RNA-Gain-of-Function Pathogenesis in SCA10

描述（由申请人提供）：脊髓小脑性共济失调10型（SCA 10）是一种常染色体显性小脑性共济失调，由ATXN 10基因中内含子ATTCT五核苷酸重复序列的大扩展突变引起。我们的研究表明，毒性RNA而不是ATXN 10蛋白产物的功能改变是SCA 10的可能原因。我们发现，扩大重复的非翻译RNA转录积累在细胞内病灶，并与异质核核糖核蛋白K（hnRNP K）相互作用。来自细胞培养物和转基因小鼠模型的初步数据表明，（AUUCU）500重复序列的表达导致有害的下游事件，类似于在SCA 10患者的细胞和脑中所见的那些。hnRNP K的敲低重演了细胞培养中的一些这些事件。本申请的目的是证明hnRNP K的螯合在SCA 10发病机制中起关键作用，并确定该机制对SCA 10中整个致病性的相对贡献。我们的中心假设是SCA 10的主要致病机制是RNA功能获得，导致hnRNP K功能丧失。为了检验这一假设，我们提出：？目的1：证明扩增的AUUCU重复RNA与hnRNP K的结合。我们将通过下拉/质谱研究和计算机生物信息学对相互作用的蛋白质进行无偏搜索，然后进行免疫共沉淀，电迁移率变化测定和高通量交联研究。?目的2：获得hnRNP K隔离导致神经元功能障碍和细胞死亡的证据。我们将概括SCA 10脑和SCA 10模型中发现的下游变化，如细胞凋亡途径的激活和各种转录物的异常选择性剪接，通过敲低野生型小鼠和细胞培养物中的hnRNP K，包括诱导多能干细胞（iPS）细胞衍生的神经细胞。?目标3：通过检查细胞培养物和转基因小鼠模型中hnRNP K过表达所实现的拯救程度，确定hnRNP K对SCA 10致病性的相对贡献。这些研究将坚定地确立hnRNP K的致病作用并提供治疗靶点。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant cerebellar ataxia, is caused by a large expansion mutation of intronic ATTCT pentanucleotide repeat in the ATXN10 gene. Our studies have suggested that toxic RNA rather than altered function of the protein product of ATXN10 is the likely cause of SCA10. We showed that the untranslated RNA transcript of the expanded repeat is accumulated in intracellular foci and interacts with heterogeneous nuclear ribonucleoprotein K (hnRNP K). Preliminary data from cell culture and transgenic mouse models suggested that expression of (AUUCU) 500 repeats causes deleterious downstream events similar to those seen in cells and a brain of SCA10 patient. Knockdown of hnRNP K recapitulates some of these events in cell culture. The goal of this application is to demonstrate that the sequestration of hnRNP K plays a key role in SCA10 pathogenesis and to determine the relative contribution of this mechanism to the entire pathogenicity in SCA10. Our central hypothesis is that the major pathogenic mechanism of SCA10 is an RNA gain-of-function that causes functional loss of hnRNP K. To examine this hypothesis we propose: ? Aim 1: Demonstrate binding of the expanded AUUCU repeat RNA to hnRNP K. We will perform unbiased searches of interacting proteins by pull-down/mass spectroscopy studies and in-silico bioinformatics, followed by co-immunoprecipitation, electromobility shift assay and high-throughput crosslinking studies. ? Aim 2: Obtain evidence that hnRNP K sequestration causes neuronal dysfunction and cell death. We will recapitulate downstream changes found in the SCA10 brain and SCA10 models, such as activation of apoptotic pathways and abnormal alternative splicing of a variety of transcripts, by knockdown of hnRNP K in wild-type mice and cell cultures including neural cells derived from induced pluripotent stem (iPS) cells. ? Aim 3: Determine the relative contribution of hnRNP K to the SCA10 pathogenicity by examining the degree of rescue achieved by over-expression of hnRNP K in cell culture and transgenic mouse models. These studies will firmly establish the pathogenic role of hnRNP K and provide a therapeutic target.