Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release.

Neurons express a variety of plasma-membrane potassium channels that play important roles in regulating neuronal excitability and synaptic transmission, but also contain mitochondrial ATP-sensitive potassium channels, the functions of which are unknown. Studies of cardiac cells suggest that similar mitochondrial ATP-sensitive potassium channels are involved in the process of ischemic preconditioning, suggesting a role in regulating cell survival. The authors report that mice given diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, exhibited a large (60% to 70%) decrease in cortical infarct size after permanent occlusion of the middle cerebral artery. Diazoxide decreases neuronal apoptosis and increases astrocyte survival and activation in the penumbral region of the ischemic cortex. The neuroprotective effect of diazoxide is abolished by 5-hydroxydecanoate, a selective antagonist of mitochondrial ATP-sensitive potassium channels. Studies of cultured hippocampal neurons reveal that diazoxide depolarizes mitochondria, prevents cytochrome c release, and protects cells against death induced by staurosporine and chemical hypoxia. Diazoxide increased the levels of Bcl2 and inhibited the association of Bax with mitochondria in neurons exposed to an apoptotic insult, suggesting that activation of mitochondrial ATP-sensitive potassium channels may stabilize mitochondrial function by differentially modulating proapoptotic and antiapoptotic proteins. Collectively, the data suggest that mitochondrial ATP-sensitive potassium channels play a key role in modulating neuronal survival under ischemic conditions, and identify agents that activate mitochondrial ATP-sensitive potassium channels as potential therapeutics for stroke and related neurodegenerative conditions.     

Systematic administration of iptakalim, an ATP-sensitive potassium channel opener, prevents rotenone-induced motor and neurochemical alterations in rats

Our previous studies revealed that iptakalim, a novel ATP-sensitive potassium channel opener, has a significant neuroprotective function against ischemia in vivo or rotenone-induced neurotoxicity in vitro. To investigate the potential pharmaceutical benefit of ATP-sensitive potassium channel openers on neurodegenerative diseases, we studied the effects of iptakalim and diazoxide, a selective mitochondrial ATP-sensitive potassium channel opener, on the rotenone-induced nigrostriatal degeneration in rats. Iptakalim (1.5 mg/kg/day, orally) or diazoxide (1.5 mg/kg/day, orally) alone was administered to rats for 3 days, and then for 4 weeks was used daily with an injection of rotenone (2.5 mg/kg/day, subcutaneously) 1 hr later each time. The results showed that rotenone-infused rats exhibited parkinsonian symptoms and had dopamine depletion in the striatum and substantia nigra. Pretreatment with iptakalim or diazoxide prevented rotenone-induced catalepsy and the reduction of striatum dopamine contents. Moreover, iptakalim and diazoxide reduced the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. These neuroprotective effects of iptakalim and diazoxide were abolished by 5-hydroxydecanoate, a selective mitochondrial ATP-sensitive potassium channel blocker. In conclusion, our data suggested that mitochondrial ATP-sensitive potassium channels might play a key role in preventing both parkinsonian symptoms and neurochemistry alterations induced by rotenone in rats. The selective activation of mitochondrial ATP-sensitive potassium channels may provide a new therapeutic strategy for prevention and treatment of neurodegenerative disorders such as Parkinson's disease.     

Poly-IC preconditioning protects against cerebral and renal ischemia-reperfusion injury

Preconditioning induces ischemic tolerance, which confers robust protection against ischemic damage. We show marked protection with polyinosinic polycytidylic acid (poly-IC) preconditioning in three models of murine ischemia-reperfusion injury. Poly-IC preconditioning induced protection against ischemia modeled in vitro in brain cortical cells and in vivo in models of brain ischemia and renal ischemia. Further, unlike other Toll-like receptor (TLR) ligands, which generally induce significant inflammatory responses, poly-IC elicits only modest systemic inflammation. Results show that poly-IC is a new powerful prophylactic treatment that offers promise as a clinical therapeutic strategy to minimize damage in patient populations at risk of ischemic injury.     

