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.     

Protective effect of BAG5 on MPP+-induced apoptosis in PC12 cells

Abstract

Objectives: Parkinson’s disease (PD) is the most common neurodegenerative disease in humans, and an abundance of evidence has implicated apoptosis signaling pathways in the neurodegeneration of PD. The purpose of this study was to assess the role of B-cell lymphoma 2 (Bcl-2)-associated athanogene 5 (BAG5) protein, which was previously confirmed to play an important role in the pathogenesis of PD, in the regulation of apoptosis induced by 1-methyl-4-phenyl-pyridinium (MPP⁺) in PC12 cells.

Methods: PC12 cells were treated with MPP⁺ for 48 hours to induce apoptosis, and activation of Bcl-2, Bcl-xl, and caspase 3 was measured by western blot.

Results: The upregulation of BAG5 in PC12 cells inhibited apoptosis and increased the expression of anti-apoptotic proteins, including Bcl-2 and Bcl-xl, after MPP⁺ treatment. In addition, downregulation of BAG5 in PC12 cells enhanced apoptosis and decreased the expression of these proteins after MPP⁺ treatment.

Discussion: The data suggest that BAG5 inhibits MPP⁺-induced apoptosis through both endogenous and mitochondria-mediated pathways of apoptosis. Through this mechanism, the upregulation of BAG5 levels may occur through its anti-apoptotic activity in PD.     

蛇床子素对MPP~＋损伤PC12细胞的保护作用研究

目的观察蛇床子素（osthole）对1-甲基-4-苯基吡啶离子（MPP＋）诱导PC12细胞损伤的神经保护作用。方法将MPP＋加入培养的PC12细胞中,建立多巴胺能神经元损伤模型,加入不同浓度的蛇床子素预处理细胞（0.01、0.05、0.1mmol/L）。处理24h后用噻唑蓝（MTT）比色法检测细胞活性;以乳酸脱氢酶（LDH）活性测定反映细胞的损伤程度;采用Westernblot法检测Bax、Bcl-2蛋白的表达,分析Bax/Bcl-2比值变化,以及检测细胞色素C的改变。结果蛇床子素可以明显减少MPP＋诱导的PC12细胞活性的降低,LDH的释放,Bax/Bcl-2比值的增高以及细胞色素C的释放（P〈0.05）。结论蛇床子素对MPP＋诱导的PC12细胞损伤具有保护作用。     

Protective effects of atypical antipsychotic drugs on PC12 cells after serum withdrawal

Atypical antipsychotic drugs are widely used in the treatment of schizophrenia, and clinical evidence has shown that early and prolonged intervention with these drugs will improve the long-term outcome. It is still unclear, however, whether the atypical antipsychotic drugs are also neuroprotective. To clarify this matter, we used PC12 cell cultures and the MTT assay for cell viability to determine whether various concentrations of the atypical antipsychotics clozapine, quetiapine, and risperidone are neuroprotective after serum withdrawal. In addition, to explore the drugs' actions, Northern blot was used to examine the gene expression of SOD1 (Cu/Zn superoxide dismutase) and p75NTR (p75 neurotrophin receptor). The results demonstrated that 1) the antipsychotic drugs can protect PC12 cells from death after serum withdrawal; cell viability in these drug treatment groups is significantly different from that in the groups without serum in the medium (P < 0.01); and 2) these drugs up-regulated the SOD1 gene expression to more than 120% (P < 0.05) and also down-regulated p75NTR mRNA levels to less than 65% of their respective control values (P < 0.05). These findings suggest that the atypical antipsychotics clozapine, quetiapine, and risperidone may exert a neuroprotective function through the modulation of SOD1 and p75NTR expression.     

Estradiol protects PC12 cells against CoCl2-induced apoptosis

In hypoxic/ischemic conditions, neuronal apoptotic events are occurred, resulting in neuronal diseases. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway leading to the inhibitory effects of estradiol against cobalt chloride (CoCl₂)-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl₂-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl₂ treatment attenuated CoCl₂-mediated the reactive oxygen species (ROS) production and limited the activities of the caspase cascades, such as caspase-8, -9 and -3. Furthermore, estradiol downregulated the Bax:Bcl-2 ratio and decreased the release of cytochrome c from the mitochondria into the cytosol in CoCl₂-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl₂-induced upregulation of Fas-ligand (Fas-L) and truncated of Bid in sequence of death receptor-mediated pathway. In addition, estradiol increased the phosphorylation of Akt in CoCl₂-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl₂-induced apoptosis in PC12 cells.Taken together, estradiol was found to have a neuroprotective effect against CoCl₂-induced apoptosis of PC12 cells by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, Fas/Fas-L as well as PI3K/Akt pathway.     

