Shang Der Chen

Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS) as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ) co-activator 1α (PGC1-α). PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis.

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress.

Promoter region methylation and reduced expression of thrombospondin-1 after oxygen-glucose deprivation in murine cerebral endothelial cells

Angiogenesis is induced in response to ischemia. Thrombospondin-1 (TSP-1) is a potent angiostatic factor. Silencing of TSP-1 expression may contribute to the postischemic angiogenesis. Upregulation of TSP-1, in contrast, may terminate the postischemic angiogenesis. A possible mechanism that silences TSP-1 expression is the DNA methylation of its promoter region. DNA methylation has been reported following cerebral ischemia. The present study aimed to explore whether methylation of the promoter region of TSP-1 regulates its expression after oxygen—glucose deprivation (OGD) in murine cerebral endothelial cells (CECs) in vitro. Sublethal OGD increased the extent of methylation of the promoter region of TSP-1 with a concurrent decrease in TSP-1 mRNA and protein expression in CECs. After reoxygenation, demethylation of the TSP-1 promoter region led to the restoration of TSP-1 mRNA and protein expression. The extent of methylation of the promoter region of TSP-1 was inversely correlated with the extent of TSP-1 gene expression at mRNA and protein levels after OGD. Oxygen—glucose deprivation-induced reduction in the TSP-1 mRNA level was not accompanied by a change in mRNA stability. These findings raise the possibility that OGD downregulation of TSP-1 expression is at least in part due to methylation of its promoter region.

Activation of calcium/calmodulin-dependent protein kinase IV and peroxisome proliferator-activated receptor γ coactivator-1α signaling pathway protects against neuronal injury and promotes mitochondrial biogenesis in the hippocampal CA1 subfield after transient global ischemia

Delayed neuronal cell death occurs in the vulnerable CA1 subfield of the hippocampus after transient global ischemia (TGI). We demonstrated previously, based on an experimental model of TGI, that the significantly increased content of oxidized proteins in hippocampal CA1 neuron was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr, as well as up‐regulation of mitochondrial uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2). Using the same animal model, the present study investigated the role of calcium/calmodulin‐dependent protein kinase IV (CaMKIV) and PGC‐1α in delayed neuronal cell death and mitochondrial biogenesis in the hippocampus. In Sprague‐Dawley rats, significantly increased expression of nuclear CaMKIV was noted in the hippocampal CA1 subfield as early as 15 min after TGI. In addition, the index of mitochondrial biogenesis, including a mitochondrial DNA‐encoded polypeptide, cytochrome c oxidase subunit 1 (COX1), and mitochondrial number significantly increased in the hippocampal CA1 subfield 4 hr after TGI. Application bilaterally into the hippocampal CA1 subfield of an inhibitor of CaMKIV, KN‐93, 30 min before TGI attenuated both CaMKIV and PGC‐1α expression, followed by down‐regulation of UCP2 and SOD2, decrease of COX1 expression and mitochondrial number, heightened protein oxidation, and enhanced hippocampal CA1 neuronal damage. This study provides correlative evidence for the neuroprotective cascade of CaMKIV/PGC‐1α which implicates at least in part the mitochondrial antioxidants UCP2 and SOD2 as well as mitochondrial biogenesis in ischemic brain injury.

Contribution of nitric oxide, superoxide anion, and peroxynitrite to activation of mitochondrial apoptotic signaling in hippocampal CA3 subfield following experimental temporal lobe status epilepticus

Purpose:u2002 One cellular consequence of status epilepticus is apoptosis in the hippocampal CA3 subfield. We evaluated the hypothesis that the repertoire of cellular events that underlie such elicited cell death entails mitochondrial dysfunction induced by an excessive production of nitric oxide synthase II (NOS II)‐derived NO, increased superoxide anion (O2−) production, and peroxynitrite formation.

