced astrocyte mitochondrial dysfunction by increasing mitochondrial complex activity besides opening mitoKATP channels

In addition to the established role of the mitochondrion in energy metabolism, regulation of cell death has been regarded as a major function of this organelle. Our previous studies have demonstrated that iptakalim (IPT), a novel ATP‐sensitive potassium channel (K_ATP channel) opener, protects against 1‐methyl‐4‐phenyl‐pyridinium ion (MPP⁺)–induced astrocyte apoptosis via mitochondria and mitogen‐activated protein kinase signal pathways. The present study aimed to investigate whether IPT can protect astrocyte mitochondria against MPP⁺‐induced mitochondrial dysfunction. We showed that treatment with IPT could ameliorate the inhibitory effect of MPP⁺ on mitochondrial respiration and ATP production by using mitochondrial complex I–supported substrates. IPT could also inhibit the increased production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria induced by MPP⁺. However, mitochondrial ATP‐sensitive potassium (mitoK_ATP) channel blocker 5‐hydroxydecanoate (5‐HD) could partly abolish all of the above effects of IPT. Because mitochondrial complex dysfunction impairs mitochondrial respiration and ATP production, a further experiment was undertaken to study the effects of IPT on the activity of mitochondrial complex (COX) I and COX IV. It was found that IPT inhibited the decrease in mitochondrial COX I and COX IV activity induced by MPP⁺, but 5‐HD failed to abolish these effects. Taken together, these findings suggest that IPT may protect astrocyte mitochondrial function by regulating complex activity in addition to opening mitoK_ATP channels. © 2008 Wiley‐Liss, Inc.     

Stat3 mediates LIF‐induced protection of astrocytes against toxic ROS by upregulating the UPC2 mRNA pool

Reactive oxygen species (ROS) have been implicated in various types of CNS damage, including stroke. We used a cultured astrocyte model to explore mechanisms of survival of CNS cells following ROS damage. We found that pretreatment with leukemia inhibitory factor (LIF) preserves astrocytes exposed to toxic levels of t‐BHP by inhibiting an increase in intracellular ROS following t‐BHP treatment. Astrocytes lacking functional Stat3 did not benefit from the pro‐survival or antioxidant effects of LIF. Inhibition of mitochondrial uncoupling protein 2 (UCP2) using a chemical inhibitor or siRNA abrogates the prosurvival effects of LIF, indicating a critical role for UCP2 in modulation of mitochondrial ROS production in survival following ROS exposure. LIF treatment of astrocytes results in increased UCP2 mRNA that is accompanied by an increase in Stat3 binding to the UCP2 promoter region. Although treatment with LIF alone did not increase UCP2 protein, a combination of LIF treatment and ROS stress led to increased UCP2 protein levels. We conclude that LIF protects astrocytes from ROS‐induced death by increasing UCP2 mRNA, allowing cells to respond to ROS stress by rapidly producing UCP2 protein that ultimately decreases endogenous mitochondrial ROS production. GLIA 2014;62:159–170     

Cytochrome c oxidase isoform IV‐2 is involved in 3‐nitropropionic acid‐induced toxicity in striatal astrocytes

Astrocyte mitochondria play an important role for energy supply and neuronal survival in the brain. Toxic and degenerative processes are largely associated with mitochondrial dysfunction. We, therefore, investigated the effect of 3‐nitropropionic acid (NPA), a mitochondrial toxin and in vitro model of Huntington's disease (HD), on mitochondrial function and viability of primary striatal astrocytes. Although NPA is known as an irreversible inhibitor of succinate dehydrogenase, we observed an increase of astrocyte ATP levels after NPA treatment. This effect could be explained by NPA‐mediated alterations of cytochrome c oxidase subunit IV isoform (COX IV) expression. The up‐regulation of COX isoform IV‐2 caused an increased enzyme activity at the expense of elevated mitochondrial peroxide production causing increased cell death. The application of a small interfering RNA against COX IV‐2 revealed the causal implication of COX isoform IV‐2 in NPA‐mediated elevation of oxidative stress and necrotic cell death. Thus, we propose a novel, additional mechanism of NPA‐induced cell stress and death which is based on structural and functional changes of astrocyte COX and which could indirectly impair neuronal survival. © 2009 Wiley‐Liss, Inc.     