MPP+诱导PC12细胞损伤的蛋白质组学研究

目的研究Mpp~ 作用PC12细胞48h后蛋白质表达谱的改变,进一步在蛋白质水平上阐明帕金森病的发病机制。方法建立Mpp 诱导的PC12细胞帕金森病模型,提取对照组与实验组的细胞总蛋白,应用荧光差异凝胶电泳(Differential Gel Electrophoresis,DIGE)系统构建双向电泳图,DeCyder软件分析蛋白表达差异信息,运用MALDI-TOF质谱鉴定差异蛋白质。结果与对照组相比,实验组共有32个蛋白点发生变化;鉴定了其中7种结构和功能各异的蛋白。结论本研究鉴定出氧化应激和线粒体损伤相关的蛋白thioredoxin、MPPs,与细胞骨架相关的蛋白NF-L、ezrin,具有分子伴侣活性的蛋白NAC、crystaillin,与免疫炎症相关的蛋白gClqBP。这些蛋白的显著改变可能与PD的发病机制密切相关。     

Olanzapine and quetiapine protect PC12 cells from beta-amyloid peptide(25-35)-induced oxidative stress and the ensuing apoptosis

We previously found that the atypical antipsychotic drugs (APDs) clozapine, olanzapine, quetiapine, and risperidone reduce PC12 cell death induced by hydrogen peroxide, N-methyl-4-phenylpyridinium ion, or beta-amyloid peptide (Abeta(25-35)). Such neurotoxic substances have in common the capability of causing oxidative stress. Atypical APDs have been used in treating schizophrenia and in treating psychotic symptoms of patients with Alzheimer's disease (AD), in which Abeta is involved by causing oxidative stress. Therefore, we hypothesized that atypical APDs might alleviate oxidative stress in PC12 cells, thus protecting them from apoptosis. PC12 cells were seeded in plates or chambers for 24 hr and cultured for another 24 hr with olanzapine or quetiapine in the medium, and then the cells were cultured in the new medium containing Abeta(25-35) and/or olanzapine, quetiapine, but not serum, for various periods. It was shown that cultures treated with olanzapine + Abeta(25-35), or quetiapine + Abeta(25-35), had significantly higher cell viabilities and lower rates of apoptosis compared with the cultures exposed only to Abeta(25-35). In addition, the drugs blocked the activation of caspase-3 caused by Abeta(25-35). Furthermore, olanzapine and quetiapine prevented Abeta(25-35)-induced overproduction of intracellular reactive oxygen species, Abeta(25-35)-induced decrease in mitochondrial membrane potential, and Abeta(25-35)-induced changes in activities of the key antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. In consideration of the wealth of evidence linking oxidative stress to the pathophysiology of schizophrenia and AD, these findings give us a new insight into the therapeutic actions of atypical antipsychotics in patients with the disorders.     

芍药苷通过分子伴侣自噬通路保护PC12细胞

目的 研究芍药苷对PC12细胞的保护作用,并探讨其对分子伴侣自噬通路的影响.方法 环境毒素MPP＋诱导PC12细胞损伤,同时给予芍药苷进行干预,检测细胞存活率、乳酸脱氢酶（LDH）漏出率及细胞凋亡率的变化,并检测分子伴侣自噬相关蛋白Lamp2a表达水平的变化.结果 MPP＋处理24h,PC12细胞的存活率降低,漏出至上清液中的LDH增多,细胞凋亡率明显升高.芍药苷（25μmol·L-1）显著提高了PC12细胞的存活率,减少了LDH的漏出,降低了细胞凋亡率.Western blot显示MPP＋导致Lamp2a的表达升高,芍药苷能够显著抑制Lamp2a的激活.结论 MPP＋导致PC12细胞损伤和分子伴侣自噬的过度激活,而芍药苷对PC12细胞具有明显的保护作用,并显著降低了分子伴侣自噬通路的活性.     

