A Genetic Approach to Study the p75 Neurotrophin Receptor

描述(申请人提供)：这项建议的总体目标是剖析p75NTR激活的不同通路的生理相关性，并利用体外和体内研究相结合的遗传学方法阐明新的功能。我们假设p75NTR有能力产生不同的，有时是相互矛盾的信号，这取决于细胞环境和激活配体的性质。为了解决p75NTR功能的复杂性，我们提出了一种结构-功能方法来分析这些不同的信号，并提出了一种遗传学方法来研究它们在体外和体内相关细胞类型中的生物学意义。为了揭示p75NTR的新功能，我们将产生该受体的功能获得等位基因，并在细胞系和原代培养中鉴定它们的功能，并通过在转基因小鼠中敲入表达来鉴定它们的功能。目前，仅有研究p75NTR在体内作用的功能丧失模型可用，其分析可能因冗余和补偿途径而变得复杂。一种功能获得的方法可能有助于揭示该受体在体内的新功能。为了剖析p75NTR激活的不同途径之间的生理相关性，我们将产生该受体的功能缺失等位基因，这些等位基因能够激活一些途径而不能激活另一些途径，并通过在转基因小鼠中敲入表达来表征它们在体外和体内的功能。通过结合单个p75NTR分子中不同的功能突变集，可以获得进一步的机制和功能洞察。我们认为，这些类型的研究将有助于将不同的信号通路和下游效应器与特定的生物学活动和表型联系起来。鉴于p75NTR的配体、信号通路和生物活性的整体医学重要性，更好地了解该受体的机制和体内功能是至关重要的，特别是由于激动剂和拮抗剂正被考虑用于神经损伤、中风和神经退行性疾病的治疗目的。

DESCRIPTION (provided by applicant): The overall aim of this proposal is to dissect the physiological relevance of the different pathways activated by p75ntr and to elucidate novel functions using a genetic approach combining in vitro and in vivo studies. We hypothesize that p75ntr has the ability to generate diverse, and sometimes contradicting, signals depending on the cell context and the nature of the activating ligand. In order to tackle the complexity of p75ntr function, we propose a structure-function approach to dissect those different signals, and a genetic approach to investigate their biological importance in relevant cell types, both in vitro and in vivo. In order to reveal novel p75ntr functions, we will generate gain-of-function alleles of this receptor and characterize their function in cell lines and primary cultures, and in vivo by knock-in expression in transgenic mice. Currently, only loss-of-function models for investigating in vivo roles of p75ntr are available, the analysis of which may be complicated by redundant and compensatory pathways. A gain-of-function approach may help to reveal novel in vivo functions of this receptor. In order to dissect the physiological relevance of the different pathways activated by p75ntr, we will generate loss-of-function alleles of this receptor capable of activating some pathways but not others, and characterize their function in vitro and in vivo by knock-in expression in transgenic mice. Further mechanistic and functional insights may be obtained by combining distinct sets of gain- and loss-of-function mutations in individual p75ntr molecules. We suggest that these types of studies will help to link distinct signaling pathways and downstream effectors to specific biological activities and phenotypes. Given the overall medical importance of the ligands, signaling pathways and biological activities of p75ntr, a better understanding of the mechanisms and in vivo functions elicited by this receptor is essential, particularly as agonists and antagonists are being considered for therapeutic purposes in nerve damage, stroke and neurodegenerative diseases.