雷帕霉素在6-羟基多巴诱导的帕金森病细胞及大鼠模型中的神经保护作用及其机制研究
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摘要
【目的】
帕金森病(Parkinson’s disease, PD)是中枢神经系统变性疾病，线粒体氧化应激和神经元凋亡在PD的病理机制中发挥关键的作用。雷帕霉素(rapamycin)为雷帕霉素靶蛋白(Target of rapamycin, TOR)信号通路特异性抑制剂，在一系列神经系统变性疾病的病理机制及治疗中具有重要的意义。本研究通过雷帕霉素预处理PD大鼠及细胞模型，揭示其在PD中的神经保护作用，并探讨其机制。
【方法】
动物实验：实验SD大鼠随机分为假手术组、PD模型组、雷帕霉素低剂量组(0.05mg/kg/d)、中剂量组(0.5mg/kg/d)及高剂量组(5mg/kg/d)进行雷帕霉素灌胃预处理。7天后，向模型组和所有雷帕霉素预处理组大鼠脑部右侧纹状体两点定向注射6-羟基多巴(6-hydroxydopamine，6-OHDA)(4μg/μl,2μl/注射点)诱导建立PD大鼠模型，假手术组大鼠注射生理盐水作为对照。术后第25天各组大鼠均用阿扑吗啡(0.5mg/kg)诱导旋转试验，检测行为学指标；在造模后第28天，各组大鼠经心脏灌注固定，取脑组织进行黑质-纹状体酪氨酸羟化酶（Tyrosine hydroxylase，TH）免疫组化染色及透射电子显微镜观察；检测各组SD大鼠中脑丙二醛(malondialdehyde, MDA)含量及超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-PX)活性；提取脑组织总RNA后，采用RT-PCR方法检测黑质-纹状体内Bax、Bcl-2和p47phox等基因mRNA的表达；同时采用western blot方法检测黑质-纹状体内Bax、Bcl-2、细胞色素c以及cleavedcaspase-9蛋白质的表达。
体外实验：采用不同浓度6-OHDA诱导24h后检测PC12细胞存活率，建立PD细胞模型。不同浓度雷帕霉素预处理PC12细胞3h、6h、12h和24h，100μM浓度6-OHDA诱导24h后，使用CCK-8细胞计数试剂盒检测PC12细胞不同时间点的存活率。采用3μM浓度雷帕霉素预处理6h，100μM浓度6-OHDA诱导24h后，使用丙啶碘化物(Propidium iodide, PI)单染试剂盒检测雷帕霉素对损伤PC12细胞周期的影响。同时使用AnnexinⅤ-FITC和PI双染试剂盒，通过流式细胞仪检测各处理组PC12细胞凋亡的变化。雷帕霉素预处理6h，6-OHDA诱导1h后，使用含DCFH-DA荧光探针的试剂盒检测各处理组PC12细胞中活性氧(reactive oxygenspecies, ROS)的生成。收集各组PC12细胞，提取细胞总RNA，通过real-time RT-PCR检测各处理组PC12细胞内P47phox、TH、血红素加氧酶-1(hemo oxygenase-1, HO-1)和转录因子NF-E2相关因子2(transcriptional factor NF-E2-related factor2，Nrf2)的mRNA表达。
【结果】
一、动物实验结果：(1)与模型组相比，各剂量雷帕霉素预处理组大鼠的旋转行为减轻(p<0.05)。(2)与假手术组相比，PD模型组损伤侧黑质部位中TH+细胞数量明显减少(p<0.001)；与模型组比较，各雷帕霉素预处理组大鼠损伤侧TH+细胞数量则明显增加(p<0.001)。(3)模型组黑质-纹状体细胞器结构不清，细胞核皱缩，染色质异常聚集；线粒体体积增大，呈空泡样变，线粒体嵴溶解。而不同剂量的雷帕霉素预处理则明显减轻细胞超微结构的异常。(4)与假手术组比较，PD模型组MDA含量明显升高(p<0.001)，而SOD和GSH-PX的活性明显降低(p<0.001)。与PD模型组比较，经雷帕霉素预处理后，各剂量组MDA的含量均显著降低(p<0.01)，而SOD和GSH-PX的活性较PD模型组升高(p<0.05)。(5)与假手术组相比，PD模型组Bcl-2mRNA水平(p=0.001)明显降低而Bax mRNA水平(p<0.001)明显升高。与PD模型组比较，雷帕霉素预处理升高Bcl-2mRNA水平(p<0.01)，同时降低BaxmRNA的表达(p<0.05)，同时Bcl-2和Bax蛋白的表达与RT-PCR的检测结果相似。(6)与假手术组比较，PD模型组P47phoxmRNA的表达明显升高(p<0.001)，而雷帕霉素预处理显著下调PD大鼠模型中P47phoxmRNA的表达(p<0.01)。在PD模型组中活性caspase-9的表达和细胞色素c的释放比假手术组均明显升高(p<0.001)。与PD模型组比较，不同剂量雷帕霉素预处理均可降低PD大鼠活性caspase-9的表达和细胞色素c的释放(p<0.05)。然而，在雷帕霉素三个剂量组之间，所有研究结果包括旋转行为、TH+细胞数量、氧化应激指标及Bcl-2、Bax、P47phox、细胞色素c和caspase-9的表达均无显著统计学差异。
二、细胞实验结果：(1)100μM浓度的6-OHDA处理24小时后，PC12细胞接近半数死亡，其存活率为57.67±9.84%。(2)0.5μM-15μM浓度的雷帕霉素单独作用24h，PC12细胞存活率与空白对照组相比均无统计学差异(p>0.05)，而雷帕霉素单独作用48h和72h则出现抑制PC12细胞存活率的现象。(3)与6-OHDA组相比，当0.5μM-3μM浓度的雷帕霉素预处理PC12细胞3h-12h，明显升高损伤细胞的存活率(p<0.05)。其中，3μM雷帕霉素预处理6h对损伤PC12细胞存活率的改善作用最显著(p<0.01)。(4)与空白对照组相比，6-OHDA使PC12细胞中处于G0/G1期的细胞明显增多(p<0.001)，S期(p=0.001)和G2/M期(p=0.009)细胞则明显减少。而3μM雷帕霉素的预处理后6h后，G0/G1期细胞与6-OHDA组比较显著减少(p=0.018)，S期细胞明显增多(p=0.028)，但G2/M期细胞数量无统计学差异。(5)单加雷帕霉素对PC12细胞的凋亡无明显影响，6-OHDA处理后细胞凋亡较正常细胞明显增多(p<0.001)，而3μM雷帕霉素预处理6h，则明显减少6-OHDA所诱导的细胞凋亡(p<0.05)。(6)单加雷帕霉素对PC12细胞内ROS的水平无明显影响，6-OHDA的诱导使PC12细胞内ROS含量明显增多(p<0.01)，而雷帕霉素预处理则抑制损伤细胞中ROS的生成(p<0.05)。(7)单用3μM雷帕霉素处理正常PC12细胞6h-12h，HO-1和Nrf2mRNA的表达明显升高(p<0.05)，而在处理24h-48h两者的表达又回落至接近0h水平；(8)与正常PC12细胞比较，6-OHDA显著升高PC12细胞中P47phox而降低TH mRNA的表达(p<0.05)；与6-OHDA组相比，3μM雷帕霉素预处理6h后，P47phoxmRNA的表达下降而TH mRNA的表达明显升高(p<0.05)。与正常对照组比较，6-OHDA可升高PC12细胞中HO-1和Nrf2mRNA的表达(p<0.05)，与6-OHDA组相比，3μM雷帕霉素预处理6h后，则进一步上调HO-1和Nrf2mRNA的表达(p<0.05)。
【结论】
雷帕霉素可有效改善PD大鼠模型的行为学症状、降低黑质多巴胺能神经元的丢失和促凋亡分子的表达。在PD细胞模型中证实，雷帕霉素具有提高损伤细胞的生存能力和减少细胞凋亡的生物学作用。细胞周期的改变和抗氧化应激能力的增强可能是其神经保护作用的重要机制。由此，恢复氧化应激中调控信号的平衡可能为PD的药物治疗拓展新的途径。
Objective
Parkinson's disease (PD) is a neurodegenerative disease of central nervous system,in which oxidative stress and neuronal apoptosis play a key role in its pathogenesis.Rapamycin, a widely used immunosuppressant, is involved in some pathophysiologicalprocesses by inactivating the target of rapamycin (TOR). In a series ofneurodegenerative diseases, rapamycin displayed crucial significance in theirtherapeutic pathogenesis. However, role of rapamycin in PD therapy is not well known.In view of this, this study pre-treated6-hydroxydopamine (6-OHDA)-induced cell andrat models of PD with rapamycin to explore its roles and mechanisms inneuroprotections.
Methods
In vivo, for the induction of PD,6-OHDA (4μg/μl,2μl/site) was injected into2different sites within the right striatum of female Sprague-Dawley (SD) rats. SD ratswere randomly divided into the following5groups: Sham-operated (group S); PDmodel (group P); Pre-treated with a low (group L-R,0.05mg/kg/d), moderate (groupM-R,0.5mg/kg/d) or high (group H-R,5mg/kg/d) dose of rapamycin. Different doses ofrapamycin or vehicle were intragastrically administered once daily to the rats from day7before the induction of PD. On day25all animals underwent rotational testing byusing apomorphine (0.5mg/kg) to evaluate the motor asymmetry caused by the unilateral nigrostriatal lesion. On day28, the rats were sacrificed by cardiac perfusionand the brains were removed. Tyrosine hydroxylase (TH)++neurons in the substantianigra were evaluated by immunohistochemistry. Neuronal and mitochondrialultrastructures were detected by transmission electron microscope (TEM). Peroxidelevels and antioxidant activities in the midbrain were measured by assay kits, includingmalondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX). After total RNA was extracted, semi-quantitative RT-PCR was performedto examine the mRNA levels of Bcl-2, Bax and P47phox. The protein expressions ofBcl-2, Bax, cytochrome c and cleaved caspase-9were detected by western blot.
In vitro, cell PD model was induced by various concentrations of6-OHDA for24hin PC12cells. The damaged PC12cells induced by6-OHDA were pre-treated withvarious concentrations of rapamycin at different time points. The viability of damagedPC12cells was determined by cell counting kit-8(CCK-8). After selection of optimalconcentration, the damaged PC12cells induced by6-OHDA were pre-treated with3μMrapamycin for6h. The cycles of damaged PC12cells were analyzed by propidiumiodide (PI) staining kit in a flow cytometer, which was also employed to detect theapoptosis of PC12cells by Annexin Ⅴ-FITC and PI double staining kit. Afterrapamycin-pretreated PC12cells were induced by6-OHDA for1h, reactive oxygenspecies (ROS) were measured by an assay kit containing a DCFH-DA probe. Followingextraction of total RNA in treated PC12cells, real-time reversetranscriptional-polymerase chain reaction (RT-PCR) was performed to determine geneexpressions of P47phox, TH, hemo oxygenase-1(HO-1) and transcriptional factorNF-E2-related factor2(Nrf2).