利培酮和氟哌啶醇对无血清诱导PC12细胞凋亡的作用

目的探讨利培酮对神经的保护作用。方法以100 mg/L神经生长因子诱导大鼠PC12细胞7 d后,将细胞随机分为无血清组、氟哌啶醇组、利培酮组(氟哌啶醇组和利培酮组的浓度均分为10,20,40,60,80μmol/L)和血清组,每组5孔。采用四甲基偶氮唑盐(MTT)法测定细胞活性,流式细胞仪检测细胞凋亡率、细胞周期,用Hoechst33342染色法观察细胞形态学变化。结果(1)给予利培酮(20,40μmol/L)72 h后,细胞活性[(64.2±4.4)%,(60.8±3.9)%]高于无血清组[(48.0±2.8)%;P<0.01],而10μmol/L、20μmol/L、40μmol/L、60μmol/L、80μmol/L各浓度氟哌啶醇组的细胞活性[分别为(31.8±3.9)、和(24.4±1.3)%、(14.3±2.6)%、(10.5±2.1)%和(4.1±1.4)%]均低于无血清组(P<0.01)。(2)流式细胞仪检测,利培酮组的凋亡率[(34.6±2.8)%]低于无血清组[(50.7±3.1)%;LSDt-=-16.0,P<0.01],而氟哌啶醇组的凋亡率[(59.3±5.2)%]高于无血清组(LSD-t=8.6,P<0.01);血清组和利培酮组细胞滞留于G1期的比例[分别为(53.5±5.4)%和(71.1±3.7)%]低于无血清组[(81.2±3.0)%]和氟哌啶醇组[(82.1±5.7)%;P<0.01]。(3)无血清组和氟哌啶醇组多见凋亡细胞,其中氟哌啶醇组更明显;而利培酮组偶见凋亡细胞,以核浓缩为主。结论利培酮对PC12细胞有抗凋亡作用,可能是其参与神经保护作用的机制之一。     

ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells

The neuroprotective effects of neuregulin (NRG), a polypeptide growth factor, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death and oxidative stress in PC12-ErbB4 cells were investigated. Treatment of PC12-ErbB4 cells with MPP+induced cell death that was markedly attenuated by NRG. The PI3K/PKB/Akt and Ras/MapK signaling pathways probably mediate the survival effect of NRG. NRG induces prolonged activation of PKB/Akt and Erk. Moreover, inhibition of the PI3K and MEK activities prevented the NRG-induced survival effect. Over-expression of constitutively active PI3K or H-Ras (12V) inhibited MPP+-mediated cell death. In addition, MPP+-mediated reactive oxygen species (ROS) elevation was also inhibited by NRG. The effect of NRG on ROS levels was blocked by PI3K and MEK inhibitors, indicating that both signaling pathways can regulate the toxic ROS levels induced by MPP+. Taken together, these results indicate that in PC12-ErbB4 cells, the NRG-induced neuroprotective effect from MPP+treatment, requires PI3K/PKB/Akt and Ras/MapK signaling networks. These authors contributed equally to this work.     

Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson's disease. To better understand the cellular insult of oxidative stress on dopaminergic neurons, we studied the cytotoxic effect of the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite, 1-methyl-4-phenyl pyridium (MPP(+)), on several parameters of cell distress using neuronal PC12 cells. We also measured the level of protein expression for the dopamine transporter and the estrogen receptors alpha and beta. Since estrogens have been reported to prevent neuronal degeneration caused by increased oxidative burden, we investigated the ability of 17beta-estradiol, the stereoisomer 17alpha-estradiol, and several phytoestrogens to rescue neuronal PC12 cells submitted to MPP(+)-induced cytotoxicity. Our results consistently show a protective effect of 17alpha-estradiol, 17beta-estradiol and certain phytoestrogens such as quercetin and resveratrol, in neuronal PC12 cells treated with MPP(+). In our cellular paradigm, phytoestrogens coumestrol, genistein, and kaempferol did not revert MPP(+)-induced cellular death. By Western blot, we demonstrated that administration of MPP(+) alone decrease dopamine transporter expression, while treatments with MPP(+) together with 17alpha-estradiol, 17beta-estradiol, quercetin, or resveratrol could restore dopamine transporter protein expression to control levels. Moreover, the same treatments did not modulate alpha estrogen receptor or beta estrogen receptor expression. By these studies, we aim to provide more evidence for the involvement of phytoestrogens in the process of neuroprotection and to test our hypothesis that some of these compounds may act as neuroprotective molecules and have a lesser hormonal effect than estrogens.     