Ionotropic glutamate receptor antagonists inhibit the proliferation of granule cell precursors in the adult brain after seizures induced by pentylenetrazol

Brief ischemia was reported to protect various cells against injury induced by subsequent ischemia-reperfusion, and this phenomenon is known as ischemic preconditioning. The aims of the present study were to clarify whether early ischemic preconditioning could be observed in the rat retina by histological examination. Male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 5-60 min before 60 min of ischemia. Additional groups of rats received 10 mg/kg 8-phenyltheophiline and 4.5 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A1 receptor antagonists, 5 mg/kg 5-hydroxydecanoate and 1 mg/kg glibenclamide, ATP-sensitive K+ channel blockers, or 2.5 mg/kg chelerythrine and 0.1 mg/kg bisindolylmaleimide I, protein kinase C inhibitors, 15 or 30 min before preconditioning. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Five minutes of preconditioning ischemia 20-40 min before 60 min of sustained ischemia completely prevented the retinal tissue damage induced by the sustained ischemia. Treatment with 8-phenyltheophylline, DPCPX, 5-hydroxydecanoate, glibenclamide, chelerythrine and bisindolylmaleimide I almost completely reduced the protective effect of early ischemic preconditioning. The results in the present study indicated that early ischemic preconditioning was demonstrated in the rat retina. Stimulation of adenosine receptors, opening of ATP-sensitive K+ channels and activation of protein kinase C might be involved in the underlying protective mechanisms.     

Endotoxin preconditioning protects against the cytotoxic effects of TNFalpha after stroke: a novel role for TNFalpha in LPS-ischemic tolerance.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-alpha (TNFalpha) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFalpha in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFalpha are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFalpha before middle cerebral artery occlusion in mice and show that TNFalpha is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFalpha are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFalpha (approximately threefold) and the proximal TNFalpha signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (approximately 2.5-fold), which may neutralize the effect of TNFalpha and reduce TNFalpha-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFalpha after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFalpha in the setting of ischemia. Our studies suggest that TNFalpha is a twin-edged sword in the setting of stroke: TNFalpha upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFalpha signaling during ischemia confers neuroprotection after LPS preconditioning.     

姜黄素通过诱导热休克蛋白70保护多巴胺能细胞

目的 探讨姜黄素通过诱导热休克蛋白70(Hsp70)高表达,抑制α-突触核蛋白的异常表达和聚集,促进蛋白酶体系统降解异常α-突触核蛋白对多巴胺能细胞的保护作用.方法 选用大鼠嗜铬细胞瘤细胞株(PCI2细胞),鱼藤酮诱导其损伤建立帕金森病细胞模型,利用姜黄素进行干预:采用MTT法检测细胞活力,荧光酶标仪检测蛋白酶体水解酶活性,Western blot检测Hsp70和α-突触核蛋白的表达,免疫荧光法检测细胞内Hsp70的表达和α-突触核蛋白的聚集.结果 鱼藤酮组PC12细胞活力及蛋白酶体水解酶活性明显降低,Hsp70的表达轻度增加,α-突触核蛋白表达和聚集明显增加,与对照组比较差异有统计学意义(P<0.05).经不同浓度姜黄素预处理4h后与0.1 μmol/L鱼藤酮共同孵育PC12细胞24 h,与鱼藤酮组比较,0.5 μmol/L和1.0 μmol/L姜黄素使细胞活力以及蛋白酶体水解酶活性明显升高,Hsp70表达明显升高,α-突触核蛋白的表达和聚集明显减少,差异有统计学意义(P<0.05);5.0 μmol/L和10 μmol/L姜黄素对鱼藤酮的拮抗作用明显减弱,细胞活力与鱼藤酮组比较差异均无统计学意义(P>0.05),胰蛋白酶、多肽-谷氨酰-多肽水解酶活性与鱼藤酮组比较差异均无统计学意义(P>0.05);10 μmol/L姜黄素组糜蛋白酶样水解酶活性进一步降低,与鱼藤酮组比较差异有统计学意义(P<0.05).结论 低浓度姜黄素能够通过诱导PC12细胞表达Hsp70,诱导蛋白酶体水解酶活性表达,进而抑制α-突触核蛋白的表达和聚集,从而拮抗鱼藤酮诱导的PC12细胞的损伤.     

ATP敏感性钾通道在帕金森病中的作用

ATP敏感性钾通道已被证实广泛分布于血管平滑肌、心肌、胰腺等组织,发挥着诸如血管舒张、心肌缺血的保护及胰岛素分泌等作用.本综述旨在阐明ATP敏感性钾通道在帕金森病发病机制中参与调控神经元电兴奋性、线粒体功能及神经递质释放的独特角色,以揭示对其进行深入研究的意义及作为帕金森病治疗靶点的可能性和潜在价值.     