The neurotoxic effect of astrocytes activated with toll-like receptor ligands

Toll-like receptors (TLRs) are key molecules in the innate immune system in the central nervous system. Although astrocytes are believed to play physiological roles in regulating neuronal activity and synaptic transmission, activated astrocytes may also be toxic to neurons. Here, we show that the ligands for TLRs 2, 4, 5 and 6 induce neuronal cell death in neuron–astrocytes co-cultures through the production of reactive oxygen species (ROS). Inhibition of ROS production by NADPH oxidase inhibitor apocynin significantly suppresses neuronal cell death. ROS induced in astrocytes via TLRs may be involved in neuroinflammation and a therapeutic target for neurotoxicity by activated astrocytes.     

Newborn neurons acquire high levels of reactive oxygen species and increased mitochondrial proteins upon differentiation from progenitors

A population of embryonic rat cortical cells cultured in the presence of FGF2 and having neuronal morphology expressed higher levels of reactive oxygen species (ROS) than did progenitor cells, astrocytes, and several cell lines of neuronal and non-neuronal origin. ROS were assessed using 5-(and-6)-chlormethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCF-DA), and high levels persisted in the presence of antioxidants or lowered levels of ambient oxygen. Greater than 95% of high ROS-producing cells, isolated by fluorescence-activated cell sorting, expressed the neuronal marker beta III tubulin. These cells did not incorporate BrdU or express nestin, unlike low ROS-producing cells, 99% of which exhibited both of these characteristics. Upon growth factor removal, low ROS-expressing cells differentiated into neurons and astrocytes and these neurons expressed high levels of ROS, indicating that ROS accumulation accompanies the differentiation of progenitors into neurons. ROS levels were decreased by added superoxide dismutase and catalase, suggesting that both superoxide and hydrogen peroxide contribute to the ROS signal. High ROS-expressing cells also contained higher levels of several mitochondrial respiratory chain components. Although ROS have been associated with conditions that lead to cell death, our results and recent studies on the role of ROS as regulators of signal pathways are consistent with the possibility that ROS play a role in the development of the neuronal phenotype. Moreover, the differential production of ROS provides a useful method to isolate from mixed populations cells that are highly enriched for either progenitor cells or neurons.     

The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes

The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.     

Release of mitochondrial Opa1 following oxidative stress in HT22 cells

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.     

P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca(2+) rise and change in mitochondrial potential

With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca(2+) level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y(1/2/4) receptor agonist in rat, caused a large cytosolic Ca(2+) increase in astrocytes and a considerably smaller Ca(2+) response in neighboring neurons. The P2Y(1) receptor antagonist MRS2179 completely blocked the ATP-induced Ca(2+) response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca(2+) level. Stimulation with UDP, a P2Y(6) receptor agonist; UTP, a P2Y(2/4) receptor agonist; 2MeSATP, a P2Y(1) receptor agonist; or 2MeSADP, a P2Y(1/12/13) receptor agonist, evoked significant Ca(2+) responses in astrocytes but small Ca(2+) responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca(2+) peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca(2+) level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain.     

Thioredoxin-Interacting Protein(TXNIP) Regulates Parkin/PINK1-mediated Mitophagy in Dopaminergic Neurons Under High-glucose Conditions: Implications for Molecular Links Between Parkinson’s Disease and Diabetes