Inhibition of glucose transport in PC12 cells by the atypical antipsychotic drugs risperidone and clozapine, and structural analogs of clozapine

Treatment of schizophrenics with some antipsychotic drugs has been associated with an increased incidence of hyperglycemia and new-onset type 2 diabetes. Some of these drugs also inhibit glucose transport in rat pheochromocytoma (PC12) cells. The current study was designed to examine the effects of the atypical antipsychotic drugs--risperidone, clozapine and analogs of clozapine on glucose uptake in PC12 cells. Glucose transport was measured in cells incubated with vehicle or drug over a range of concentrations (0.2-100 microM). Uptake of 3H-2-deoxyglucose was measured over 5 min and the data were normalized on the basis of total cell protein. Risperidone and clozapine inhibited glucose transport in a dose-dependent fashion with IC(50)'s estimated to be 35 and 20 microM, respectively. The clozapine metabolite, desmethylclozapine, was considerably more potent than the parent drug, whereas clozapine N-oxide was essentially inactive. The structural analogs of clozapine, loxapine and amoxapine, both inhibited glucose transport with amoxapine being the least potent. The ability of the drugs to inhibit glucose transport was significantly decreased by including 2-deoxyglucose (5 mM) in the uptake medium. Schild analysis of the glucose sensitivity of clozapine, loxapine and risperidone indicated that 2-deoxyglucose non-competitively antagonized the inhibitory effects of these drugs. Moreover, clozapine and fluphenazine inhibited glucose transport in the rat muscle cell line, L6. These studies suggest that the drugs may block glucose accumulation directly at the level of the glucose transporter (GLUT) protein in cells derived from both peripheral and brain tissue. Furthermore, this work may provide clues about how the antipsychotic drugs produce hyperglycemia in vivo.     

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells

Heme oxygenase-1 (HO-1) catalyses the rate-limiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP⁺) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP⁺ and whether HO-1 has a protective role against the MPP⁺-induced cytotoxicity in PC-12 cells. MPP⁺ triggered a relatively rapid induction of HO-1. The MPP⁺-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP⁺-induced cytotoxicity.     

氧化应激是MPP+诱导SHSY5Y细胞凋亡的可能机制

探讨MPP+诱导SHSY5Y细胞凋亡的可能机制.采用流式细胞仪测定SHSY5Y细胞的凋l亡率、Bcl-2/Bax蛋白的表达率、活性氧的生成量,免疫细胞化学染色法观察活化型CPP32的表达.结果显示,MPP+诱导SHSY5Y细胞凋亡,两者间呈明显的量效和时效关系.在凋亡过程中Bcl-2蛋白表达显著降低,Bax蛋白表达随之增高.随着MPP+作用浓度的增加和作用时间的延长,ROS的生成逐渐增加,活化型Caspase-3阳性细胞的百分比也逐渐增加,均有明显的量效和时效关系.提示Bcl-2/Bax蛋白是MPP+诱导SHSY5Y细胞凋亡的重要调节蛋白,ROS和CPP32在凋亡过程中可能具有重要作用.     

Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death

Alpha-synuclein is a pre-synaptic protein of unknown function that has been implicated in the pathogenesis of Parkinson's disease (PD). Recently, we demonstrated that 1-methyl-4-phenylpyridinium (MPP+) induces caspase-3-dependent proteolytic activation of PKCdelta, which subsequently contributes to neuronal apoptotic cell death in mesencephalic dopaminergic neuronal cells. In the present study, we examined whether PKCdelta interacts with alpha-synuclein to modulate MPP+-induced dopaminergic degeneration. Over-expression of wild-type human alpha-synuclein in mesencephalic dopaminergic neuronal cells (N27 cells) attenuated MPP+-induced (300 microM) cytotoxicity, release of mitochondrial cytochrome c, and subsequent caspase-3 activation, without affecting reactive oxygen species (ROS) generation. Wild-type alpha-synuclein over-expression also dramatically reduced MPP+-induced caspase-3-mediated proteolytic cleavage of PKCdelta, whereas over-expression of the mutant human alpha-synucleinA53T did not alter the PKCdelta cleavage under similar conditions. Immunoprecipitation-kinase assay revealed reduced PKCdelta kinase activity in wild-type alpha-synuclein over-expressing cells in response to MPP+ treatment. Wild-type alpha-synuclein over-expression also rescued mesencephalic dopaminergic neuronal cells from MPP+-induced apoptotic cell death, while alpha-synucleinA53T exacerbated the MPP+-induced DNA fragmentation. Furthermore, co-immunoprecipitation studies revealed that alpha-synuclein interacts with the pro-apoptotic proteins PKCdelta and BAD, but not with the anti-apoptotic protein Bcl-2 following MPP+ treatment. We also observed that the interaction between PKCdelta and alpha-synuclein does not involve direct phosphorylation. Together, our results demonstrate that wild-type alpha-synuclein interacts with the pro-apoptotic molecules BAD and PKCdelta to protect dopaminergic neuronal cells against neurotoxic insults.     

