Modeling Parkinson's Disease with Isogenic hiPSC-Derived Dopaminergic Neurons

描述(申请人提供)：这项R01拨款旨在确定新的分子途径和治疗方法，以防止帕金森病(PD)中基于线粒体的损伤和多巴胺(DA)神经元细胞死亡。作为工具，我们将利用帕金森病突变基因和暴露于环境风险因素(如某些杀虫剂)的潜在相互作用，这些因素可能在一定程度上作为线粒体毒素导致疾病。虽然一些流行病学研究表明杀虫剂，特别是百草枯(PQ)和甘露醇(MB)的组合与帕金森病的病因有关，但关于它们对人类DA神经元的直接作用的证据仍然很少。其中一个原因是无法在人类细胞中有效地模拟疾病，部分原因是帕金森病表现的性质(即，发病晚和进展缓慢)，部分原因是由以下原因引起的并发症 可能影响暴露后结果的患者遗传背景的上位性效应。为了克服这些问题，我们使用了帕金森病的人类IPSC模型，在该模型中，“对照”细胞和突变细胞在基因上是相同的(等基因)，除了突触核蛋白基因座(A53T)上的一个致病基因突变。这个模型给了我们一个前所未有的机会来研究人类A9型DA神经元在农药暴露后的遗传背景的脆弱性。我们获得的初步数据表明，MEF2的活性降低，这是一种参与神经发生和神经保护的转录因子，可能在环境或遗传损伤导致的帕金森病的发病中发挥重要作用。我们发现杀虫剂PQ、MB或鱼藤酮影响DA神经元的线粒体功能，产生过量的一氧化氮(NO)和活性氧(ROS)。NO/ROS导致MeF_2的S亚硝化/氧化异常(形成SnO-MEF_2和SOH-MEF_2)。这些翻译后修饰会削弱MEF2的转录活性。我们确定了MEF2亚硝化/氧化引起的潜在下游细胞事件，包括转录共激活分子PGC1的减少，其基因由MEF2调控，是线粒体功能的关键调节因子。接下来，我们将尝试通过(A)通过对MEF2的遗传修饰来阻止MEF2的亚硝化/氧化，或(B)提高PGC1的活性，从而将HiPSC来源的神经元从PD相关的细胞死亡中拯救出来。最后，在我们的HTS中心，作为潜在的治疗药物，我们将识别能够增加MEF2活性或防止其氧化的小分子。因此，这些研究将阐明帕金森病中遗传和环境风险因素之间的分子联系，我们将利用这些信息开发新的治疗靶点，用于在人类环境中治疗帕金森病的药物筛选(通过使用HiPSC衍生的DA神经元)。作为特定的目标，我们计划：目的1.表征环境风险因素诱导的同基因HiPSC A53T-β-突触核蛋白与WTDA神经元(以及非DA神经元)的易损性。目的2.阐明SOH-MEF2对SNO-MEF2易感性的途径。目的3.筛选新的药物以保护帕金森病患者免受这种遗传或环境诱导的神经元损伤。

DESCRIPTION (provided by applicant): This R01 grant aims to identify new molecular pathways and treatments to prevent mitochondrial-based injury and dopaminergic (DA) neuronal cell death in Parkinson disease (PD). As tools, we will take advantage of the potential interplay of genes mutated in PD and exposure to environmental risk factors, such as certain pesticides, that might contribute to disease in part as mitochondrial toxins. Although several epidemiological studies have suggested an association of pesticides, particularly the combination of paraquat (PQ) and maneb (MB), to the etiology of PD, evidence for a direct role of their effect on human DA neurons remains poorly studied. One reason for this is the inability to effectively model the disease in human cells, in part due to the nature of PD manifestations (i.e., late onset and slow progression of pathology), and in part due to complications arising from epistatic effects of the patient's genetic background that might influence the outcome after exposure. To overcome these problems, we are using a human iPSC model of PD in which the "control" and mutant cells are genetically identical (isogenic) except for a single pathogenic poin mutation in the ¿-synuclein locus (A53T). This model gives us an unprecedented opportunity to examine the vulnerability of human A9-type DA neurons after pesticide exposure with regard to genetic background. We mount preliminary data that decreased activity of MEF2, a transcription factor involved in both neurogenesis and neuroprotection, may play a contributory role in PD pathogenesis due to environmental or genetic insult. We find that the pesticides PQ, MB, or rotenone affect mitochondrial function in DA neurons, producing excessive nitric oxide (NO) and reactive oxygen species (ROS). NO/ROS lead to aberrant S-nitrosylation/oxidation of MEF2 (forming SNO-MEF2 and SOH-MEF2). These posttranslational modifications impair MEF2 transcriptional activity. We identify potential downstream cellular events resulting from nitrosylation/oxidation of MEF2, including a decrease in the transcriptional co-activator molecule PGC1¿, whose gene is regulated by MEF2 and is a key regulator of mitochondrial function. We will next attempt to rescue hiPSC-derived neurons from PD-related cell death by (a) preventing nitrosylation/oxidation of MEF2 via genetic modification of MEF2, or (b) boosting PGC1¿ activity. Finally, in our HTS Center, as potential therapeutics we will identify small molecules tht increase MEF2 activity or prevent its oxidation. Thus, these studies will elucidate molecular events linking genetic and environmental risk factors in PD, and we will use this information to develop novel therapeutic targets for drug screening for the treatment of PD in the human context (by using hiPSC-derived DA neurons). As Specific Aims we plan: Aim 1. To characterize environmental risk factor- induced vulnerability in isogenic hiPSC A53T-¿-synuclein vs. WT DA neurons (and vs. non-DA neurons). Aim 2. To elucidate pathways for this susceptibility involving SNO-MEF2 of SOH-MEF2. Aim 3. To screen for novel agents that protect from this genetic or environmentally-induced neuronal injury in PD.