Molecular Dissection of Synucleinopathies Using Yeast, Rodent and Human iPS Cells

描述（由申请人提供）：突触核蛋白病，包括帕金森病和路易体痴呆，是常见的与a-突触核蛋白神经元聚集相关的衰老依赖性神经退行性疾病。体内大鼠突触核蛋白病模型描述了明显的神经元变性之前的早期关键病理改变。这些可能代表关键的病理生理变化，可能导致疾病进展，而不会与细胞死亡相关事件混淆。我假设这些在退行性前阶段受到干扰的通路是突触核蛋白病发病机制的重要贡献者，并将为疾病修饰治疗提供新的靶点。两个互补的体外模型将用于确定这些退化前变化与a-突触核蛋白毒性之间的关系。在酵母中过度表达人类a-syn概括了人类突触核蛋白病中所见的细胞缺陷，从而深入了解了由a-syn错误折叠引起的路径生物学，并创建了一个适合高通量分析的模型。利用无可比拟的遗传工具，我将使用酵母模型来建立这些变化与a-syn依赖性细胞毒性之间的因果关系，并在建立这种联系时调查这种联系的机制基础。其次，我将描述来自家族性a- synnucleinopathy患者的诱导多能干细胞（iPS），包括a-syn的A53T和增殖（复制、三倍）突变。最近的突破性发现使人类体细胞重编程成为多能iPS细胞，为人类疾病的研究提供了前所未有的方法。这些细胞可以稳定地分化为神经元，为验证和扩展大鼠AAV和酵母突触核蛋白病模型的发现提供了高度相关的背景，并使用无偏倚的基因表达谱产生新的退行性前变化。利用酵母、大鼠和人类iPS细胞的多模型方法将有助于验证重要的发病途径，为药物开发和其他治疗策略提供新的治疗靶点。

DESCRIPTION (provided by applicant): Synucleinopathies, including Parkinson's disease and dementia with Lewy bodies, are common aging-dependent neurodegenerative diseases associated with neuronal aggregation of a-synuclein. An in vivo rat synucleinopathy model delineated early key pathological changes preceding overt neuronal degeneration. These may represent critical pathophysiological changes potentially causal to disease progression, without being confounded by cell death-related events. I hypothesized that these pathways perturbed in the pre-degenerative stages are important contributors to the pathogenesis of synucleinopathies, and will yield novel targets for disease-modifying therapies. Two complementary in vitro models will be used to determine the relationships between these pre-degenerative changes and a-synuclein toxicity. Over expressing human a-syn in yeast recapitulates cellular defects seen in the human synucleinopathies, yielding insights into the path biology caused by a-syn misfolding and creating a model amenable to high throughput analyses. Taking advantage of unparalleled genetic tools available, I will use the yeast model to establish causal relationships between these changes and a-syn-dependent cytotoxicity, and to investigate the mechanistic underpinning of such connections when established. Second, I will characterize induced pluripotent stem (iPS) cells derived from familial a-synucleinopathy patients including the A53T and multiplication (duplication, triplication) mutations of a-syn. The recent groundbreaking discovery enabling reprogramming of human somatic cells into pluripotent iPS cells offers an unprecedented approach to the study of human diseases. These cells can be robustly differentiated into neurons, providing a highly relevant context in which to validate and extend findings from the rat AAV and yeast synucleinopathy models, and to generate novel pre-degenerative changes using unbiased gene expression profiling. The multi- model approach using yeast, rat and human iPS cells will facilitate the validation of important pathogenetic pathways, offering novel therapeutic targets for drug development and other therapeutic strategies. PUBLIC HEALTH RELEVANCE: Synucleinopathies are common aging-dependent neurodegenerative diseases associated with neuronal aggregation of a-synuclein, including Parkinson's disease and dementia with Lewy bodies. An in vivo rat synucleinopathy model delineated early key pathological changes preceding overt neuronal degeneration, potentially yielding novel therapeutic targets. Two complementary in vitro models will be used to address the relationships between these pre-degenerative changes and a-synuclein toxicity. First, with powerful genetic tools, a yeast model of synucleinopathy will be used to establish causal relationships and delineate mechanisms of these changes in mediating a- synuclein toxicity. Second, yeast findings will be validated in human neurons derived from patients with familial synucleinopathies using the induced pluripotent stem cell technology. Gene expression profiling in these neurons will reveal new disease-relevant changes in this novel human disease model.