Peroxisome proliferator-activated receptors γ/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus

BackgroundStatus epilepticus induces subcellular changes that may lead to neuronal cell death in the hippocampus. However, the mechanism of seizure-induced neuronal cell death remains unclear. The mitochondrial uncoupling protein 2 (UCP2) is expressed in selected regions of the brain and is emerged as an endogenous neuroprotective molecule in many neurological disorders. We evaluated the neuroprotective role of UCP2 against seizure-induced hippocampal neuronal cell death under experimental status epilepticus.MethodsIn Sprague–Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Oxidized protein level, translocation of Bcl-2, Bax and cytochrome c between cytosol and mitochondria, and expression of peroxisome proliferator-activated receptors γ (PPARγ) and UCP2 were examined in the hippocampal CA3 subfield following KA-induced status epilepticus. The effects of microinjection bilaterally into CA3 area of a PPARγ agonist, rosiglitazone or a PPARγ antagonist, GW9662 on UCP2 expression, induced superoxide anion (O2· -) production, oxidized protein level, mitochondrial respiratory chain enzyme activities, translocation of Bcl-2, Bax and cytochrome c, and DNA fragmentation in bilateral CA3 subfields were examined.ResultsIncreased oxidized proteins and mitochondrial or cytosol translocation of Bax or cytochrome c in the hippocampal CA3 subfield was observed 3–48u2009h after experimental status epilepticus. Expression of PPARγ and UCP2 increased 12–48u2009h after KA-induced status epilepticus. Pretreatment with rosiglitazone increased UCP2 expression, reduced protein oxidation, O2· - overproduction and dysfunction of mitochondrial Complex I, hindered the translocation of Bax and cytochrome c, and reduced DNA fragmentation in the CA3 subfield. Pretreatment with GW9662 produced opposite effects.ConclusionsActivation of PPARγ upregulated mitochondrial UCP2 expression, which decreased overproduction of reactive oxygen species, improved mitochondrial Complex I dysfunction, inhibited mitochondrial translocation of Bax and prevented cytosolic release of cytochrome c by stabilizing the mitochondrial transmembrane potential, leading to amelioration of apoptotic neuronal cell death in the hippocampus following status epilepticus.

ATM Gene Regulates Oxygen-Glucose Deprivation–Induced Nuclear Factor-κB DNA-Binding Activity and Downstream Apoptotic Cascade in Mouse Cerebrovascular Endothelial Cells

Background and Purpose— Cells lacking the ATM (ataxia telangectasia mutated) gene are hypersensitive to DNA damage caused by a variety of insults. ATM may regulate oxidative stress–induced signaling cascades involving nuclear factor-&kgr;B (NF-&kgr;B), a transcription factor that is upstream of a wide variety of stress-responsive genes. We investigated the potential interaction of ATM and NF-&kgr;B after oxygen-glucose deprivation (OGD) in cerebral endothelial cells (CECs). Methods— Primary cultures of mouse CECs were subjected to OGD in the absence or presence of ATM antisense oligonucleotides or the NF-&kgr;B inhibitor SN50. ATM expression was determined with the use of reverse transcription–polymerase chain reaction and Western blot, and NF-&kgr;B activity was assessed by electrophoretic mobility shift assay. Cells were assessed for mitochondrial DNA damage with the use of long polymerase chain reaction and were assessed for caspase-3 and caspase-8 activity with the use of fluorogenic substrates. Cell death was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and LDH release. Results— OGD stimulated ATM gene expression at the mRNA and protein level in CECs as early as 1 hour after OGD initiation. ATM gene knockdown with the use of an antisense oligonucleotide suppressed OGD-induced ATM protein expression, which was accompanied by an attenuation of NF-&kgr;B activation and the subsequent expression of downstream genes, including the antiapoptotic gene c-IAP2. ATM knockdown also accentuated OGD-induced mitochondrial DNA damage and the activation of caspase-3 and caspase-8, leading to enhanced CEC death. The specific NF-&kgr;B inhibitor SN50 mimicked the effects of ATM knockdown. Conclusions— We conclude that ATM may play a cytoprotective role in OGD-induced CEC death via a NF-&kgr;B–dependent signaling pathway.