Results
In vivo experiment:(1) Pre-treatment with various doses of rapamycin significantlyreduced the number of contralateral rotations as compared with those in group P(p<0.05).(2) The number of remaining TH+neurons in the substantia nigra from groupP rats was significantly lower as compared with that from group S rats (p<0.001). Pre-treatment with rapamycin significantly increased the number of TH+neurons in thesubstantia nigra as compared to group P (p<0.001).(3) TEM detections demonstratedcondensed chromatin in shrunken nuclei in the neurons of PD model rats. Themitochondria were evidently swelled and vacuolated, and the cristae were lessened,distorted or even disappeared. However, in the rats pre-treated with rapamycin，ultrastructual injuries were significantly alleviated.(4) In group P rats, MDA levelswere elevated but SOD and GSH-PX activities were decreased as compared with thosein group S rats (p<0.001). Following pre-treatment with rapamycin, a reduction in MDAlevels and an increase in SOD and GSH-PX activities were observed as compared withthose in group P (p<0.05).(5) In group P rats, RT-PCR analyses showed lower Bcl-2but higher Bax mRNA levels than those in group S rats (p<0.01). Pre-treatment withrapamycin significantly increased Bcl-2and decreased Bax mRNA levels as comparedto pre-treatment with the vehicle (group P)(p<0.01). Western blot analyses revealedsimilar results to RT-PCR detection in protein expression of Bcl-2and Bax (p<0.05).(6)In group P rats, RT-PCR analyses showed that the P47phoxmRNA levels weresignificant elevated as compared with those in group S rats (p<0.001). Pre-treatmentwith rapamycin significantly reduced P47phoxmRNA expression as compared topre-treatment with vehicle (group P)(p<0.01). Western blot analyses demonstrated thatthe release of cytochrome c and the expression of cleaved caspase-9were increased ingroup P rats, as compared with those in group S rats (p<0.001). Pre-treatment withrapamycin significantly suppressed cytochrome c release and caspase-9expression ascompared with pre-treatment with the vehicle (group P)(p<0.05). In addition, all theresults including behavioral rotations, remaining TH+neurons, oxidative stressindicators, as well as expressions of Bcl-2, Bax, P47phox, cytochrome c and cleavedcaspase-9showed no statistical differences among all doses of rapamycin pre-treatedgroups.
In vitro experiment:(1) PD cell models were successfully induced by100μMconcentration of6-OHDA for24h in PC12cells, which resulted in57.67±9.84%of cellviability.(2) As compared with blank control, mono-treatment with0.5μM-15μM rapamycin for24h exhibited no impact on the viability of normal PC12cells (p>0.05).However, mono-treatment with rapamycin for48h and72h showed inhibition of theviability of PC12cells.(3) Pre-treatment with0.5μM-3μM rapamycin for3h-12hsignificantly elevated the viability of damaged PC12cells induced by6-OHDA(p<0.05), of which pre-treatment with3μM rapamycin for6h offered an optimalcondition (p<0.01).(4) Cycle analyses demonstrated that6-OHDA remarkablyincreased the percentage of PC12cells in G0/G1phase (p<0.001), and decreased thepercentage of PC12cells in S and G2/M phase (p<0.01). Following pre-treatment with3μM rapamycin for6h, in damaged PC12cells, the percentage of G0/G1phase wasobviously reduced (p=0.018) and the percentage of S phase was elevated (p=0.028) ascompared with6-OHDA group.(5) Mono-treatment with rapamycin had no effect onapoptosis of normal PC12cells. However,6-OHDA induction significantly promotedapoptosis of PC12cells (p<0.001), which could be remarkably alleviated bypre-treatment with3μM rapamycin for6h (p<0.05).(6) There was no differencebetween normal and rapamycin mono-treated cells on ROS levels, and6-OHDAsignificantly promoted the production of ROS in damaged PC12cells (p<0.01).However, pre-treatment of rapamycin resulted in a reduction of ROS levels in6-OHDA-induced PC12cells (p<0.05).(7) After mono-treatment with3μM rapamycinfor6h-12h, mRNA expressions of HO-1and Nrf2were higher than in normal PC12cells (p<0.05), which fell to baseline levels in24h-48h.(8)6-OHDA inductionobviously increase P47phoxbut reduced TH mRNA expressions in PC12cells (p<0.05),which were reversed by pre-treatment with3μM rapamycin for6h (p<0.05). HO-1andNrf2mRNA expressions in damaged PC12cells induced by6-OHDA were higher thanthose in normal PC12cells (p<0.05), which were further up-regulated by pre-treatmentwith rapamycin (p<0.05).
Conclusions
Rapamycin could provide behavioral improvements, protect against the loss ofdopaminergic neurons and reduce expressions of pro-apoptotic markers in PD rats, which was consistent with enhanced viability and reduced apoptosis of damaged PC12cells in rapamycin-pretreated PD cell models. In PD, enhanced capabilities againstoxidative stress and changes in cell cycles may play a key role in the neuroprotectivemechanisms of rapamycin. In view of this, recovery of equilibrium between pro-andanti-oxidative stress signals may provide a promising strategy in PD therapy.
帕金森病(Parkinson’s disease, PD)是中枢神经系统变性疾病，线粒体氧化应激和神经元凋亡在PD的病理机制中发挥关键的作用。雷帕霉素(rapamycin)为雷帕霉素靶蛋白(Target of rapamycin, TOR)信号通路特异性抑制剂，在一系列神经系统变性疾病的病理机制及治疗中具有重要的意义。本研究通过雷帕霉素预处理PD大鼠及细胞模型，揭示其在PD中的神经保护作用，并探讨其机制。
【方法】
动物实验：实验SD大鼠随机分为假手术组、PD模型组、雷帕霉素低剂量组(0.05mg/kg/d)、中剂量组(0.5mg/kg/d)及高剂量组(5mg/kg/d)进行雷帕霉素灌胃预处理。7天后，向模型组和所有雷帕霉素预处理组大鼠脑部右侧纹状体两点定向注射6-羟基多巴(6-hydroxydopamine，6-OHDA)(4μg/μl,2μl/注射点)诱导建立PD大鼠模型，假手术组大鼠注射生理盐水作为对照。术后第25天各组大鼠均用阿扑吗啡(0.5mg/kg)诱导旋转试验，检测行为学指标；在造模后第28天，各组大鼠经心脏灌注固定，取脑组织进行黑质-纹状体酪氨酸羟化酶（Tyrosine hydroxylase，TH）免疫组化染色及透射电子显微镜观察；检测各组SD大鼠中脑丙二醛(malondialdehyde, MDA)含量及超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-PX)活性；提取脑组织总RNA后，采用RT-PCR方法检测黑质-纹状体内Bax、Bcl-2和p47phox等基因mRNA的表达；同时采用western blot方法检测黑质-纹状体内Bax、Bcl-2、细胞色素c以及cleavedcaspase-9蛋白质的表达。
体外实验：采用不同浓度6-OHDA诱导24h后检测PC12细胞存活率，建立PD细胞模型。不同浓度雷帕霉素预处理PC12细胞3h、6h、12h和24h，100μM浓度6-OHDA诱导24h后，使用CCK-8细胞计数试剂盒检测PC12细胞不同时间点的存活率。采用3μM浓度雷帕霉素预处理6h，100μM浓度6-OHDA诱导24h后，使用丙啶碘化物(Propidium iodide, PI)单染试剂盒检测雷帕霉素对损伤PC12细胞周期的影响。同时使用AnnexinⅤ-FITC和PI双染试剂盒，通过流式细胞仪检测各处理组PC12细胞凋亡的变化。雷帕霉素预处理6h，6-OHDA诱导1h后，使用含DCFH-DA荧光探针的试剂盒检测各处理组PC12细胞中活性氧(reactive oxygenspecies, ROS)的生成。收集各组PC12细胞，提取细胞总RNA，通过real-time RT-PCR检测各处理组PC12细胞内P47phox、TH、血红素加氧酶-1(hemo oxygenase-1, HO-1)和转录因子NF-E2相关因子2(transcriptional factor NF-E2-related factor2，Nrf2)的mRNA表达。
【结果】
一、动物实验结果：(1)与模型组相比，各剂量雷帕霉素预处理组大鼠的旋转行为减轻(p<0.05)。(2)与假手术组相比，PD模型组损伤侧黑质部位中TH+细胞数量明显减少(p<0.001)；与模型组比较，各雷帕霉素预处理组大鼠损伤侧TH+细胞数量则明显增加(p<0.001)。(3)模型组黑质-纹状体细胞器结构不清，细胞核皱缩，染色质异常聚集；线粒体体积增大，呈空泡样变，线粒体嵴溶解。而不同剂量的雷帕霉素预处理则明显减轻细胞超微结构的异常。(4)与假手术组比较，PD模型组MDA含量明显升高(p<0.001)，而SOD和GSH-PX的活性明显降低(p<0.001)。与PD模型组比较，经雷帕霉素预处理后，各剂量组MDA的含量均显著降低(p<0.01)，而SOD和GSH-PX的活性较PD模型组升高(p<0.05)。(5)与假手术组相比，PD模型组Bcl-2mRNA水平(p=0.001)明显降低而Bax mRNA水平(p<0.001)明显升高。与PD模型组比较，雷帕霉素预处理升高Bcl-2mRNA水平(p<0.01)，同时降低BaxmRNA的表达(p<0.05)，同时Bcl-2和Bax蛋白的表达与RT-PCR的检测结果相似。(6)与假手术组比较，PD模型组P47phoxmRNA的表达明显升高(p<0.001)，而雷帕霉素预处理显著下调PD大鼠模型中P47phoxmRNA的表达(p<0.01)。在PD模型组中活性caspase-9的表达和细胞色素c的释放比假手术组均明显升高(p<0.001)。与PD模型组比较，不同剂量雷帕霉素预处理均可降低PD大鼠活性caspase-9的表达和细胞色素c的释放(p<0.05)。然而，在雷帕霉素三个剂量组之间，所有研究结果包括旋转行为、TH+细胞数量、氧化应激指标及Bcl-2、Bax、P47phox、细胞色素c和caspase-9的表达均无显著统计学差异。
二、细胞实验结果：(1)100μM浓度的6-OHDA处理24小时后，PC12细胞接近半数死亡，其存活率为57.67±9.84%。(2)0.5μM-15μM浓度的雷帕霉素单独作用24h，PC12细胞存活率与空白对照组相比均无统计学差异(p>0.05)，而雷帕霉素单独作用48h和72h则出现抑制PC12细胞存活率的现象。(3)与6-OHDA组相比，当0.5μM-3μM浓度的雷帕霉素预处理PC12细胞3h-12h，明显升高损伤细胞的存活率(p<0.05)。其中，3μM雷帕霉素预处理6h对损伤PC12细胞存活率的改善作用最显著(p<0.01)。(4)与空白对照组相比，6-OHDA使PC12细胞中处于G0/G1期的细胞明显增多(p<0.001)，S期(p=0.001)和G2/M期(p=0.009)细胞则明显减少。而3μM雷帕霉素的预处理后6h后，G0/G1期细胞与6-OHDA组比较显著减少(p=0.018)，S期细胞明显增多(p=0.028)，但G2/M期细胞数量无统计学差异。(5)单加雷帕霉素对PC12细胞的凋亡无明显影响，6-OHDA处理后细胞凋亡较正常细胞明显增多(p<0.001)，而3μM雷帕霉素预处理6h，则明显减少6-OHDA所诱导的细胞凋亡(p<0.05)。(6)单加雷帕霉素对PC12细胞内ROS的水平无明显影响，6-OHDA的诱导使PC12细胞内ROS含量明显增多(p<0.01)，而雷帕霉素预处理则抑制损伤细胞中ROS的生成(p<0.05)。(7)单用3μM雷帕霉素处理正常PC12细胞6h-12h，HO-1和Nrf2mRNA的表达明显升高(p<0.05)，而在处理24h-48h两者的表达又回落至接近0h水平；(8)与正常PC12细胞比较，6-OHDA显著升高PC12细胞中P47phox而降低TH mRNA的表达(p<0.05)；与6-OHDA组相比，3μM雷帕霉素预处理6h后，P47phoxmRNA的表达下降而TH mRNA的表达明显升高(p<0.05)。与正常对照组比较，6-OHDA可升高PC12细胞中HO-1和Nrf2mRNA的表达(p<0.05)，与6-OHDA组相比，3μM雷帕霉素预处理6h后，则进一步上调HO-1和Nrf2mRNA的表达(p<0.05)。
【结论】
雷帕霉素可有效改善PD大鼠模型的行为学症状、降低黑质多巴胺能神经元的丢失和促凋亡分子的表达。在PD细胞模型中证实，雷帕霉素具有提高损伤细胞的生存能力和减少细胞凋亡的生物学作用。细胞周期的改变和抗氧化应激能力的增强可能是其神经保护作用的重要机制。由此，恢复氧化应激中调控信号的平衡可能为PD的药物治疗拓展新的途径。
Objective
Parkinson's disease (PD) is a neurodegenerative disease of central nervous system,in which oxidative stress and neuronal apoptosis play a key role in its pathogenesis.Rapamycin, a widely used immunosuppressant, is involved in some pathophysiologicalprocesses by inactivating the target of rapamycin (TOR). In a series ofneurodegenerative diseases, rapamycin displayed crucial significance in theirtherapeutic pathogenesis. However, role of rapamycin in PD therapy is not well known.In view of this, this study pre-treated6-hydroxydopamine (6-OHDA)-induced cell andrat models of PD with rapamycin to explore its roles and mechanisms inneuroprotections.
