Mitochondrial Aconitase and Parkinson's Disease

描述（由申请人提供）：本提案的长期目标是阐明帕金森病（PD）中线粒体氧化应激导致多巴胺能神经元死亡的机制。线粒体氧化应激、生物能下降和铁超载引起并协同产生帕金森病中与年龄相关的神经元死亡的确切机制尚不清楚。据推测，帕金森氏病的神经元损伤的结果，部分直接超氧自由基毒性由于线粒体顺乌头酸酶的氧化失活。该假说预测，由复合物I抑制或异常多巴胺代谢引起的超氧化物产生使含[4Fe-4S]Z+的线粒体顺乌头酸酶失活，导致顺乌头酸酶活性丧失并释放Fe z+和H2 O2。因此，这种关键的TCA循环酶的翻译后修饰可导致铁负荷增加、氧化剂负荷和生物能量下降。线粒体顺乌头酸酶mRNA 5'端非翻译区的铁反应元件（IRE）的存在为帕金森病中的铁失调提供了另外的机制。该提案将利用人类PD样本、PD动物模型（1-甲基-4-苯基-1，2，3，6-四氢吡啶和6-羟基多巴胺）和多巴胺能细胞培养模型，并结合多种工具和技术，包括生化分析、共聚焦显微镜、分子生物学和转基因/敲除/衰老小鼠。具体目标1将确定线粒体顺乌头酸酶是否在人类和实验性帕金森病中失活。衰老和慢性线粒体氧化应激的影响将使用MnSOD（一种关键的线粒体抗氧化剂）缺乏的小鼠来确定。具体目标2将确定线粒体顺乌头酸酶失活是否有助于受损的铁稳态。具体目标3将确定使用天然或合成的抗氧化剂（例如MnSOD转基因小鼠或金属卟啉）清除线粒体超氧化物是否以与实验性帕金森病中的铁超负荷减少和神经元死亡相关的方式防止线粒体顺乌头酸酶失活。具体目标4将确定实验性帕金森病中线粒体顺乌头酸酶失活的下游后果。具体而言，将检查通过其mRNA中的5' IRE对脑线粒体顺乌头酸酶合成的调节、对TCA循环能力的影响以及顺乌头酸酶基因沉默的直接神经毒性。这些研究可以推进我们对帕金森病神经元死亡的氧化机制的理解，并提出新的治疗策略，以拯救神经元从年龄相关的神经退行性变。此外，这一研究路线可以解释帕金森病是由衰老和环境因素揭示的遗传因素引起的。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to elucidate the mechanism by which mitochondrial oxidative stress produces dopaminergic neuronal death in Parkinson's Disease (PD). The precise mechanism by which mitochondrial oxidative stress, bioenergetic decline and iron overload arise and collaborate to produce age-related neuronal death in Parkinson's disease remains unclear. It is hypothesized that neuronal damage in Parkinson's disease results, in part from direct superoxide radical toxicity due to oxidative inactivation of mitochondrial aconitase. The hypothesis predicts that superoxide production, arising from Complex I inhibition or abnormal dopamine metabolism, inactivates [4Fe-4S]Z+-containing mitochondrial aconitase, resulting in loss of aconitase activity and release Fe z+ and H202. Posttranslational modification of this key TCA cycle enzyme can therefore result in an increased iron load, oxidant burden and bioenergetic decline. The presence of an iron responsive element (IRE) in the 5' untranslated region of the mitochondrial aconitase Mrna provides an additional mechanism for iron dysregulation in Parkinson's disease. The proposal will utilize human PD samples, animal models of PD (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and 6-hydroxydopamine) and dopaminergic cell culture models in conjunction with a diversity of tools and techniques that include biochemical analyses, confocal microscopy, molecular biology and transgenic/knockout/aging mice. Specific Aim 1 will determine whether mitochondrial aconitase is inactivated in human and experimental Parkinson's disease. The influence of aging and chronic mitochondrial oxidative stress will be determined using mice deficient in MnSOD, a critical mitochondrial antioxidant. Specific Aim 2 will determine whether mitochondrial aconitase inactivation contributes to impaired iron homeostasis. Specific Aim 3 will determine whether scavenging mitochondrial superoxide using native or synthetic antioxidants (e.g. MnSOD transgenic mice or metalloporphyrins) protect against mitochondrial aconitase inactivation in a manner that correlates with decreased iron overload and neuronal death in experimental Parkinson's disease. Specific Aim 4 will determine the downstream consequences of mitochondrial aconitase inactivation in experimental Parkinson's disease. Specifically, regulation of brain mitochondrial aconitase synthesis by the 5' IRE in its mRNA, impact on the TCA cycle capacity and direct neurotoxicity of aconitase gene silencing will be examined. These studies can advance our understanding of the oxidative mechanisms of neuronal death in Parkinson's disease and suggest novel therapeutic strategies for rescuing neurons from age-related neurodegeneration. Additionally, this line of investigation may explain Parkinson's disease arising from genetic factors uncovered by aging as well as environmental factors.