The potential role of mitochondrial dysfunction in seizure-associated cell death in the hippocampus and epileptogenesis

Epilepsy is considered one of the most common neurological disorders worldwide. The burst firing neurons associated with prolonged epileptic discharges could lead to a large number of changes with events of cascades at the cellular level. From its role as the cellular powerhouse, mitochondria also play a crucial role in the mechanisms of cell death. Emerging evidence has shown that prolonged seizures may result in mitochondrial dysfunction and increase of oxidative and nitrosative stress in the hippocampus that precede neuronal cell death and cause subsequent epileptogenesis. The selective dysfunction of mitochondrial respiratory chain Complex I has been suggested to be a biochemical hallmark of seizure-induced neuronal cell death and epileptogenesis. Therefore, protection of mitochondria from bioenergetic failure and oxidative stress in the hippocampus may open a new vista to the development of effective neuroprotective strategies against seizure-induced brain damage and to the design of novel treatment perspectives against therapy-resistant forms of epilepsy.

Carbamoylating chemoresistance induced by cobalt pretreatment in C6 glioma cells: putative roles of hypoxia-inducible factor-1

We tested whether pretreatment of reagents known to induce hypoxia‐inducible factor‐1 (HIF‐1) may confer chemoresistance against cytotoxicity of 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) to rat C6 glioma cells. We also studied which cytotoxic mechanism(s) of chloroethylnitrosoureas could be neutralized by cobalt preconditioning. Preconditioning of rat C6 glioma cells with cobalt chloride (300 μM, 2 h) induced HIF‐1 binding activity based on electrophoretic mobility shift assay (EMSA). Results from Western blotting confirmed a heightened HIF‐1α level upon cobalt chloride exposure (300–400 μM, 2 h). Cobalt chloride (300 μM) pretreatment for 2 h substantially neutralized BCNU toxicity, leading to increases in glioma cell survival based on MTT assay. In addition, pre‐exposure of C6 cells with desferrioxamine (DFO; 400 μM, 3 h), an iron chelator known to activate HIF‐1, also induced HIF‐1 binding and rendered the glioma cells resistant to cytotoxicity of BCNU. Pre‐incubation with cobalt chloride abolished the cytotoxicity of several carbamoylating agents including 2‐chloroethyl isocyanate and cyclohexyl isocyanate, the respective carbamoylating metabolites of BCNU and 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea. The protective effect of cobalt exposure, however, was not observed when cells were challenged with alkylating agents including temozolomide. Cadmium chloride (50 μM) effectively reversed cobalt‐induced HIF‐1 activation. Correspondingly, cadmium chloride suppressed carbamoylating chemoresistance mediated by cobalt chloride pretreatment. Furthermore, both double‐stranded oligodeoxynucleotide (ODN) decoy with HIF‐1 cognate sequence and antisense phosphorothioate ODNs against HIF‐1α partially abolished the carbamoylating chemoresistance associated with cobalt preconditioning. Our results suggest that cobalt‐ or DFO‐preconditioning may enhance glioma carbamoylating chemoresistance that is dependent, at least in part, on induction of HIF‐1.

Combination therapy for ischemic stroke: Potential of neuroprotectants plus thrombolytics

Thrombolysis improves clinical outcome in patients with acute ischemic stroke. However, only a small fraction of patients receive thrombolytic therapy due to the narrow therapeutic time window available for the treatment in patients with ischemic stroke. A better understanding of the mechanisms underlying ischemic injury may lead to the development of novel therapeutic strategies to reduce brain damage after stroke. Cerebral ischemia triggers a number of pathophysiological and biochemical changes in the brain that present potential targets for therapeutic intervention. Candidate pathways include those regulating cellular calcium influx, excitatory neurotransmitter uptake, and generation of reactive oxygen species, as well as activation of enzymes including kinases, proteases, and lipases. The end result of these pathophysiological pathways may be apoptosis (programmed cell death) or necrosis. The activation of inflammatory cascades following ischemia also contributes to brain injury. Several neuroprotective agents which block cell death pathways have been proposed to have therapeutic potential in patients with stroke including calcium channel antagonists, glutamate receptor antagonists, free radical scavengers, anti-inflammatory strategies, inhibitors for nitric oxide synthase, and growth factors. Although results from clinical trials to date have been disappointing, there is reason to believe that combination therapy involving both thrombolytics and neuroprotectants holds promise for stroke treatment and warrants further investigation.