Methods
In vivo, for the induction of PD,6-OHDA (4μg/μl,2μl/site) was injected into2different sites within the right striatum of female Sprague-Dawley (SD) rats. SD ratswere randomly divided into the following5groups: Sham-operated (group S); PDmodel (group P); Pre-treated with a low (group L-R,0.05mg/kg/d), moderate (groupM-R,0.5mg/kg/d) or high (group H-R,5mg/kg/d) dose of rapamycin. Different doses ofrapamycin or vehicle were intragastrically administered once daily to the rats from day7before the induction of PD. On day25all animals underwent rotational testing byusing apomorphine (0.5mg/kg) to evaluate the motor asymmetry caused by the unilateral nigrostriatal lesion. On day28, the rats were sacrificed by cardiac perfusionand the brains were removed. Tyrosine hydroxylase (TH)++neurons in the substantianigra were evaluated by immunohistochemistry. Neuronal and mitochondrialultrastructures were detected by transmission electron microscope (TEM). Peroxidelevels and antioxidant activities in the midbrain were measured by assay kits, includingmalondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX). After total RNA was extracted, semi-quantitative RT-PCR was performedto examine the mRNA levels of Bcl-2, Bax and P47phox. The protein expressions ofBcl-2, Bax, cytochrome c and cleaved caspase-9were detected by western blot.
In vitro, cell PD model was induced by various concentrations of6-OHDA for24hin PC12cells. The damaged PC12cells induced by6-OHDA were pre-treated withvarious concentrations of rapamycin at different time points. The viability of damagedPC12cells was determined by cell counting kit-8(CCK-8). After selection of optimalconcentration, the damaged PC12cells induced by6-OHDA were pre-treated with3μMrapamycin for6h. The cycles of damaged PC12cells were analyzed by propidiumiodide (PI) staining kit in a flow cytometer, which was also employed to detect theapoptosis of PC12cells by Annexin Ⅴ-FITC and PI double staining kit. Afterrapamycin-pretreated PC12cells were induced by6-OHDA for1h, reactive oxygenspecies (ROS) were measured by an assay kit containing a DCFH-DA probe. Followingextraction of total RNA in treated PC12cells, real-time reversetranscriptional-polymerase chain reaction (RT-PCR) was performed to determine geneexpressions of P47phox, TH, hemo oxygenase-1(HO-1) and transcriptional factorNF-E2-related factor2(Nrf2).
Results
In vivo experiment:(1) Pre-treatment with various doses of rapamycin significantlyreduced the number of contralateral rotations as compared with those in group P(p<0.05).(2) The number of remaining TH+neurons in the substantia nigra from groupP rats was significantly lower as compared with that from group S rats (p<0.001). Pre-treatment with rapamycin significantly increased the number of TH+neurons in thesubstantia nigra as compared to group P (p<0.001).(3) TEM detections demonstratedcondensed chromatin in shrunken nuclei in the neurons of PD model rats. Themitochondria were evidently swelled and vacuolated, and the cristae were lessened,distorted or even disappeared. However, in the rats pre-treated with rapamycin，ultrastructual injuries were significantly alleviated.(4) In group P rats, MDA levelswere elevated but SOD and GSH-PX activities were decreased as compared with thosein group S rats (p<0.001). Following pre-treatment with rapamycin, a reduction in MDAlevels and an increase in SOD and GSH-PX activities were observed as compared withthose in group P (p<0.05).(5) In group P rats, RT-PCR analyses showed lower Bcl-2but higher Bax mRNA levels than those in group S rats (p<0.01). Pre-treatment withrapamycin significantly increased Bcl-2and decreased Bax mRNA levels as comparedto pre-treatment with the vehicle (group P)(p<0.01). Western blot analyses revealedsimilar results to RT-PCR detection in protein expression of Bcl-2and Bax (p<0.05).(6)In group P rats, RT-PCR analyses showed that the P47phoxmRNA levels weresignificant elevated as compared with those in group S rats (p<0.001). Pre-treatmentwith rapamycin significantly reduced P47phoxmRNA expression as compared topre-treatment with vehicle (group P)(p<0.01). Western blot analyses demonstrated thatthe release of cytochrome c and the expression of cleaved caspase-9were increased ingroup P rats, as compared with those in group S rats (p<0.001). Pre-treatment withrapamycin significantly suppressed cytochrome c release and caspase-9expression ascompared with pre-treatment with the vehicle (group P)(p<0.05). In addition, all theresults including behavioral rotations, remaining TH+neurons, oxidative stressindicators, as well as expressions of Bcl-2, Bax, P47phox, cytochrome c and cleavedcaspase-9showed no statistical differences among all doses of rapamycin pre-treatedgroups.
In vitro experiment:(1) PD cell models were successfully induced by100μMconcentration of6-OHDA for24h in PC12cells, which resulted in57.67±9.84%of cellviability.(2) As compared with blank control, mono-treatment with0.5μM-15μM rapamycin for24h exhibited no impact on the viability of normal PC12cells (p>0.05).However, mono-treatment with rapamycin for48h and72h showed inhibition of theviability of PC12cells.(3) Pre-treatment with0.5μM-3μM rapamycin for3h-12hsignificantly elevated the viability of damaged PC12cells induced by6-OHDA(p<0.05), of which pre-treatment with3μM rapamycin for6h offered an optimalcondition (p<0.01).(4) Cycle analyses demonstrated that6-OHDA remarkablyincreased the percentage of PC12cells in G0/G1phase (p<0.001), and decreased thepercentage of PC12cells in S and G2/M phase (p<0.01). Following pre-treatment with3μM rapamycin for6h, in damaged PC12cells, the percentage of G0/G1phase wasobviously reduced (p=0.018) and the percentage of S phase was elevated (p=0.028) ascompared with6-OHDA group.(5) Mono-treatment with rapamycin had no effect onapoptosis of normal PC12cells. However,6-OHDA induction significantly promotedapoptosis of PC12cells (p<0.001), which could be remarkably alleviated bypre-treatment with3μM rapamycin for6h (p<0.05).(6) There was no differencebetween normal and rapamycin mono-treated cells on ROS levels, and6-OHDAsignificantly promoted the production of ROS in damaged PC12cells (p<0.01).However, pre-treatment of rapamycin resulted in a reduction of ROS levels in6-OHDA-induced PC12cells (p<0.05).(7) After mono-treatment with3μM rapamycinfor6h-12h, mRNA expressions of HO-1and Nrf2were higher than in normal PC12cells (p<0.05), which fell to baseline levels in24h-48h.(8)6-OHDA inductionobviously increase P47phoxbut reduced TH mRNA expressions in PC12cells (p<0.05),which were reversed by pre-treatment with3μM rapamycin for6h (p<0.05). HO-1andNrf2mRNA expressions in damaged PC12cells induced by6-OHDA were higher thanthose in normal PC12cells (p<0.05), which were further up-regulated by pre-treatmentwith rapamycin (p<0.05).
Conclusions
Rapamycin could provide behavioral improvements, protect against the loss ofdopaminergic neurons and reduce expressions of pro-apoptotic markers in PD rats, which was consistent with enhanced viability and reduced apoptosis of damaged PC12cells in rapamycin-pretreated PD cell models. In PD, enhanced capabilities againstoxidative stress and changes in cell cycles may play a key role in the neuroprotectivemechanisms of rapamycin. In view of this, recovery of equilibrium between pro-andanti-oxidative stress signals may provide a promising strategy in PD therapy.
引文
1. Orth M, Schapira AH. Mitochondrial involvement in Parkinson’s disease.Neurochem Int2002;40(6):533–541.
2. Rajasankar S, Manivasagam T, Surendran S. Ashwagandha leaf extract: a potentialagent in treating oxidative damage and physiological abnormalities seen in a mousemodel of Parkinson’s disease. Neurosci Lett2009;454(1):11–15.
3. Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease:a mechanism of pathogenic and therapeutic significance. Ann N Y Acad Sci2008;1147:93-104.
4. Brenner-Lavie H, Klein E, Zuk R, et al. Dopamine modulates mitochondrialfunction in viable SH—SY5Y cells possibly via its interaction with complex I：relevance to dopamine pathologyin schizophrenia．Biochim Biophys Acta2008；1777(2)：173—185.
5. Mitsumoto A, Nakagawa Y, Takeuchi A, et al. Oxidized forms of peroxiredoxins andDJ-1on two-dimensional gels increased in response to sublethal levels of paraquat.Free Radic Res2001;35(3):301–310.
6. Taira T, Saito Y, Niki T, et al. DJ-1has a role in antioxidative stress to prevent celldeath. EMBO Rep2004;5(4):213–218.
7. Eriksen JL, Dawson TM, Dickson DW, et al. Caught in the act: α-synuclein is theculprit in Parkinson’s disease. Neuron2003;40(3):453-456.
8. Ethell DW, Fei Q. Parkinson-linked genes and toxins that affect neuronal cell deaththrough the Bcl-2family. Antioxid Redox Signal2009;11(3):529-540.
9. Agid Y．Aging，disease and nerve cell death．Bull Acad Natl Med l995;179(6):l193-l203.
10. Fiskum G, S tarkov A, Polster BM, et al. Mitochondrial mechanisms of neural celldeath and neuroprotective interventions in Parkinson’s disease. Ann N Y Acd Sci2003;991:111-119.
11. Shoulson I, Oakes D, Fahn S, et al. Impact of sustained deprenyl (selegiline) inlevodopa-treated Parkinson’s disease: A randomized placebo-controlled extension ofthe deprenyl and tocopherol antioxidative therapy of Parkinsonism trial. Ann Neurol2002;51(5):604–612.
12. P lhagen S, Heinonen E, H gglund J, et al. Swedish Parkinson StudyGroup.Selegiline slows the progression of the symptoms of Parkinson disease. Neurology2006;66(8):1200–1206.
13. Parkinson Study Group. A controlled, randomized, delayed-start study of rasagilinein early Parkinson disease. Arch Neurol2004;61(4):561–566.
14. Olanow CW, Rascol O, Hauser R, et al. ADAGIO Study Investigators. Adouble-blind, delayed-start trial of rasagiline in Parkinson’s disease. N Engl J Med2009;361(13):1268–1278.
15. Smeding HM, Speelman JD, Huizenga HM, et al. Predictors of cognitive andpsychosocial outcome after STN DBS in Parkinson’s disease. J Neurol NeurosurgPsychiatry20l1;82(7):754-760.
16. Murakami M, Ichisaka T, Maeda M, et al. mTOR is essential for growth andproliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol2004;24(15),6710–6718.
17. Gangloff YG, Mueller M, Dann SG, et al. Disruption of the mouse mTOR gene leadsto early postimplantation lethality and prohibits embryonic stem cell development.Mol Cell Biol2004;24(21):9508–9516.
18. Sudarsanam S, Johnson DE. Functional consequences of mTOR inhibition. CurrOpin Drug Discov Devel2010;13(1):31-40.
19. Graziani EI. Recent advances in the chemistry, biosynthesis and pharmacology ofrapamycin analogs. Nat Prod Rep2009;26(5):602-609.
20. Jia K, Chen D, Riddle DL. The TOR pathway interacts with the insulin signalingpathway to regulate C. elegans larval development, metabolism and life span.Development2004;131(16):3897–3906.
21. Selman C, Tullet JM, Wieser D, et al. Ribosomal protein S6kinase1signalingregulates mammalian life span. Science2009;326(5949):140–144.
22. Bonawitz ND, Chatenay-Lapointe M, Pan Y, et al. Reduced TOR signaling extendschronological life span via increased respiration and upregulation of mitochondrialgene expression. Cell Metab2007;5(4):265-277.
23. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespanin genetically heterogeneous mice. Nature2009;460(7253):392-395.
