Pathogenic Mechanism of Spinocerebellar Ataxia Type 10

描述(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遗传性疾病，以共济失调、癫痫和期待为特征。编码E46L蛋白的SCA10基因内含子9中ATTCT重复序列的大量扩增是导致这种疾病的突变。扩张性突变导致该病表型的机制尚不清楚。本项目的目的是阐明SCA10的致病机制。长期目标是根据SCA10的发病机制开发合理的治疗方法。中心假说是SCA10的主要致病机制是由于E46L RNA中的大片段AUUCU重复序列扰乱了多核苷束结合蛋白(PTB)的正常细胞功能。这一假说基于初步数据，即(I)SCA10细胞中没有显示出E46L mRNA和蛋白的水平改变；(Ii)E46L蛋白功能的丧失并不会导致小鼠出现类似SCA10的表型；(Iii)扩增的ATTCT重复序列能在细胞核中转录和积累；(Iv)体外有3种PTB(PTB、nPTB和hnRNP K)与AUCU重复序列结合，并且至少有1种(HnRNP K)与SCA10患者来源的细胞的核病灶中存在共定位。以下两个具体目标将用来检验我们的假设。具体目的1是证明带有扩展的AUUCU重复序列的转录本通过结合和干扰SCA10细胞中PTB的功能而获得毒性功能。预计(A)在SCA10细胞中存在可检测到的扩展的AUUCU重复序列，以及(B)在SCA10细胞中扩展的AUUCU重复序列与PTB结合并改变PTB的细胞功能。目的2建立SCA10遗传性小鼠模型。目的2将通过(A)在表达扩增的AUUCU重复序列的转基因小鼠中鉴定SCA10样表型，以及(B)建立缺乏编码hnRNP K的Hnrpk基因和编码脑特异性nPTB的Ptbp2基因的SCA10小鼠遗传模型。表达扩增的AUUCU重复序列的转基因小鼠有望通过缺失这些PTB而通过RNA介导的功能获得表现出类似SCA10的表型。阐明SCA10的发病机制具有创新性，因为SCA10是已知的唯一由五核苷酸重复序列扩张引起的人类疾病。在SCA10中建立这一机制将填补目前的认识空白，促进合理治疗的发展。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by ataxia, epilepsy and anticipation. A large expansion of the ATTCT repeat in intron 9 of the SCA10 gene encoding the E46L protein is the mutation responsible for this disease. The mechanism by which the expansion mutation leads to the disease phenotype is unknown. The goal of this project is to elucidate the pathogenic mechanism of SCA10. The long-term goal is to develop a rational therapy for SCA10 based on the pathogenic mechanism. The central hypothesis is that the major pathogenic mechanism of SCA10 is due to the disruption of the normal cellular function of polyprimidine tract binding proteins (PTBs) by the large expanded AUUCU repeat in the E46L RNA. This hypothesis is based on preliminary data that (i) SCA10 cells do not show altered levels of E46L mRNA and protein, (ii) loss of function of the E46L protein does not lead to a SCA10-like phenotype in mice, (iii), expanded ATTCT repeats are transcribed and accumulated in nuclear foci, and (iv) 3 species of PTBs (PTB, nPTB and hnRNP K) bind to AUUCU repeats in vitro and at least 1 (hnRNP K) co-localizes in the nuclear foci in cells derived from an SCA10 patient. The following 2 Specific Aims will be used to test our hypothesis. Specific Aim 1 is to demonstrate that the transcript with an expanded AUUCU repeat gains toxic functions by binding and perturbing the function of PTBs in SCA10 cells. It is expected that (a) there is a detectable level of the expanded AUUCU repeat in SCA10 cells, and (b) expanded AUUCU repeats in SCA10 cells bind to PTBs and alter the cellular functions of PTBs. Specific Aim 2 is to establish genetic mouse models of SCA10. Aim 2 will be accomplished by (a) identifying the SCA10-like phenotype in the transgenic mice that express expanded AUUCU repeats, and (b) establishing genetic mouse models of SCA10 that lack the Hnrpk gene encoding hnRNP K and the Ptbp2 gene encoding the brain-specific nPTB. Transgenic mice expressing expanded AUUCU repeats are expected to exhibit an SCA10-like phenotype by the RNA-mediated gain of function through deficiency of these PTBs. Elucidating the disease mechanism of SCA10 is innovative because SCA10 is the only human disease known to be caused by an expansion of a pentanucleotide repeat. Establishing the mechanism in SCA10 will fill the current gap of knowledge and facilitate the development of rational therapy.