RNA-Gain-of-Function Pathogenesis in SCA10

SCA10 中 RNA 功能获得的发病机制

基本信息

批准号：
8557439
负责人：
TETSUO ASHIZAWA
金额：
$ 32.32万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8557439
关键词：
Alternative Splicing Apoptosis Apoptotic Autopsy Behavior Binding Bioinformatics Biological Assay Brain Cell Culture Techniques Cell Death Cell Survival Cells Cerebellar Ataxia Chromosomes Co-Immunoprecipitations Computer Simulation Cultured Cells Data Disease Epilepsy Event Family Fibroblasts Fluorescent in Situ Hybridization Future Genes Genetic Goals Heterogeneous-Nuclear Ribonucleoprotein K Human In Vitro Individual Introns Knockout Mice Knowledge Mass Spectrum Analysis Mediating Messenger RNA Microsatellite Repeats Mitochondria Modeling Motor Mus Mutation Neurodegenerative Disorders Neurologic Neuronal Dysfunction Neurons PC12 Cells Pathogenesis Pathogenicity Pathway interactions Patients Pentanucleotide Repeats Phenotype Play Process Proteins RNA RNA Splicing RNA-Binding Proteins Relative (related person)Research Personnel Role Small Interfering RNA Sodium Chloride Spinocerebellar Ataxias Transcript Transgenic Mice Untranslated RNA Western Blotting Wild Type Mouse adeno-associated viral vector brain cell caspase-3 clinical phenotype crosslink functional loss gain of function high throughput screening in vivo induced pluripotent stem cell interest loss of function member mouse model mutant nervous system disorder protein function protein kinase C-delta prototype public health relevance solute therapeutic development therapeutic target translational study

项目摘要

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.

描述（申请人提供）：脊髓小脑共济失调10型（SCA10），一种常染色体显性遗传小脑共济失调，由ATXN10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，SCA10 的可能原因是有毒 RNA，而不是 ATXN10 蛋白质产物的功能改变。我们发现，扩展重复序列的非翻译 RNA 转录物在细胞内聚集，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 重复的表达会导致有害的下游事件，类似于在 SCA10 患者的细胞和大脑中看到的情况。 hnRNP K 的敲低再现了细胞培养中的一些此类事件。本申请的目的是证明 hnRNP K 的隔离在 SCA10 发病机制中发挥关键作用，并确定该机制对 SCA10 整个致病性的相对贡献。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 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。？目标 2：获得 hnRNP K 隔离导致神经元功能障碍和细胞死亡的证据。我们将重述在 SCA10 大脑和 SCA10 模型中发现的下游变化，例如通过敲低野生型小鼠和细胞培养物（包括诱导多能神经细胞）中的 hnRNP K，激活细胞凋亡途径和各种转录物的异常选择性剪接。干细胞（iPS）。？目标 3：通过检查细胞培养物和转基因小鼠模型中 hnRNP K 过度表达所实现的拯救程度，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。