24. Schieke SM, Phillips D, McCoy JP Jr, et al. The mammalian target of rapamycin(mTOR) pathway regulates mitochondrial oxygen consumption and oxidativecapacity. J Biol Chem2006;281(37):27643-27652.
25. Cunningham JT, Rodgers JT, Arlow DH, et al. mTOR controls mitochondrialoxidative function through a YY1-PGC-1α transcriptional complex. Nature2007;450(7170):736–740.
26. D’Antona G, Ragni M, Cardile A, et al: Branched-chain amino acid supplementationpromotes survival and supports cardiac and skeletal muscle mitochondrialbiogenesis in middle-aged mice. Cell Metab2010;12(4):362–372.
27. Polak P, Cybulski N, Feige JN,et al. Adipose-specific knockout of raptor results inlean mice with enhanced mitochondrial respiration. Cell Metab2008;8(5):399–410.
28. Pan Y, Shadel GS. Extension of chronological life span by reduced TOR signalingrequires down-regulation of Sch9p and involves increased mitochondrial OXPHOScomplex density. Aging (Albany NY)2009;1(1):131–145.
29. Jing CH, Wang L, Liu PP, et al. Autophagy activation is associatedwith neuroprotection against apoptosis via a mitochondrial pathway in a rat modelof subarachnoid hemorrhage. Neuroscience.2012;213:144-153.
30. Tun o′n MJ, Sa′nchez-Campos S, Gutie′rrez B, et al. Effects of FK506andrapamycin on generation of reactive oxygen species, nitric oxide production andnuclear factor kappa B activation in rat hepatocytes. Biochem Pharmacol2003;66(3):439–445.
31. Kim JH, Chu SC, Gramlich JL, et al. Activation of the PI3K/mTOR pathway byBCRABL contributes to increased production of reactive oxygen species. Blood2005;105(4):1717–1723.
32. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci2006;119(pt20):4285–4292.
33. Pan Y, Schroeder EA, Ocampo A, et al. Regulation of yeast chronological lifespanby TORC1via adaptive mitochondrial ROS signaling. Cell Metab2011;13(6):668–678.
34. Wu JJ, Quijano C, Chen E, et al. Mitochondrial dysfunction and oxidative stressmediate the physiological impairment induced by the disruption of autophagy.Aging (Albany NY)2009;1(4):425–437.
35. Chen L, Xu B, Liu L, et al. Hydrogen peroxide inhibits mTOR signaling byactivation of AMPKalpha leading to apoptosis of neuronal cells. Lab Invest2010;90(5):762-773.
36. Chong ZZ, Li F, Maiese K. The pro-survival pathways of mTOR and protein kinaseB target glycogen synthase kinase-3beta and nuclear factor-kappaB to fosterendogenous microglial cell protection. Int J Mol Med2007;19(2):263-272.
37. Choi KC, Kim SH, Ha JY, et al. A novel mTOR activating protein protectsdopamine neurons against oxidative stress by repressing autophagy related celldeath. J Neurochem2010;112(2):366-376.
38. Ravikumar B, Berger Z, Vacher C, et al. Rapamycin pre-treatment protectsagainst apoptosis. Hum Mol Genet2006;15(7):1209-1216.
39. Carloni S, Buonocore G, Longini M, et al. Inhibition of rapamycin-inducedautophagy causes necrotic cell death associated with Bax/Bad mitochondrialtranslocation. Neuroscience2012;203:160-169.
40. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins withpolyglutamine and polyalanine expansions are degraded by autophagy. Hum MolGenet2002;11(9):1107-17.
41. Tsvetkov AS, Miller J, Arrasate M, et al. A small-molecule scaffold inducesautophagy in primary neurons and protects against toxicity in a Huntington diseasemodel. Proc Natl Acad Sci USA2010;107(39):16982–16987.
42. Ravikumar B, Vacher C, Berger Z, et al. Inhibition of mTOR induces autophagy andreduces toxicity of polyglutamine expansions in fly and mouse models ofHuntington disease. Nature Genet2004;36(6):585–595.
43. Sarkar S, Krishna G, Imarisio S, et al. A rational mechanism for combinationtreatment of Huntington’s disease using lithium and rapamycin. Hum MolGenet2008;17(2):170–178.
44. Fox JH, Connor T, Chopra V, et al. The mTOR kinase inhibitor Everolimusdecreases S6kinase phosphorylation but fails to reduce mutant huntingtin levels inbrain and is not neuroprotective in the R6/2mouse model of Huntington’s disease.Mol Neurodegener2010;5:26.
45. Caccamo A, Majumder S, Richardson A, et al. Molecular interplay betweenmammalian target of rapamycin (mTOR), amyloid-β, and Tau: effects on cognitiveimpairments. J Biol Chem2010;285(17):13107–13120.
46. Caccamo A, Maldonado MA, Majumder S, et al. Naturally secreted amyloid-βincreases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediatedmechanism. J Biol Chem2011;286(11):8924–8932.
47. Spilman P, Podlutskaya N, Hart MJ, et al. Inhibition of mTOR by rapamycinabolishes cognitive deficits and reduces amyloid-β levels in a mouse model ofAlzheimer’s disease. PLoS ONE2010;5(4): e9979.
48. Zhang S, Salemi J, Hou H, et al. Rapamycin promotes β-amyloid production viaADAM-10inhibition. Biochem Biophys Res Commun2010;398(3):337–341.
49. Menzies FM, Huebener J, Renna M, et al. Autophagy induction reduces mutantataxin-3levels and toxicity in a mouse model of spinocerebellar ataxia type3. Brain2010;133(pt1):93–104.
50. Zhao C, Yasumura D, Li X, et al. mTOR-mediated dedifferentiation of the retinalpigment epithelium initiates photoreceptor degeneration in mice. J Clin Invest2011;121(1):369–383.
51. Zhang X, Li L, Chen S, et al. Rapamycin treatment augments motor neurondegeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis.Autophagy2011;7(4):412–425.
52. Dehay B, Bové J, Rodríguez-Muela N, et al. Pathogenic lysosomal depletionin Parkinson's disease. J Neurosci2010;30(37):12535-12544.
53. Chu Y, Dodiya H, Aebischer P, et al. Alterations in lysosomal and proteasomalmarkers in Parkinson’s disease: relationship to α-synuclein inclusions. NeurobiolDis2009;35(3):385–398.
54. Cullen V, Sardi SP, Ng J, et al. Acid β-glucosidase mutants linked to gaucher disease,Parkinson disease, and lewy body dementia alter α-synuclein processing. AnnNeurol2011;69(6):940–953.
55. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-induced apoptosisthrough autophagy induction. Neuroscience2009;164(2):541-551.
56. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis2008;32(1):16–25.
57. Yu WH, Dorado B, Figueroa HY, et al. Metabolic activity determines efficacy ofmacroautophagic clearance of pathological oligomeric α-synuclein. Am J Pathol2009;175(2):736–747.
58. Crews L, Spencer B, Desplats P, et al. Selective molecular alterations in theautophagy pathway in patients with Lewy body disease and in models ofα-synucleinopathy. PLoS ONE2010;5(2): e9313.
59. Sarkar S, Floto RA, Berger Z, et al. Lithium induces autophagy by inhibitinginositol monophosphatase. J Cell Biol2005;170(7):1101–1111.
60. Sarkar S, Davies JE, Huang Z, et al. Trehalose, a novel mTOR-independentautophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein.J Biol Chem2007;282(8):5641–5652.
61. Malagelada C, Ryu EJ, Biswas SC, et al. RTP801is elevated in Parkinson brainsubstantia nigral neurons and mediates death in cellular models of Parkinson’sdisease by a mechanism involving mammalian target of rapamycin inactivation. JNeurosci2006;26(39):9996–10005.
62. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci2010;30(3):1166–1175.
63. Yue Z, Horton A, Bravin M, et al. A novel protein complex linking the delta2glutamate receptor and autophagy: implications for neurodegeneration in lurchermice. Neuron2002;35(5):921–933.
64. Bauvy C, Gane P, Arico S, et al. Autophagy delays sulindac sulfide-inducedapoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res2001;268(2):139–149.
65. Boya P, González-Polo RA, Casares N, et al. Inhibition of macroautophagy triggersapoptosis. Mol Cell Biol2005;25(3):1025–1040.
66. Bjedov I, Toivonen JM, Kerr F, et al. Mechanisms of life span extension byrapamycin in the fruit fly Drosophila melanogaster. Cell Metab2010;11(1):35–46.
67. Webb JL, Ravikumar B, Atkins J, et al. Alpha-Synuclein is degraded byboth autophagy and the proteasome. J Biol Chem2003;278(27):25009-25013.
68. Siddiqui A, Hanson I, Andersen JK. Mao-B elevation decreases parkin's ability toefficiently clear damaged mitochondria: protective effects of rapamycin. Free RadicRes2012;46(8):1011-1018.
69. Liu K, Shi N, Sun Y, et al. Therapeutic effects of rapamycin on MPTP-inducedParkinsonism in mice. Neurochem Res2013;38(1):201-207.
1. Ravikumar B, Duden R and Rubinsztein DC. Aggregate-prone proteins withpolyglutamine and polyalanine expansions are degraded by autophagy. Hum MolGenet.2002;11(9):1107-1117.
2. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis.2008;32(1):16-25.
3. Cortes CJ, Qin K, Cook J, et al. Rapamycin delays disease onset and prevents PrPplaque deposition in a mouse model of Gerstmann-Str ussler-Scheinker disease. JNeurosci.2012;32(36):12396-12405.
4. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci.2010;30(3):1166–1175.
5. Caccamo A, Majumder S, Richardson A, et al. Molecular interplay betweenmammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects oncognitive impairments. J Biol Chem.2010;285(17):13107-13120.
6. Harrison DE, Strong R, Sharp ZD, et a1. Rapamycin fed late in life extends lifespanin genetically heterogeneous mice．Nature.2009;460(7253):392—395.
7. Spilman P, Padlutskaya N, Hart M J, et a1. Inhibition of mTOR by rapamycinabolishes cognitive deficits and reduces amyloid-beta levels in a mouse model ofAlzheimer’s disease．PLoS One,2010;5(4): e9979．
8. Ravikumar B, Vacher C, Berqer Z, et a1. Inhibition of mTOR induces autophagyand reduces toxicity of polyglutamine expansions in fly and mouse models ofHuntington disease. Nat Genet.2004;36(6):585—595.
9. Sarkar S, Krishna G, Imarisio S, et al. A rational mechanism for combinationtreatment of Huntington’s disease using lithium and rapamycin. Hum. Mol. Genet.2008;17(2):170–178.
10. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-inducedapoptosis through autophagy induction. Neuroscience.2009;164(2):541-551.
11. Webb JL, Ravikumar B, Atkins J, et a1. Alpha-synuelein is degraded by bothautophagy and the proteasome. J Biol Chem.2003;278(27):25009—25013.
12. Tain LS, Mortiboys H, Tao RN, et a1. Rapamycin activation of4E-BP preventsParkinsonian dopaminergic neuron loss. Nat Neurosci.2009;12(9):1129—1135.
13. Przedborski S&Vila M. The1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinemouse model: a tool to explore the pathogenesis of Parkinson’s disease. Ann N YAcad Sci.2003;991:189–198.
14. Liu K, Liu C, Shen L, et al. Therapeutic effects of rapamycin on MPTP-inducedParkinsonism in mice. Neurochem Res.2013;38(1):201-207.
15. Dabbeni SF, Di SS, Franceschini D, et al. Melatonin protects against6-OHDA-induced neurotoxicity in rats：a role for mitochondfial complex I activity.FASEB J.2001;15(1):164-170.
16. Acton PD, Mozley PD. Single photon emission tomography imaging inparkinsonian disorders：a review. Behav Neurol.2000;12(1，2):11-27.
17. Kirik D, Rosenblad C, Bjorklund A. Characterization of behavioral andneurodegenerative changes following partial lesions of the nigrostriatal dopaminesystem induced by intrastriatal6-hydroxydopamine in the rat. Exp Neurol.1998;152(2):259-277.
18. Hall ED, Detloff MR, Johnson K, et al. Peroxynitrite-mediated protein nitration andlipid peroxidation in a mouse model of traumatic brain injury. J Neurotrauma.2004;21(1):9-20.
