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

描述（由申请人提供）：该R 01资助旨在确定新的分子途径和治疗方法，以预防帕金森病（PD）中基于神经元的损伤和多巴胺能（DA）神经元细胞死亡。作为工具，我们将利用PD中突变基因的潜在相互作用和暴露于环境风险因素（如某些杀虫剂）的可能性，这些因素可能部分导致疾病，如线粒体毒素。虽然一些流行病学研究表明，农药，特别是百草枯（PQ）和代森锰（MB）的组合，PD的病因，其对人类DA神经元的影响的直接作用的证据仍然很少研究。其中一个原因是不能在人细胞中有效地模拟疾病，部分原因是PD表现的性质（即，迟发和缓慢进展的病理），部分原因是由于并发症引起的 患者遗传背景的上位效应可能影响暴露后的结果。为了克服这些问题，我们正在使用PD的人iPSC模型，其中“对照”和突变细胞在遗传上是相同的（同基因的），除了在突触核蛋白基因座（A53 T）中的单个致病点突变。该模型为我们提供了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。我们安装的初步数据表明，MEF 2，参与神经发生和神经保护的转录因子的活性降低，可能在PD发病机制中发挥了贡献作用，由于环境或遗传损伤。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的异常S-亚硝基化/氧化（形成SNO-MEF 2和SOH-MEF 2）。这些翻译后修饰损害MEF 2转录活性。我们确定了MEF 2的亚硝基化/氧化导致的潜在下游细胞事件，包括转录共激活分子PGC 1？的减少，其基因受MEF 2调节，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MEF 2的遗传修饰防止MEF 2的亚硝基化/氧化，或（B）增强PGC 1？活性来拯救hiPSC衍生的神经元免于PD相关的细胞死亡。最后，在我们的HTS中心，作为潜在的治疗方法，我们将确定增加MEF 2活性或防止其氧化的小分子。因此，这些研究将阐明PD中遗传和环境风险因素的分子事件，我们将利用这些信息开发新的治疗靶点，用于在人类背景下治疗PD的药物筛选（通过使用hiPSC衍生的DA神经元）。具体目标：目标1。表征环境风险因素诱导的同基因hiPSC A53 T-突触核蛋白与WT DA神经元（和与非DA神经元）的脆弱性。目标2.阐明涉及SOH-MEF 2的SNO-MEF 2的这种易感性的途径。 目标3。筛选新的药物，保护这种遗传或环境诱导的神经元损伤的PD。

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.