Endogenous neuroprotection in the retina

Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows severe ischemia. The retina is protected against the damage following severe ischemia for up to 72h after the application of IPC. However, there is no early preconditioning, i.e. protective effects starting within hours of preconditioning. The IPC stimulus consists of a brief, non-damaging period of ischemia. It results in complete preservation of retinal structure and function following ischemia, and is thus the most robust neuroprotection demonstrated in the retina to date. Release of adenosine, de novo protein synthesis, and mediators such as protein kinase C and K(+) ATP channels are required for IPC protection. Both the adenosine A1 and A2a receptors are involved. However, the molecular mechanisms for neuroprotection have not been completely described. It appears that both increased expression of protective proteins and decreased expression of pro-apoptotic proteins are involved. In addition, IPC prevents hypoperfusion following severe ischemia. Further study of the IPC phenomenon could lead to an enhanced understanding of the mechanisms of ischemic damage and its prevention in the retina.     

脑缺血耐受机制的研究进展

2－3min的脑缺血对随后的严重脑缺血具有明显保护作用，即脑缺血耐受，目前发现蛋白合成、不同类型的离子通道的变化以及胶质细胞的支持参与了脑缺血耐受，研究缺血耐受机制能够为进一步研究脑损伤与保护机制提供新的视角。     

Heat shock protects PC12 cells against MPP+ toxicity

A mild heat shock preconditioning has been shown to induce thermotolerance and protection against a number of cytotoxic agents that may induce cell death by either apoptosis or necrosis. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that selectively targets dopaminergic cells of the substantia nigra and, as such, it is often used to induce neuronal cell death in models of Parkinson's disease. PC12 cells were heat-shocked for 1 h at 41.5 °C. This led to a rapid induction of Hsp25 and Hsp70. Levels of these proteins remained elevated for at least 24 h post heat shock. Treatment of PC12 cells with 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of MPTP, resulted in cell death. Morphological analysis and the lack of caspase activity suggested that cell death was by necrosis. Heat shocking the cells 6 h prior to addition of MPP⁺ significantly inhibited the induction of cell death by MPP⁺. These results indicated that heat shock is protective against MPP⁺ neurotoxicity in PC12 cells.     

Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

BACKGROUND： Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson＇s disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson＇s disease remains unclear. OBJECTIVE： To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING： A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS： PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS： PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco＇s modified eagle＇s medium （DMEM）, was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES： Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS） was used. RESULTS： In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition （P 〈 0.01）. Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition （P 〈 0.01）, as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots     

Targeting mitochondrial ATP-sensitive potassium channels—a novel approach to neuroprotection

Mitochondrial responses to ischemic stress play an important role in necrosis and apoptosis of brain cells. Recent studies using several different experimental preparations have shown that activation of ATP-sensitive potassium channels in mitochondria (mitoK_ATP channels) is able to protect neurons and astroglia against injury and death. Thus, targeting of mitoK_ATP channels appears to be a novel approach to neuroprotection. However, little is known about the mechanisms involved. The purpose of this review is to detail the current state of knowledge about this important, emerging area of investigation, and to provide suggestions for future studies.     

Toll-like receptor 9: a new target of ischemic preconditioning in the brain.

Preconditioning with lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, provides neuroprotection against subsequent cerebral ischemic brain injury, through a tumor necrosis factor (TNF)alpha-dependent process. Here, we report the first evidence that another TLR, TLR9, can induce neuroprotection. We show that the TLR9 ligand CpG oligodeoxynucleotide (ODN) can serve as a potent preconditioning stimulus and provide protection against ischemic brain injury. Our studies show that systemic administration of CpG ODN 1826 in advance of brain ischemia (middle cerebral artery occlusion (MCAO)) reduces ischemic damage up to 60% in a dose- and time-dependent manner. We also offer evidence that CpG ODN preconditioning can provide direct protection to cells of the central nervous system, as we have found marked neuroprotection in modeled ischemia in vitro. Finally, we show that CpG preconditioning significantly increases serum TNFalpha levels before MCAO and that TNFalpha is required for subsequent reduction in damage, as mice lacking TNFalpha are not protected against ischemic injury by CpG preconditioning. Our studies show that preconditioning with a TLR9 ligand induces neuroprotection against ischemic injury through a mechanism that shares common elements with LPS preconditioning via TLR4.     

Diazoxide preconditioning antagonizes cytotoxicity induced by epileptic seizures

Diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, can protect neurons and astrocytes against oxidative stress and apoptosis. In this study, we established a cellular model of epilepsy by culturing hippocampal neurons in magnesium-free medium, and used this to investigate effects of diazoxide preconditioning on the expression of inwardly rectifying potassium channel (Kir) subunits of the ATP-sensitive potassium. We found that neuronal viability was significantly reduced in the epileptic cells, whereas it was enhanced by diazoxide preconditioning. Double immunofluorescence and western blot showed a significant increase in the expression of Kir6.1 and Kir6.2 in epileptic cells, especially at 72 hours after seizures. Diazoxide pretreatment completely reversed this effect at 24 hours after seizures. In addition, Kir6.1 expression was significantly upregulated compared with Kir6.2 in hippocampal neurons after seizures. These findings indicate that diazoxide pretreatment may counteract epileptiform discharge-induced cytotoxicity by suppressing the expression of Kir subunits.     