19. Smith MP and Cass WA. Oxidative stress and dopamine depletion in an intrastriatal6-hydroxydopamine model of Parkinson's disease. Neuroscience.2007;144(3):1057-1066.
20. Smith MP and Cass WA. GDNF reduces oxidative stress in a6-hydroxydopaminemodel of Parkinson's disease. Neurosci Lett.2007;412(3):259-263.
21. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci.2006;119(Pt20):4285-4292.
22. Marobbio CM, Pisano I, Porcelli V, et al. Rapamycin reduces oxidative stress infrataxin-deficient yeast cells. Mitochondrion.2012;12(1):156-161.
23. Long J, Liu C, Sun L, et al. Neuronal mitochondrial toxicity of malondialdehyde:inhibitory effects on respiratory function and enzyme activities in rat brainmitochondria. Neurochem Res2009;34(4):786-794.
24. Onyango IG. Mitochondrial dysfunction and oxidative stress in Parkinson's disease.Neurochem Res.2008;33(3):589-597.
25. Martinou JC and Youle RJ. Mitochondria in apoptosis: Bcl-2family members andmitochondrial dynamics. Dev Cell.2011;21(1):92-101.
26. Kirkland RA and Franklin JL. Bax, reactive oxygen, and cytochrome c release inneuronal apoptosis. Antioxid Redox Signal2003;5(5):589-596.
27. Zhang C, Tian X, Luo Y,et al. Ginkgolide B attenuates ethanol-inducedneurotoxicity through regulating NADPH oxidases. Toxicology.2011;287(1-3):124-130.
28. Santini E, Heiman M, Greengard P, et al. Inhibition of mTOR signaling inParkinson's disease prevents L-DOPA-induced dyskinesia. Sci Signal2009;2(80):ra36.
29. Bhagwat SV and Crew AP. Novel inhibitors of mTORC1and mTORC2. Curr OpinInvestig Drugs.2010;11(6):638-645.
1. Maurer M，Tsai M，Metz M，et a1．A role for Bax in the regulation of apoptosis inmouse mast cells．J Invest Dermatol.2000;114(6):1205—1206.
2. Sch IM． Brain iron deposition and the free radical mitochondrial theory ofageing．Ageing Res Rev.2004;3(3):265—301．
3. Qian L，Flood PM．Microglial cells and Parkinson’s disease．Immunol Res.2008;41(3):155—164.
4. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci.2006;119(Pt20):4285-4292.
5. Marobbio CM, Pisano I, Porcelli V, et al. Rapamycin reduces oxidative stress infrataxin-deficient yeast cells. Mitochondrion.2012;12(1):156-61.
6. Bové J, Martínez-Vicente M, Vila M. Fighting neurodegeneration with rapamycin:mechanistic insights. Nat Rev Neurosci.2011;12(8):437-452.
7. David B， Sakina T， Nathalie L， et a1． Molecular pathway involved in theneurotoxicity of6-OHDA, dopamine and MPTP：contribution to the apoptotictheory in Parkinson’s disease．Prog Neurobiol.2001;65(2):135—172．
8. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-induced apoptosisthrough autophagy induction. Neuroscience.2009;164(2):541-551.
9. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibitsmTORC2assembly and Akt/PKB. Mol Cell.2006;22(2):159-168.
10. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis.2008;32(1):16–25.
11. Yang F, Yang YP, Mao CJ,et al. Role of autophagy and proteasome degradationpathways in apoptosis of PC12cells overexpressing human alpha-synuclein.Neurosci Lett.2009;454(3):203-208.
12. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci.2010;30(3):1166–1175.
13. Siddiqui A, Hanson I, Andersen JK. Mao-B elevation decreases parkin's ability toefficiently clear damaged mitochondria: protective effects of rapamycin. Free RadicRes.2012;46(8):1011-1018.
14. Armstrong JS, Homung B, Lecane P, et a1. Rotenone—induced G2／M cell cyclearrest and apoptosis in a human B lymphoma cell line PW. Biochem and BiophysRes Commun.2001;289(5):973—978.
15. Badisa RB, Darling-Reed SF, Soliman KF. The protective role of D-glucose against1-methyl-4-phenylpyridiniumion (MPP+): induced mitochondrial dysfunction in C6astroglial cells. Neurochem Res.2010;35(9):1413-1421.
16. Gao QG, Xie JX, Wong MS, et al. IGF-I receptor signaling pathway is involved inthe neuroprotective effect of genistein in the neuroblastoma SK-N-SH cells. Eur JPharmacol.2012;677(1-3):39-46.
17. Lanzi C, G Cassinelli, G Cuccuru, et al. Cell cycle checkpoint efficiency andcellular response to paclitaxel in prostate cancer cells. Prostate.2001;48(4):254-264.
18. Miyake N, Chikumi H, Takata M, et al. Rapamycin induces p53-independentapoptosis through the mitochondrial pathway in non-small cell lung cancercells.Oncol Rep.2012;28(3):848-854.
19. Dai ZJ, Gao J, Ma XB,et al. Antitumor effects of rapamycin in pancreatic cancercells by inducing apoptosis and autophagy. Int J Mol Sci.2012;14(1):273-285.
20. Moad AI, Tengku Muhammad TS, Oon CE, et al. Rapamycin Induces Apoptosis WhenAutophagy is inhibited in T-47D Mammary Cells and Both Processes are Regulated byPhlda1. Cell Biochem Biophys.2013;66(3):567-587.
21. Raaz U, Kuhn H, Wirtz H,et al. Rapamycin reduces high-amplitude, mechanicalstretch-induced apoptosis in pulmonary microvascular endothelial cells. MicrovascRes.2009;77(3):297-303.
22. Farrelly AM, Kilbride SM, Bonner C, et al. Rapamycin protects against dominantnegative-HNF1A-induced apoptosis in INS-1cells. Apoptosis.2011;16(11):1128-1137.
23. Bai J, Meng Z. Expression of apoptosis-related genes in liver from rats exposed tosulfur dioxide. Toxicology.2005;216(2-3):253–260.
24. Kim J, Cha YN, Surh YJ. A protective role of nuclear factor erythroid2-relatedfactor-2(Nrf2) in inflammatory disorders. Mutat. Res.2010;690(1-2):12–23.
25. Choi AM, Alam J. Heme oxygenase-1: function, regulation, and implication of anovel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell MolBiol.1996;15(1):9-19.
26. Kim Y, Li E, Park S. Insulin-like growth factor-1inhibits6-hydroxydopamine-mediated endoplasmic reticulum stress-induced apoptosis viaregulation of heme oxygenase-1and Nrf2expression in PC12cells. Int JNeurosci.2012;122(11):641-649.
27. Tobón-Velasco JC, Vázquez-Victorio G, Macías-Silva M, et al. S-allyl cysteineprotects against6-hydroxydopamine-induced neurotoxicity in the rat striatum:involvement of Nrf2transcription factor activation and modulation of signalingkinase cascades. Free Radic Biol Med.2012;53(5):1024-1040.
28. Cicora F, Lausada N, Vasquez DN, et al. Protective effect of immunosuppressivetreatment before orthotopic kidney autotransplantation. Transpl Immunol.2011;24(2):107-112.
29. Kist A, Wakkie J, Madu M, et al. Rapamycin induces heme oxygenase-1in liver butinhibits bile flow recovery after ischemia. J Surg Res.2012;176(2):468-475.
30. Visner G, Lu F, ZhouH, et al. Rapamycin induces heme oxygenase-1in humanpulmonary vascular cells: Implications in the antiproliferative response torapamycin. Circulation.2003;107(6):911-916.
31. Zoncu R, Efeyan A, Sabatini D. mTOR: From growth signal integration to cancer,diabetes and ageing. Nat Rev Mol Cell Biol.2011;12(1):21-35.
32. Hwang YP, Jeong HG. Ginsenoside Rb1protects against6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathwayin human dopaminergic cells. Toxicol. Appl. Pharmacol.2010;242(1):1828.
33. Gao HM, Liu B, and Hong JS. Critical role for microglial NADPH oxidase inrotenone-induced degeneration of dopaminergic neurons. Journal of Neuroscience.2003;23(15):6181–6187.
34. Koppula S, Kumar H, Kim IS, et al. Reactive oxygen species and inhibitors ofinflammatory enzymes, NADPH oxidase, and iNOS in experimental modelsof Parkinson's disease. Mediators Inflamm.2012;2012:823902.
35. Li X, Schwacha MG, Chaudry IH, et al. Heme oxygenase-1protects againstneutrophil-mediated intestinal damage by down-regulation of neutrophil p47phoxand p67phox activity and O2-production in a two-hit model of alcohol intoxicationand burn injury. J Immunol.2008;180(10):6933-6940.
36. Yu H, Huang J, Wang S, et al. Overexpression of Smad7suppressedROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1.Mol Biol Rep.2013;[Epub ahead of print].
[1] The Parkinson Study Group. Pramipexole vs Levodopa as Initial Treatment forParkinson Disease: A4-Year Randomized Controlled Trial. Arch Neurol.2004;61(7):1044-1053.
[2] Jenner P. A novel dopamine agonist for the transdermal treatment of Parkinson’sdisease. Neurology.2005;65(2Suppl1): S3-S5.
[3] LeWitt PA, Lyons KE, Pahwa R. Advanced Parkinson disease treated with rotigotinetransdermal system. Neurology.2007;68(16):1262-1267.
[4] Splinter MY. Rotigotine: Transdermal Dopamine Agonist Treatment of Parkinson'sdisease and Restless Legs Syndrome. Ann Pharmacother.2007;41(2):285-295.
[5] Baufreton J, Garret M, Rivera A, et al. D5(Not D1) Dopamine Receptors PotentiateBurst-Firing in Neurons of the Subthalamic Nucleus by Modulating an L-TypeCalcium Conductance. The Journal of Neuroscience.2003;23(3):816-825.
[6] Chergui K, Svenningsson P, Greengard P. Cyclin-dependent kinase5regulatesdopaminergic and glutamatergic transmission in the striatum. Proc Natl Acad SciU S A.2004;101(7):2191-2196.
[7] Chase TN, Bibbiani F,Bara-Jimenez W,et al. Translating A2Aantagonist KW6002from animal models to parkinsonian patients. Neurology.2003;61(11Suppl6):S107-S111.
[8] Bara-Jimenez W, Sherzai A, Dimitrova T, et al. Adenosine A2Areceptor antagonisttreatment of Parkinson’s disease. Neurology.2003;61(3):293-296.
[9] Weiss SM, Benwell K, Cliffe IA, et al. Discovery of nonxanthine adenosine A2Areceptor antagonists for the treatment of Parkinson’s disease. Neurology.2003;61(11Suppl6): S101-S106.
[10] Mihara T, Mihara K, Yarimizu J,et al. Pharmacological characterization of a novel,potent adenosine A1and A2A receptor dual antagonist, ASP5854, in models ofParkinson’s disease and cognition. J Pharmacol Exp Ther.2007;323(2):708-719.
[11] Marino MJ, Williams DL Jr, O'Brien JA, et al. Allosteric modulation of group IIImetabotropic glutamate receptor4: A potential approach to Parkinson’s diseasetreatment. Proc Natl Acad Sci U S A.2003;100(23):13668-13673.
[12] Lopez S, Turle-Lorenzo N, Acher F, et al. Targeting Group III MetabotropicGlutamate Receptors Produces Complex Behavioral Effects in Rodent Models ofParkinson's Disease. J Neurosci.2007;27(25):6701-6711.
[13] Li G, Cui G, Tzeng NS, et al. Femtomolar concentrations of dextromethorphanprotect mesencephalic dopaminergic neurons from inflammatory damage. FASEBJ.2005;19(6):489-496.
[14] Hernán MA, Logroscino G, García Rodríguez LA. Nonsteroidal anti-inflammatorydrugs and the incidence of Parkinson disease. Neurology.2006;66(7):1097-1099.
[15] Chen H, Zhang SM, Hernán MA, et al. Nonsteroidal Anti-inflammatory Drugs andthe Risk of Parkinson Disease. Arch Neurol.2003;60(8):1059-1064.