美满霉素对MPP+诱导的PC12细胞凋亡的保护作用

目的在离体细胞培养中探讨美满霉素(Minocycline,MC)对1-甲基-4-苯基吡啶离子(MPP )诱导的帕金森病细胞凋亡模型的保护作用.方法将MC或MPP 加入培养的PC12细胞中,建立多巴胺神经元凋亡模型,四甲基偶氮唑盐法(MTT法)检测细胞代谢活性,电泳法检测细胞凋亡,流式细胞术检测细胞凋亡率.结果MPP 浓度为10μmol/L时建立多胺神经元凋亡模型;MC 100 μmol/L预处理可明显升高MPP 处理的PC12细胞活性;MC MPP 组细胞凋亡率显著低于MPP 组(P＜0.01),但仍明显高于阴性对照组(P＜0.05).结论MC对MPP 诱导的细胞凋亡有一定的保护作用.     

The effect of chronic atypical antipsychotic drugs and haloperidol on amphetamine-induced dopamine release in vivo.

The effect of chronic administration of antipsychotic drugs (21 days in drinking water followed by 3 days drug washout) on the D-amphetamine (1.0 mg/kg, s.c.)-induced increase in dopamine (DA) release in the striatum and the nucleus accumbens of awake, freely-moving rats was investigated with microdialysis. Chronic administration of haloperidol, a typical antipsychotic, (0.5 mg/kg/day), decreased basal extracellular DA release in the striatum and the nucleus accumbens but did not affect D-amphetamine-induced DA release in either region. In marked contrast, chronic administration of three atypical antipsychotic drugs: amperozide (2 mg/kg/day), clozapine (10 mg/kg/day) and melperone (2 mg/kg/day) increased basal extracellular DA and enhanced D-amphetamine-induced DA release in the striatum. In the nucleus accumbens, basal extracellular DA was decreased by chronic amperozide, unchanged by chronic clozapine and increased by chronic melperone. Most significantly, D-amphetamine-induced DA release was inhibited by chronic amperozide or clozapine, but unaffected by chronic melperone in this region. These results suggest that atypical antipsychotic drugs can alter DA release in a region specific manner. In particular, attenuation of amphetamine-like stimulation of DA release with reduced basal DA release in the nucleus accumbens could contribute to the antipsychotic action of amperozide which has a very weak affinity for D2 DA receptors.     

活化素A对MPP^＋所诱导的PC12细胞损伤的保护作用

目的观察重组人活化素A(rhAcT)对MPP+所诱导的PC12细胞损伤的保护作用.方法将MMP+或活化素A,L-deprenyl加入体外培养的PC12细胞,用四甲基偶氮唑盐(MTT)法检测细胞活力的变化;用免疫细胞化学法和RT-PCR评价细胞的酪氨酸羟化酶和bcl-2蛋白及mRNA含量的变化,用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测细胞凋亡的变化,比较各组的差异.结果预先给予rhAcT和L-deprenyl的两组细胞活力明显高于MPP+损害组,酪氨酸羟化酶和bcl-2的蛋白及mRNA表达强于损害组,同时两组的凋亡细胞明显减少,rhAcT和L-deprenyl两组间无显著差异.结论rhAcT和L-deprenyl通过上调bcl-2的表达,抑制凋亡的发生,而对MMP+所诱导的PC12细胞的损伤具有保护作用.     

Effects of atypical antipsychotic drugs vs. haloperidol on expression of heat shock protein in the discrete brain regions of phencyclidine-treated rats

Mu and delta opioid receptors were labeled in enkephalin knockout mice by quantitative autoradiography. Discrete, large increases (100-300%) were found in limbic forebrain structures for mu binding and striatum and pallidum for delta binding. The up-regulation of opioid receptors may reflect a form of 'denervation supersensitivity. ' The receptor up-regulation in limbic areas is consistent with the increased emotional and aggressive behaviors observed in the enkephalin knockout mice.