Diazoxide pretreatment induces delayed preconditioning in astrocytes against oxygen glucose deprivation and hydrogen peroxide-induced toxicity

Recent studies suggest that activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) with diazoxide can protect neurons against ischemic stress. However, it is not yet known whether astrocytes, which are more resilient against ischemia, respond similarly to diazoxide. We exposed cultured astrocytes to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) with or without pretreatment with the mK(ATP) opener diazoxide. Marked decreases in astrocyte viability were evident after 9 and 12 hr of OGD [76% +/- 3% (n = 50) and 60% +/- 1% (n = 50)] and 400 and 600 microM H2O2 [40% +/- 2% (n = 16) and 25% +/- 2% (n = 16)], respectively, compared with no treatment (100% +/- 1%). Diazoxide treatment (3 days of sequential application) dramatically reversed the negative effects of OGD and H2O2, resulting in complete blockade of astrocyte cell death. Effects of diazoxide were blocked by the mK(ATP) blocker 5-hydroxydecanoic acid (5-HD). Furthermore, incubation of astrocytes with diazoxide resulted in loss of mitochondrial membrane potential monitored by tetramethylrhodamineethylester fluorescence. Additionally, generation of reactive oxygen species was observed in response to diazoxide, assessed using the oxidation-sensitive dye hydroethidine, and this effect was abolished by antioxidants, catalase, and a superoxide dismutase mimetic, M40401. Finally, diazoxide increased the protein level of phosphorylated protein kinase C (PKC) revealed by immunoblot analysis. Our findings demonstrate that opening of mK(ATP) by diazoxide identifies a delayed preconditioning effect that is protective against two types of injury in astrocytes and that diazoxide may deliver protection via mitochondrial depolarization, free radical production, and PKC activation.     

Stroke and its modification in Parkinson's disease

Previous studies have not agreed on the incidence of ischemic stroke in persons with Parkinson's disease. There are epidemiologic and neurochemical facets of Parkinson's disease that might confer some benefit or protection against ischemic stroke. We used a case-control method to determine the lifetime history of ischemic stroke in 200 patients with Parkinson's disease and 200 controls of a similar age range. Analysis was also carried out for myocardial infarction as a marker of generalized atherosclerotic disease and for stroke risk factors. The cumulative incidence of ischemic stroke was significantly less in the patients with Parkinson's disease than in the controls, as was the cumulative incidence of myocardial infarction. Among risk factors, significantly fewer patients with Parkinson's disease used tobacco than controls. The decreased incidence of ischemic stroke in the patients with Parkinson's disease appears to be related to their less severe generalized atherosclerosis, possibly due to their lower incidence of tobacco use. In view of the known potential for dopamine to exacerbate experimental ischemic tissue damage, the possibility that the dopamine deficiency in the central nervous system of persons with Parkinson's disease confers an additional specific protective benefit against ischemic stroke cannot be excluded and requires further study.     

Iptakalim hydrochloride protects cells against neurotoxin-induced glutamate transporter dysfunction in in vitro and in vivo models

Iptakalim hydrochloride (Ipt), a novel antihypertensive drug, exhibits K(ATP) channel activation. Here, we report that Ipt remarkably protects cells against neurotoxin-induced glutamate transporter dysfunction in in vitro and in vivo models. Chronic exposure of cultured PC12 cells to neurotoxins, such as 6-OHDA, MPP+, or rotenone, decreased overall [3H]-glutamate uptake in a concentration-dependent manner. Pre-treatment using 10 microM Ipt significantly protected cells against neurotoxin-induced glutamate uptake diminishment, and this protection was abolished by the K(ATP) channel blocker glibenclamide (20 microM), suggesting that the protective mechanisms may involve the opening of K(ATP) channels. In 6-OHDA-treated rats (as an in vivo Parkinson's disease model), [3H]-glutamate uptake was significantly lower in synaptosomes isolated from the striatum and cerebral cortex, but not the hippocampus. Pre-conditioning using 10, 50, and 100 microM Ipt significantly restored glutamate uptake impairment and these protections were abolished by blockade of K(ATP) channels. It is concluded that Ipt exhibits substantial protection of cells against neurotoxicity in in vitro and in vivo models. The cellular mechanisms of this protective effect may involve the opening of K(ATP) channels. Collectively, Ipt may serve as a novel and effective drug for PD therapy.