[16] Teismann P, Tieu K, Choi DK, et al. Cyclooxygenase-2is instrumental inParkinson’s disease neurodegeneration. Proc Natl Acad Sci U S A.2003;100(9):5473-5478.
[17] Wang T, Zhang W, Pei Z, et al. Reactive microgliosis participates inMPP+-induced dopaminergic neurodegeneration: role of67kDa laminin receptor.FASEB J.2006;20(7):906-915.
[18] Kulkarnl AP, Kellaway LA, Lahiri DK, et al. Neuroprotection fromComplement-Mediated Inflammatory Damage. Ann N Y Acad Sci.2004;1035:147-164.
[19] Shen J, Cookson MR. Mitochondria and dopamine: new insights into recessiveparkinsonism. Neuron.2004;43(3):301-304.
[20] Shults CW, Oakes D, Kieburtz K, et al. Effects of coenzyme Q10in earlyParkinson’s disease: evidence of slowing of the functional decline. Arch Neurol.2002;59(10):1541-1550.
[21] Bonuccelli U, Dotto DP. New pharmacologic horizons in the treatment ofParkinson disease.Neurology.2006;67(7Suppl2): S30-S38.
[22] Matthews RT, Ferrante RJ, Klivenyi P, et al. Creatine and cyclocreatine attenuateMPTP neurotoxicity. Exp Neurol.1999;157(1):142-149.
[23] Elm JJ, Goetz CG, Ravina B, et al. A responsive outcome for Parkinson’s diseaseneuroprotection futility studies. Ann Neurol.2005;57(2):197-203.
[24] Devos D, Krystkowiak P, Clement F, et al. Improvement of gait by chronic, highdoses of methylphenidate in patients with advanced Parkinson’s disease. J NeurolNeurosurg Psychiatry.2007;78(5):470-475.
[25] Ravina BM, Fagan SC, Hart RG, et al. Neuroprotective agents for clinical trials inParkinson’s disease. Neurology.2003;60(8):1234-1240.
[26] Iwashita A, Yamazaki S, Mihara K, et al. Neuroprotective Effects of a NovelPoly(ADP-Ribose) Polymerase-1Inhibitor,2-{3-[4-(4-Chlorophenyl)-1-piperazinyl] propyl}-4(3H)-quinazolinone(FR255595), in an in Vitro Model of Cell Death and in Mouse1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Model of Parkinson’s Disease. JPharmacol Exp Ther.2004;309(3):1067-1078.
[27] Han JM, Lee YJ, Lee SY, et al. Protective Effect of Sulforaphane againstDopaminergic Cell Death. J Pharmacol Exp Ther.2007;321(1):249-256.
[28] Weinreb O, Amit T, Bar-Am O, et al. Novel Neuroprotective Mechanism of Actionof Rasagiline Is Associated with Its Propargyl Moiety: Interaction of Bcl-2FamilyMembers with PKC Pathway. Ann N Y Acad Sci.2005;1053:348-355.
[29] Caccia C, Maj R, Calabresi M, et al. Safinamide: From molecular targets to a newanti-Parkinson drug. Neurology.2006;67(7Suppl2):S18-S23.
[30] Masliah E, Rockenstein E, Veinbergs I, et al. Dopaminergic loss and inclusion bodyformation in alpha-synuclein mice: implications for neurodegenerative disorders.Science.2000;287(5456):1265-1269.
[31] Feany MB, Bender WW. A Drosophila model of Parkinson's disease. Nature.2000;404(6776):394-398.
[32] El-Agnaf OM, Paleologou KE, Greer B, et al. A strategy for designing inhibitors ofα-synuclein aggregation and toxicity as a novel treatment for Parkinson's diseaseand related disorders. FASEB J.2004;18(11):1315-1317.
2. Rajasankar S, Manivasagam T, Surendran S. Ashwagandha leaf extract: a potentialagent in treating oxidative damage and physiological abnormalities seen in a mousemodel of Parkinson’s disease. Neurosci Lett2009;454(1):11–15.
3. Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease:a mechanism of pathogenic and therapeutic significance. Ann N Y Acad Sci2008;1147:93-104.
4. Brenner-Lavie H, Klein E, Zuk R, et al. Dopamine modulates mitochondrialfunction in viable SH—SY5Y cells possibly via its interaction with complex I：relevance to dopamine pathologyin schizophrenia．Biochim Biophys Acta2008；1777(2)：173—185.
5. Mitsumoto A, Nakagawa Y, Takeuchi A, et al. Oxidized forms of peroxiredoxins andDJ-1on two-dimensional gels increased in response to sublethal levels of paraquat.Free Radic Res2001;35(3):301–310.
6. Taira T, Saito Y, Niki T, et al. DJ-1has a role in antioxidative stress to prevent celldeath. EMBO Rep2004;5(4):213–218.
7. Eriksen JL, Dawson TM, Dickson DW, et al. Caught in the act: α-synuclein is theculprit in Parkinson’s disease. Neuron2003;40(3):453-456.
8. Ethell DW, Fei Q. Parkinson-linked genes and toxins that affect neuronal cell deaththrough the Bcl-2family. Antioxid Redox Signal2009;11(3):529-540.
9. Agid Y．Aging，disease and nerve cell death．Bull Acad Natl Med l995;179(6):l193-l203.
10. Fiskum G, S tarkov A, Polster BM, et al. Mitochondrial mechanisms of neural celldeath and neuroprotective interventions in Parkinson’s disease. Ann N Y Acd Sci2003;991:111-119.
11. Shoulson I, Oakes D, Fahn S, et al. Impact of sustained deprenyl (selegiline) inlevodopa-treated Parkinson’s disease: A randomized placebo-controlled extension ofthe deprenyl and tocopherol antioxidative therapy of Parkinsonism trial. Ann Neurol2002;51(5):604–612.
12. P lhagen S, Heinonen E, H gglund J, et al. Swedish Parkinson StudyGroup.Selegiline slows the progression of the symptoms of Parkinson disease. Neurology2006;66(8):1200–1206.
13. Parkinson Study Group. A controlled, randomized, delayed-start study of rasagilinein early Parkinson disease. Arch Neurol2004;61(4):561–566.
14. Olanow CW, Rascol O, Hauser R, et al. ADAGIO Study Investigators. Adouble-blind, delayed-start trial of rasagiline in Parkinson’s disease. N Engl J Med2009;361(13):1268–1278.
15. Smeding HM, Speelman JD, Huizenga HM, et al. Predictors of cognitive andpsychosocial outcome after STN DBS in Parkinson’s disease. J Neurol NeurosurgPsychiatry20l1;82(7):754-760.
16. Murakami M, Ichisaka T, Maeda M, et al. mTOR is essential for growth andproliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol2004;24(15),6710–6718.
17. Gangloff YG, Mueller M, Dann SG, et al. Disruption of the mouse mTOR gene leadsto early postimplantation lethality and prohibits embryonic stem cell development.Mol Cell Biol2004;24(21):9508–9516.
18. Sudarsanam S, Johnson DE. Functional consequences of mTOR inhibition. CurrOpin Drug Discov Devel2010;13(1):31-40.
19. Graziani EI. Recent advances in the chemistry, biosynthesis and pharmacology ofrapamycin analogs. Nat Prod Rep2009;26(5):602-609.
20. Jia K, Chen D, Riddle DL. The TOR pathway interacts with the insulin signalingpathway to regulate C. elegans larval development, metabolism and life span.Development2004;131(16):3897–3906.
21. Selman C, Tullet JM, Wieser D, et al. Ribosomal protein S6kinase1signalingregulates mammalian life span. Science2009;326(5949):140–144.
22. Bonawitz ND, Chatenay-Lapointe M, Pan Y, et al. Reduced TOR signaling extendschronological life span via increased respiration and upregulation of mitochondrialgene expression. Cell Metab2007;5(4):265-277.
23. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespanin genetically heterogeneous mice. Nature2009;460(7253):392-395.
24. Schieke SM, Phillips D, McCoy JP Jr, et al. The mammalian target of rapamycin(mTOR) pathway regulates mitochondrial oxygen consumption and oxidativecapacity. J Biol Chem2006;281(37):27643-27652.
25. Cunningham JT, Rodgers JT, Arlow DH, et al. mTOR controls mitochondrialoxidative function through a YY1-PGC-1α transcriptional complex. Nature2007;450(7170):736–740.
26. D’Antona G, Ragni M, Cardile A, et al: Branched-chain amino acid supplementationpromotes survival and supports cardiac and skeletal muscle mitochondrialbiogenesis in middle-aged mice. Cell Metab2010;12(4):362–372.
27. Polak P, Cybulski N, Feige JN,et al. Adipose-specific knockout of raptor results inlean mice with enhanced mitochondrial respiration. Cell Metab2008;8(5):399–410.
28. Pan Y, Shadel GS. Extension of chronological life span by reduced TOR signalingrequires down-regulation of Sch9p and involves increased mitochondrial OXPHOScomplex density. Aging (Albany NY)2009;1(1):131–145.
29. Jing CH, Wang L, Liu PP, et al. Autophagy activation is associatedwith neuroprotection against apoptosis via a mitochondrial pathway in a rat modelof subarachnoid hemorrhage. Neuroscience.2012;213:144-153.
30. Tun o′n MJ, Sa′nchez-Campos S, Gutie′rrez B, et al. Effects of FK506andrapamycin on generation of reactive oxygen species, nitric oxide production andnuclear factor kappa B activation in rat hepatocytes. Biochem Pharmacol2003;66(3):439–445.
31. Kim JH, Chu SC, Gramlich JL, et al. Activation of the PI3K/mTOR pathway byBCRABL contributes to increased production of reactive oxygen species. Blood2005;105(4):1717–1723.
32. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci2006;119(pt20):4285–4292.
33. Pan Y, Schroeder EA, Ocampo A, et al. Regulation of yeast chronological lifespanby TORC1via adaptive mitochondrial ROS signaling. Cell Metab2011;13(6):668–678.
34. Wu JJ, Quijano C, Chen E, et al. Mitochondrial dysfunction and oxidative stressmediate the physiological impairment induced by the disruption of autophagy.Aging (Albany NY)2009;1(4):425–437.
35. Chen L, Xu B, Liu L, et al. Hydrogen peroxide inhibits mTOR signaling byactivation of AMPKalpha leading to apoptosis of neuronal cells. Lab Invest2010;90(5):762-773.
36. Chong ZZ, Li F, Maiese K. The pro-survival pathways of mTOR and protein kinaseB target glycogen synthase kinase-3beta and nuclear factor-kappaB to fosterendogenous microglial cell protection. Int J Mol Med2007;19(2):263-272.
37. Choi KC, Kim SH, Ha JY, et al. A novel mTOR activating protein protectsdopamine neurons against oxidative stress by repressing autophagy related celldeath. J Neurochem2010;112(2):366-376.
38. Ravikumar B, Berger Z, Vacher C, et al. Rapamycin pre-treatment protectsagainst apoptosis. Hum Mol Genet2006;15(7):1209-1216.
39. Carloni S, Buonocore G, Longini M, et al. Inhibition of rapamycin-inducedautophagy causes necrotic cell death associated with Bax/Bad mitochondrialtranslocation. Neuroscience2012;203:160-169.
40. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins withpolyglutamine and polyalanine expansions are degraded by autophagy. Hum MolGenet2002;11(9):1107-17.
41. Tsvetkov AS, Miller J, Arrasate M, et al. A small-molecule scaffold inducesautophagy in primary neurons and protects against toxicity in a Huntington diseasemodel. Proc Natl Acad Sci USA2010;107(39):16982–16987.
42. Ravikumar B, Vacher C, Berger Z, et al. Inhibition of mTOR induces autophagy andreduces toxicity of polyglutamine expansions in fly and mouse models ofHuntington disease. Nature Genet2004;36(6):585–595.
43. Sarkar S, Krishna G, Imarisio S, et al. A rational mechanism for combinationtreatment of Huntington’s disease using lithium and rapamycin. Hum MolGenet2008;17(2):170–178.
