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

描述(申请人提供)：突触核病，包括帕金森氏病和路易体痴呆，是常见的与a-突触核蛋白神经元聚集有关的衰老相关性神经退行性疾病。体内大鼠共核病模型描绘了在显性神经元变性之前的早期关键病理变化。这些可能代表了可能导致疾病进展的关键病理生理变化，而不会被与细胞死亡相关的事件所混淆。我推测，这些在退变前阶段受到干扰的通路是联体核病发病机制的重要贡献者，并将为疾病修改疗法产生新的靶点。两个互补的体外模型将被用来确定这些前退变变化和α-突触核蛋白毒性之间的关系。在酵母中过度表达人a-syn重述了在人类突触核病中看到的细胞缺陷，对a-syn错误折叠引起的路径生物学产生了洞察，并创建了一个适合高通量分析的模型。利用现有的无与伦比的遗传工具，我将使用酵母模型来建立这些变化和依赖a-syn的细胞毒性之间的因果关系，并在建立后调查这种联系的机制基础。其次，我将描述来自家族性a-突触核苷酸病患者的诱导多能干细胞(IPS)的特征，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新编程成为多能多能干细胞，为人类疾病的研究提供了一种前所未有的方法。这些细胞可以被强有力地分化为神经元，提供了一个高度相关的环境，在其中验证和扩展来自大鼠AAV和酵母共核病模型的发现，并使用无偏见的基因表达谱产生新的变性前变化。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致病途径，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.