Molecular mechanisms underlying direct conversion of fibroblast to neurons

许多神经系统疾病的分子病因，如自闭症或阿尔茨海默病，知之甚少。这些疾病功能研究的一个主要障碍是获取人类神经元细胞的途径有限。因此，最近发现，前神经转录因子的过表达可以直接将各种细胞类型转化为诱导神经元（iN）细胞，这为研究和治疗神经系统疾病提供了一个新的、令人着迷的方法。然而，只有当我们在分子水平上完全理解重编程过程并能够提高人类细胞的转换效率（目前约为2%）时，人类iN细胞在基础研究和再生医学方面的全部潜力才会实现。本研究计划的目的是利用进化保守但更有效的小鼠模型系统（转换效率约为20%）来定义iN细胞生成的分子机制，以便了解和改进重编程过程，以用于未来的医学应用。本研究将阐明人类和小鼠细胞转化所需的三种转录因子（Ascl1、Brn2和Myt1l）的层次结构，并确定每种因子的重要功能域。我将采用生化和功能分析来识别和表征转录因子及其功能域的基本相互作用伙伴，获得对iN细胞重编程机制的有价值的见解。最后，我将利用我的结构-功能和机制研究的信息来设计和测试合成重编程因子，以提高iN细胞转换效率，例如，通过融合重编程因子域与相互作用的交互激活子。我相信，这项工作不仅将提高我们对控制细胞命运转换的一般机制的理解，而且还将有助于使人类信息成为一种有效和可靠的工具，有可能导致新的生物医学应用。

The molecular etiologies of many neurological disorders, such as autism or Alzheimers disease, are poorly understood. A major hurdle in the functional study of these diseases is the limited access to human neuronal cells. Therefore, the recent discovery that overexpression of proneural transcription factors can directly convert various cell types into induced neuronal (iN) cells has provided a new and fascinating approach to study and potentially treat neurological diseases. Nevertheless, the full potential of human iN cells for basic research and regenerative medicine will only be realized once we fully understand the reprogramming process at the molecular level and are able to increase the conversion efficiency of human cells (currently around 2%).The aim of this research proposal is to define the molecular mechanisms of iN cell generation using the evolutionary conserved but more efficient mouse model system (conversion efficiency around 20%) in order to understand and improve the reprogramming process for future medical applications. This study will elucidate the hierarchy of the three transcription factors required to convert human and mouse cells (Ascl1, Brn2 and Myt1l), as well as determine the important functional domains of each factor. I will employ biochemical and functional assays to identify and characterize essential interaction partners of the transcription factors and their functional domains, gaining valuable insight into the mechanisms of iN cell reprogramming. Finally, I will use the information from my structure- function and mechanistic studies to design and test synthetic reprogramming factors for their capacity to enhance iN cell conversion efficiencies, e.g. by fusing reprogramming factor domains with interacting transactivators. I am confident that this work will not only improve our understanding of the general mechanisms that govern cell fate switches, but will also help to make human iN formation an efficient and reliable tool, potentially leading to new biomedical applications.