44. Fox JH, Connor T, Chopra V, et al. The mTOR kinase inhibitor Everolimusdecreases S6kinase phosphorylation but fails to reduce mutant huntingtin levels inbrain and is not neuroprotective in the R6/2mouse model of Huntington’s disease.Mol Neurodegener2010;5:26.
45. Caccamo A, Majumder S, Richardson A, et al. Molecular interplay betweenmammalian target of rapamycin (mTOR), amyloid-β, and Tau: effects on cognitiveimpairments. J Biol Chem2010;285(17):13107–13120.
46. Caccamo A, Maldonado MA, Majumder S, et al. Naturally secreted amyloid-βincreases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediatedmechanism. J Biol Chem2011;286(11):8924–8932.
47. Spilman P, Podlutskaya N, Hart MJ, et al. Inhibition of mTOR by rapamycinabolishes cognitive deficits and reduces amyloid-β levels in a mouse model ofAlzheimer’s disease. PLoS ONE2010;5(4): e9979.
48. Zhang S, Salemi J, Hou H, et al. Rapamycin promotes β-amyloid production viaADAM-10inhibition. Biochem Biophys Res Commun2010;398(3):337–341.
49. Menzies FM, Huebener J, Renna M, et al. Autophagy induction reduces mutantataxin-3levels and toxicity in a mouse model of spinocerebellar ataxia type3. Brain2010;133(pt1):93–104.
50. Zhao C, Yasumura D, Li X, et al. mTOR-mediated dedifferentiation of the retinalpigment epithelium initiates photoreceptor degeneration in mice. J Clin Invest2011;121(1):369–383.
51. Zhang X, Li L, Chen S, et al. Rapamycin treatment augments motor neurondegeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis.Autophagy2011;7(4):412–425.
52. Dehay B, Bové J, Rodríguez-Muela N, et al. Pathogenic lysosomal depletionin Parkinson's disease. J Neurosci2010;30(37):12535-12544.
53. Chu Y, Dodiya H, Aebischer P, et al. Alterations in lysosomal and proteasomalmarkers in Parkinson’s disease: relationship to α-synuclein inclusions. NeurobiolDis2009;35(3):385–398.
54. Cullen V, Sardi SP, Ng J, et al. Acid β-glucosidase mutants linked to gaucher disease,Parkinson disease, and lewy body dementia alter α-synuclein processing. AnnNeurol2011;69(6):940–953.
55. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-induced apoptosisthrough autophagy induction. Neuroscience2009;164(2):541-551.
56. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis2008;32(1):16–25.
57. Yu WH, Dorado B, Figueroa HY, et al. Metabolic activity determines efficacy ofmacroautophagic clearance of pathological oligomeric α-synuclein. Am J Pathol2009;175(2):736–747.
58. Crews L, Spencer B, Desplats P, et al. Selective molecular alterations in theautophagy pathway in patients with Lewy body disease and in models ofα-synucleinopathy. PLoS ONE2010;5(2): e9313.
59. Sarkar S, Floto RA, Berger Z, et al. Lithium induces autophagy by inhibitinginositol monophosphatase. J Cell Biol2005;170(7):1101–1111.
60. Sarkar S, Davies JE, Huang Z, et al. Trehalose, a novel mTOR-independentautophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein.J Biol Chem2007;282(8):5641–5652.
61. Malagelada C, Ryu EJ, Biswas SC, et al. RTP801is elevated in Parkinson brainsubstantia nigral neurons and mediates death in cellular models of Parkinson’sdisease by a mechanism involving mammalian target of rapamycin inactivation. JNeurosci2006;26(39):9996–10005.
62. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci2010;30(3):1166–1175.
63. Yue Z, Horton A, Bravin M, et al. A novel protein complex linking the delta2glutamate receptor and autophagy: implications for neurodegeneration in lurchermice. Neuron2002;35(5):921–933.
64. Bauvy C, Gane P, Arico S, et al. Autophagy delays sulindac sulfide-inducedapoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res2001;268(2):139–149.
65. Boya P, González-Polo RA, Casares N, et al. Inhibition of macroautophagy triggersapoptosis. Mol Cell Biol2005;25(3):1025–1040.
66. Bjedov I, Toivonen JM, Kerr F, et al. Mechanisms of life span extension byrapamycin in the fruit fly Drosophila melanogaster. Cell Metab2010;11(1):35–46.
67. Webb JL, Ravikumar B, Atkins J, et al. Alpha-Synuclein is degraded byboth autophagy and the proteasome. J Biol Chem2003;278(27):25009-25013.
68. Siddiqui A, Hanson I, Andersen JK. Mao-B elevation decreases parkin's ability toefficiently clear damaged mitochondria: protective effects of rapamycin. Free RadicRes2012;46(8):1011-1018.
69. Liu K, Shi N, Sun Y, et al. Therapeutic effects of rapamycin on MPTP-inducedParkinsonism in mice. Neurochem Res2013;38(1):201-207.
1. Ravikumar B, Duden R and Rubinsztein DC. Aggregate-prone proteins withpolyglutamine and polyalanine expansions are degraded by autophagy. Hum MolGenet.2002;11(9):1107-1117.
2. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis.2008;32(1):16-25.
3. Cortes CJ, Qin K, Cook J, et al. Rapamycin delays disease onset and prevents PrPplaque deposition in a mouse model of Gerstmann-Str ussler-Scheinker disease. JNeurosci.2012;32(36):12396-12405.
4. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci.2010;30(3):1166–1175.
5. Caccamo A, Majumder S, Richardson A, et al. Molecular interplay betweenmammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects oncognitive impairments. J Biol Chem.2010;285(17):13107-13120.
6. Harrison DE, Strong R, Sharp ZD, et a1. Rapamycin fed late in life extends lifespanin genetically heterogeneous mice．Nature.2009;460(7253):392—395.
7. Spilman P, Padlutskaya N, Hart M J, et a1. Inhibition of mTOR by rapamycinabolishes cognitive deficits and reduces amyloid-beta levels in a mouse model ofAlzheimer’s disease．PLoS One,2010;5(4): e9979．
8. Ravikumar B, Vacher C, Berqer Z, et a1. Inhibition of mTOR induces autophagyand reduces toxicity of polyglutamine expansions in fly and mouse models ofHuntington disease. Nat Genet.2004;36(6):585—595.
9. Sarkar S, Krishna G, Imarisio S, et al. A rational mechanism for combinationtreatment of Huntington’s disease using lithium and rapamycin. Hum. Mol. Genet.2008;17(2):170–178.
10. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-inducedapoptosis through autophagy induction. Neuroscience.2009;164(2):541-551.
11. Webb JL, Ravikumar B, Atkins J, et a1. Alpha-synuelein is degraded by bothautophagy and the proteasome. J Biol Chem.2003;278(27):25009—25013.
12. Tain LS, Mortiboys H, Tao RN, et a1. Rapamycin activation of4E-BP preventsParkinsonian dopaminergic neuron loss. Nat Neurosci.2009;12(9):1129—1135.
13. Przedborski S&Vila M. The1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinemouse model: a tool to explore the pathogenesis of Parkinson’s disease. Ann N YAcad Sci.2003;991:189–198.
14. Liu K, Liu C, Shen L, et al. Therapeutic effects of rapamycin on MPTP-inducedParkinsonism in mice. Neurochem Res.2013;38(1):201-207.
15. Dabbeni SF, Di SS, Franceschini D, et al. Melatonin protects against6-OHDA-induced neurotoxicity in rats：a role for mitochondfial complex I activity.FASEB J.2001;15(1):164-170.
16. Acton PD, Mozley PD. Single photon emission tomography imaging inparkinsonian disorders：a review. Behav Neurol.2000;12(1，2):11-27.
17. Kirik D, Rosenblad C, Bjorklund A. Characterization of behavioral andneurodegenerative changes following partial lesions of the nigrostriatal dopaminesystem induced by intrastriatal6-hydroxydopamine in the rat. Exp Neurol.1998;152(2):259-277.
18. Hall ED, Detloff MR, Johnson K, et al. Peroxynitrite-mediated protein nitration andlipid peroxidation in a mouse model of traumatic brain injury. J Neurotrauma.2004;21(1):9-20.
19. Smith MP and Cass WA. Oxidative stress and dopamine depletion in an intrastriatal6-hydroxydopamine model of Parkinson's disease. Neuroscience.2007;144(3):1057-1066.
20. Smith MP and Cass WA. GDNF reduces oxidative stress in a6-hydroxydopaminemodel of Parkinson's disease. Neurosci Lett.2007;412(3):259-263.
21. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci.2006;119(Pt20):4285-4292.
22. Marobbio CM, Pisano I, Porcelli V, et al. Rapamycin reduces oxidative stress infrataxin-deficient yeast cells. Mitochondrion.2012;12(1):156-161.
23. Long J, Liu C, Sun L, et al. Neuronal mitochondrial toxicity of malondialdehyde:inhibitory effects on respiratory function and enzyme activities in rat brainmitochondria. Neurochem Res2009;34(4):786-794.
24. Onyango IG. Mitochondrial dysfunction and oxidative stress in Parkinson's disease.Neurochem Res.2008;33(3):589-597.
25. Martinou JC and Youle RJ. Mitochondria in apoptosis: Bcl-2family members andmitochondrial dynamics. Dev Cell.2011;21(1):92-101.
26. Kirkland RA and Franklin JL. Bax, reactive oxygen, and cytochrome c release inneuronal apoptosis. Antioxid Redox Signal2003;5(5):589-596.
27. Zhang C, Tian X, Luo Y,et al. Ginkgolide B attenuates ethanol-inducedneurotoxicity through regulating NADPH oxidases. Toxicology.2011;287(1-3):124-130.
28. Santini E, Heiman M, Greengard P, et al. Inhibition of mTOR signaling inParkinson's disease prevents L-DOPA-induced dyskinesia. Sci Signal2009;2(80):ra36.
29. Bhagwat SV and Crew AP. Novel inhibitors of mTORC1and mTORC2. Curr OpinInvestig Drugs.2010;11(6):638-645.
1. Maurer M，Tsai M，Metz M，et a1．A role for Bax in the regulation of apoptosis inmouse mast cells．J Invest Dermatol.2000;114(6):1205—1206.
2. Sch IM． Brain iron deposition and the free radical mitochondrial theory ofageing．Ageing Res Rev.2004;3(3):265—301．
3. Qian L，Flood PM．Microglial cells and Parkinson’s disease．Immunol Res.2008;41(3):155—164.
4. Patel PH, Tamanoi F. Increased Rheb-TOR signaling enhances sensitivity of thewhole organism to oxidative stress. J Cell Sci.2006;119(Pt20):4285-4292.
5. Marobbio CM, Pisano I, Porcelli V, et al. Rapamycin reduces oxidative stress infrataxin-deficient yeast cells. Mitochondrion.2012;12(1):156-61.
6. Bové J, Martínez-Vicente M, Vila M. Fighting neurodegeneration with rapamycin:mechanistic insights. Nat Rev Neurosci.2011;12(8):437-452.
7. David B， Sakina T， Nathalie L， et a1． Molecular pathway involved in theneurotoxicity of6-OHDA, dopamine and MPTP：contribution to the apoptotictheory in Parkinson’s disease．Prog Neurobiol.2001;65(2):135—172．
8. Pan T, Rawal P, Wu Y, et al. Rapamycin protects against rotenone-induced apoptosisthrough autophagy induction. Neuroscience.2009;164(2):541-551.
9. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibitsmTORC2assembly and Akt/PKB. Mol Cell.2006;22(2):159-168.
10. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-inducedneurodegeneration via autophagy enhancement. Neurobiol Dis.2008;32(1):16–25.
11. Yang F, Yang YP, Mao CJ,et al. Role of autophagy and proteasome degradationpathways in apoptosis of PC12cells overexpressing human alpha-synuclein.Neurosci Lett.2009;454(3):203-208.
12. Malagelada C, Jin ZH, Jackson-Lewis V, et al. Rapamycin protects against neurondeath in in vitro and in vivo models of Parkinson’s disease. J Neurosci.2010;30(3):1166–1175.
13. Siddiqui A, Hanson I, Andersen JK. Mao-B elevation decreases parkin's ability toefficiently clear damaged mitochondria: protective effects of rapamycin. Free RadicRes.2012;46(8):1011-1018.