Patients with diabetes mellitus have a higher risk of developing Parkinson’s disease(PD). However, the molecular links between PD and diabetes remain unclear.In this study, we investigated the roles of thioredoxininteracting protein(TXNIP) in Parkin/PINK1-mediated mitophagy in dopaminergic(DA) cells under high-glucose(HG) conditions. In streptozotocin-induced diabetic mice,TXNIP was upregulated and autophagy was inhibited in the midbrain, while the loss of DA neurons was accelerated by hyperglycemia. In cultured PC12 cells under HG, TXNIP expression was upregulated and the intracellular reactive oxygen species(ROS) levels increased, leading to cell death. Autophagic flux was further blocked and PINK1 expression was decreased under HG conditions. Parkin expression in the mitochondrial fraction and carbonyl cyanide 3-chlorophenylhydrazone(CCCP)-induced co-localization of COX IV(marker for mitochondria) and LAMP1(marker for lysosomes) were also significantly decreased by HG. Overexpression of TXNIP was sufficient to decrease the expression of both PINK1 and Parkin in PC12 cells, while knockdown of the expression of TXNIP by si RNA decreased intracellular ROS and attenuated cellular injury under HG. Moreover, inhibition of TXNIP improved the CCCP-induced co-localization of COX IV and LAMP1 in PC12 cells under HG. Together, these results suggest that TXNIP regulates Parkin/PINK1-mediated mitophagy under HG conditions, and targeting TXNIP may be a promising therapeutic strategy for reducing the risk of PD under hyperglycemic conditions.     

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.     

Inhibition of mitochondrial cytochrome c oxidase potentiates Aβ-induced ER stress and cell death in cortical neurons

Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca^2 + transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer's disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca^2 + homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aβ-induced depletion of ER Ca^2 + content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca^2 + levels triggered by Aβ and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca^2 + release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aβ or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aβ- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca^2 + signalling and UPR. Furthermore, in neurons treated with Aβ, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-induced cell death occurs independently of Ca^2 + release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD.     

Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kappaB activation

We previously reported that incubation of cultured astrocytes in Ca2 + -containing medium after exposure to Ca2 + -free medium caused Ca2 + influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2 + -mediated injury of cultured astrocytes. Our results show that Ca2 + reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase-3 inhibitors. Paradoxical Ca2 + challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2 + reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1-5 days, cell toxicity including apoptosis was observed. Ca2 + reperfusion injury induced by Ca2 + depletion or H2O2 exposure was blocked by the iron chelator 1, 10-phenanthroline, the NF-kappaB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF-kappaB p65 subunit, and the effect was blocked by 1,10-phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2 + reperfusion-induced apoptosis is mediated at least partly by ROS production and ROS cause NF-kappaB activation in cultured astrocytes.     

Deletion of mitochondrial uncoupling protein-2 increases ischemic brain damage after transient focal ischemia by altering gene expression patterns and enhancing inflammatory cytokines

Mitochondrial hyperpolarization inhibits the electron transport chain and increases incomplete reduction of oxygen, enabling production of reactive oxygen species (ROS). The consequence is mitochondrial damage that eventually causes cell death. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that dissipate the mitochondrial proton gradient by transporting H⁺ across the inner membrane, thereby stabilizing the inner mitochondrial membrane potential and reducing the formation of ROS. The role of UCP2 in neuroprotection is still in debate. This study seeks to clarify the role of UCP2 in transient focal ischemia (tFI) and to further understand the mechanisms of ischemic brain damage. Both wild-type and UCP2-knockout mice were subjected to tFI. Knocking out UCP2 significantly increased the infarct volume to 61% per hemisphere as compared with 18% in wild-type animals. Knocking out UCP2 suppressed antioxidant, cell-cycle, and DNA repair genes, including Sod1 and Sod2, Gstm1, and cyclins. Furthermore, knocking out UCP2 significantly upregulated the protein levels of the inflammatory cytokines, including CTACK, CXCL16, Eotaxin-2, fractalkine, and BLC. It is concluded that knocking out the UCP2 gene exacerbates neuronal death after cerebral ischemia with reperfusion and this detrimental effect is mediated by alteration of antioxidant genes and upregulation of inflammatory mediators.     

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.     

Cyanide enhancement of dopamine-induced apoptosis in mesencephalic cells involves mitochondrial dysfunction and oxidative stress

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.     

Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis

In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.