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（SCA10）是一种常染色体显性小脑性共济失调，由ATXN10基因中内含子ATTCT五核苷酸重复大量扩增突变引起。我们的研究表明，毒性RNA而不是ATXN10蛋白产物功能的改变可能是SCA10的原因。我们发现扩增重复序列的未翻译RNA转录物在细胞内灶中积累，并与异质核核糖核蛋白K （hnRNP K）相互作用。来自细胞培养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，类似于在细胞和SCA10患者的大脑中看到的事件。hnRNP K的敲低在细胞培养中再现了其中的一些事件。本应用的目的是证明hnRNP K的隔离在SCA10的发病机制中起关键作用，并确定该机制在SCA10整个致病性中的相对贡献。我们的中心假设是，SCA10的主要致病机制是RNA功能获得，导致hnRNP k的功能丧失。为了检验这一假设，我们提出：？目标1：证明扩展的AUUCU重复RNA与hnRNP k的结合。我们将通过拉下/质谱研究和硅生物信息学对相互作用蛋白进行无偏搜索，随后进行共免疫沉淀、电迁移转移试验和高通量交联研究。？ 目的2：获得hnRNP K封存导致神经元功能障碍和细胞死亡的证据。我们将概括在SCA10脑和SCA10模型中发现的下游变化，如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包括来自诱导多能干细胞（iPS）细胞的神经细胞。？ 目的3：通过检测细胞培养和转基因小鼠模型中过表达hnRNP K对SCA10致病性的拯救程度，确定hnRNP K对SCA10致病性的相对贡献。这些研究将牢固地确立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.