14. Armstrong JS, Homung B, Lecane P, et a1. Rotenone—induced G2／M cell cyclearrest and apoptosis in a human B lymphoma cell line PW. Biochem and BiophysRes Commun.2001;289(5):973—978.
15. Badisa RB, Darling-Reed SF, Soliman KF. The protective role of D-glucose against1-methyl-4-phenylpyridiniumion (MPP+): induced mitochondrial dysfunction in C6astroglial cells. Neurochem Res.2010;35(9):1413-1421.
16. Gao QG, Xie JX, Wong MS, et al. IGF-I receptor signaling pathway is involved inthe neuroprotective effect of genistein in the neuroblastoma SK-N-SH cells. Eur JPharmacol.2012;677(1-3):39-46.
17. Lanzi C, G Cassinelli, G Cuccuru, et al. Cell cycle checkpoint efficiency andcellular response to paclitaxel in prostate cancer cells. Prostate.2001;48(4):254-264.
18. Miyake N, Chikumi H, Takata M, et al. Rapamycin induces p53-independentapoptosis through the mitochondrial pathway in non-small cell lung cancercells.Oncol Rep.2012;28(3):848-854.
19. Dai ZJ, Gao J, Ma XB,et al. Antitumor effects of rapamycin in pancreatic cancercells by inducing apoptosis and autophagy. Int J Mol Sci.2012;14(1):273-285.
20. Moad AI, Tengku Muhammad TS, Oon CE, et al. Rapamycin Induces Apoptosis WhenAutophagy is inhibited in T-47D Mammary Cells and Both Processes are Regulated byPhlda1. Cell Biochem Biophys.2013;66(3):567-587.
21. Raaz U, Kuhn H, Wirtz H,et al. Rapamycin reduces high-amplitude, mechanicalstretch-induced apoptosis in pulmonary microvascular endothelial cells. MicrovascRes.2009;77(3):297-303.
22. Farrelly AM, Kilbride SM, Bonner C, et al. Rapamycin protects against dominantnegative-HNF1A-induced apoptosis in INS-1cells. Apoptosis.2011;16(11):1128-1137.
23. Bai J, Meng Z. Expression of apoptosis-related genes in liver from rats exposed tosulfur dioxide. Toxicology.2005;216(2-3):253–260.
24. Kim J, Cha YN, Surh YJ. A protective role of nuclear factor erythroid2-relatedfactor-2(Nrf2) in inflammatory disorders. Mutat. Res.2010;690(1-2):12–23.
25. Choi AM, Alam J. Heme oxygenase-1: function, regulation, and implication of anovel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell MolBiol.1996;15(1):9-19.
26. Kim Y, Li E, Park S. Insulin-like growth factor-1inhibits6-hydroxydopamine-mediated endoplasmic reticulum stress-induced apoptosis viaregulation of heme oxygenase-1and Nrf2expression in PC12cells. Int JNeurosci.2012;122(11):641-649.
27. Tobón-Velasco JC, Vázquez-Victorio G, Macías-Silva M, et al. S-allyl cysteineprotects against6-hydroxydopamine-induced neurotoxicity in the rat striatum:involvement of Nrf2transcription factor activation and modulation of signalingkinase cascades. Free Radic Biol Med.2012;53(5):1024-1040.
28. Cicora F, Lausada N, Vasquez DN, et al. Protective effect of immunosuppressivetreatment before orthotopic kidney autotransplantation. Transpl Immunol.2011;24(2):107-112.
29. Kist A, Wakkie J, Madu M, et al. Rapamycin induces heme oxygenase-1in liver butinhibits bile flow recovery after ischemia. J Surg Res.2012;176(2):468-475.
30. Visner G, Lu F, ZhouH, et al. Rapamycin induces heme oxygenase-1in humanpulmonary vascular cells: Implications in the antiproliferative response torapamycin. Circulation.2003;107(6):911-916.
31. Zoncu R, Efeyan A, Sabatini D. mTOR: From growth signal integration to cancer,diabetes and ageing. Nat Rev Mol Cell Biol.2011;12(1):21-35.
32. Hwang YP, Jeong HG. Ginsenoside Rb1protects against6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathwayin human dopaminergic cells. Toxicol. Appl. Pharmacol.2010;242(1):1828.
33. Gao HM, Liu B, and Hong JS. Critical role for microglial NADPH oxidase inrotenone-induced degeneration of dopaminergic neurons. Journal of Neuroscience.2003;23(15):6181–6187.
34. Koppula S, Kumar H, Kim IS, et al. Reactive oxygen species and inhibitors ofinflammatory enzymes, NADPH oxidase, and iNOS in experimental modelsof Parkinson's disease. Mediators Inflamm.2012;2012:823902.
35. Li X, Schwacha MG, Chaudry IH, et al. Heme oxygenase-1protects againstneutrophil-mediated intestinal damage by down-regulation of neutrophil p47phoxand p67phox activity and O2-production in a two-hit model of alcohol intoxicationand burn injury. J Immunol.2008;180(10):6933-6940.
36. Yu H, Huang J, Wang S, et al. Overexpression of Smad7suppressedROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1.Mol Biol Rep.2013;[Epub ahead of print].
[1] The Parkinson Study Group. Pramipexole vs Levodopa as Initial Treatment forParkinson Disease: A4-Year Randomized Controlled Trial. Arch Neurol.2004;61(7):1044-1053.
[2] Jenner P. A novel dopamine agonist for the transdermal treatment of Parkinson’sdisease. Neurology.2005;65(2Suppl1): S3-S5.
[3] LeWitt PA, Lyons KE, Pahwa R. Advanced Parkinson disease treated with rotigotinetransdermal system. Neurology.2007;68(16):1262-1267.
[4] Splinter MY. Rotigotine: Transdermal Dopamine Agonist Treatment of Parkinson'sdisease and Restless Legs Syndrome. Ann Pharmacother.2007;41(2):285-295.
[5] Baufreton J, Garret M, Rivera A, et al. D5(Not D1) Dopamine Receptors PotentiateBurst-Firing in Neurons of the Subthalamic Nucleus by Modulating an L-TypeCalcium Conductance. The Journal of Neuroscience.2003;23(3):816-825.
[6] Chergui K, Svenningsson P, Greengard P. Cyclin-dependent kinase5regulatesdopaminergic and glutamatergic transmission in the striatum. Proc Natl Acad SciU S A.2004;101(7):2191-2196.
[7] Chase TN, Bibbiani F,Bara-Jimenez W,et al. Translating A2Aantagonist KW6002from animal models to parkinsonian patients. Neurology.2003;61(11Suppl6):S107-S111.
[8] Bara-Jimenez W, Sherzai A, Dimitrova T, et al. Adenosine A2Areceptor antagonisttreatment of Parkinson’s disease. Neurology.2003;61(3):293-296.
[9] Weiss SM, Benwell K, Cliffe IA, et al. Discovery of nonxanthine adenosine A2Areceptor antagonists for the treatment of Parkinson’s disease. Neurology.2003;61(11Suppl6): S101-S106.
[10] Mihara T, Mihara K, Yarimizu J,et al. Pharmacological characterization of a novel,potent adenosine A1and A2A receptor dual antagonist, ASP5854, in models ofParkinson’s disease and cognition. J Pharmacol Exp Ther.2007;323(2):708-719.
[11] Marino MJ, Williams DL Jr, O'Brien JA, et al. Allosteric modulation of group IIImetabotropic glutamate receptor4: A potential approach to Parkinson’s diseasetreatment. Proc Natl Acad Sci U S A.2003;100(23):13668-13673.
[12] Lopez S, Turle-Lorenzo N, Acher F, et al. Targeting Group III MetabotropicGlutamate Receptors Produces Complex Behavioral Effects in Rodent Models ofParkinson's Disease. J Neurosci.2007;27(25):6701-6711.
[13] Li G, Cui G, Tzeng NS, et al. Femtomolar concentrations of dextromethorphanprotect mesencephalic dopaminergic neurons from inflammatory damage. FASEBJ.2005;19(6):489-496.
[14] Hernán MA, Logroscino G, García Rodríguez LA. Nonsteroidal anti-inflammatorydrugs and the incidence of Parkinson disease. Neurology.2006;66(7):1097-1099.
[15] Chen H, Zhang SM, Hernán MA, et al. Nonsteroidal Anti-inflammatory Drugs andthe Risk of Parkinson Disease. Arch Neurol.2003;60(8):1059-1064.
[16] Teismann P, Tieu K, Choi DK, et al. Cyclooxygenase-2is instrumental inParkinson’s disease neurodegeneration. Proc Natl Acad Sci U S A.2003;100(9):5473-5478.
[17] Wang T, Zhang W, Pei Z, et al. Reactive microgliosis participates inMPP+-induced dopaminergic neurodegeneration: role of67kDa laminin receptor.FASEB J.2006;20(7):906-915.
[18] Kulkarnl AP, Kellaway LA, Lahiri DK, et al. Neuroprotection fromComplement-Mediated Inflammatory Damage. Ann N Y Acad Sci.2004;1035:147-164.
[19] Shen J, Cookson MR. Mitochondria and dopamine: new insights into recessiveparkinsonism. Neuron.2004;43(3):301-304.
[20] Shults CW, Oakes D, Kieburtz K, et al. Effects of coenzyme Q10in earlyParkinson’s disease: evidence of slowing of the functional decline. Arch Neurol.2002;59(10):1541-1550.
[21] Bonuccelli U, Dotto DP. New pharmacologic horizons in the treatment ofParkinson disease.Neurology.2006;67(7Suppl2): S30-S38.
[22] Matthews RT, Ferrante RJ, Klivenyi P, et al. Creatine and cyclocreatine attenuateMPTP neurotoxicity. Exp Neurol.1999;157(1):142-149.
[23] Elm JJ, Goetz CG, Ravina B, et al. A responsive outcome for Parkinson’s diseaseneuroprotection futility studies. Ann Neurol.2005;57(2):197-203.
[24] Devos D, Krystkowiak P, Clement F, et al. Improvement of gait by chronic, highdoses of methylphenidate in patients with advanced Parkinson’s disease. J NeurolNeurosurg Psychiatry.2007;78(5):470-475.
[25] Ravina BM, Fagan SC, Hart RG, et al. Neuroprotective agents for clinical trials inParkinson’s disease. Neurology.2003;60(8):1234-1240.
[26] Iwashita A, Yamazaki S, Mihara K, et al. Neuroprotective Effects of a NovelPoly(ADP-Ribose) Polymerase-1Inhibitor,2-{3-[4-(4-Chlorophenyl)-1-piperazinyl] propyl}-4(3H)-quinazolinone(FR255595), in an in Vitro Model of Cell Death and in Mouse1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Model of Parkinson’s Disease. JPharmacol Exp Ther.2004;309(3):1067-1078.
[27] Han JM, Lee YJ, Lee SY, et al. Protective Effect of Sulforaphane againstDopaminergic Cell Death. J Pharmacol Exp Ther.2007;321(1):249-256.
[28] Weinreb O, Amit T, Bar-Am O, et al. Novel Neuroprotective Mechanism of Actionof Rasagiline Is Associated with Its Propargyl Moiety: Interaction of Bcl-2FamilyMembers with PKC Pathway. Ann N Y Acad Sci.2005;1053:348-355.
[29] Caccia C, Maj R, Calabresi M, et al. Safinamide: From molecular targets to a newanti-Parkinson drug. Neurology.2006;67(7Suppl2):S18-S23.
[30] Masliah E, Rockenstein E, Veinbergs I, et al. Dopaminergic loss and inclusion bodyformation in alpha-synuclein mice: implications for neurodegenerative disorders.Science.2000;287(5456):1265-1269.
[31] Feany MB, Bender WW. A Drosophila model of Parkinson's disease. Nature.2000;404(6776):394-398.
[32] El-Agnaf OM, Paleologou KE, Greer B, et al. A strategy for designing inhibitors ofα-synuclein aggregation and toxicity as a novel treatment for Parkinson's diseaseand related disorders. FASEB J.2004;18(11):1315-1317.
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