The toxicity of the RNA CGG repeats in FXTAS

描述（由申请方提供）：脆性X相关震颤/共济失调综合征（FXTAS）是一种进行性神经退行性疾病，特征为震颤伴共济失调、脑萎缩和认知缺陷。FXTAS由FMR 1基因的5' UTR中的CGG扩增引起，其长度从约55至200个重复（CGG 55 -200）变化。同一基因中超过200个重复的CGG扩展会导致脆性X综合征（FXS），这是一种与FXTAS不同的智力障碍。长CGG扩增沉默FMR 1基因的转录;而短扩增提高FMR 1的转录。许多模型表明，具有CGG 55 -200重复序列的FMR 1 mRNA或FMR 1 mRNA外的RNA CGG 55 -200重复序列导致神经变性。 FXTAS的主要病理特征是形成含有蛋白酶体、RNA结合蛋白和与应激反应相关的蛋白的泛素（Ub）阳性包涵体。这些包涵体在FXTAS发病机制中的作用还不清楚。我们已经检查了FXTAS小鼠模型rCGG ki小鼠中的泛素-蛋白酶体系统（UPS）的活性，并且发现这些小鼠中的Ub-蛋白缀合物在小脑中积累。我们还观察到rCGG重复序列增加了S302形式的RNA结合蛋白CUGBP 1（命名为CUGBP 1 REP）的非磷酸化水平。CUGBP 1 REP的已知功能之一是通过将mRNA招募到应激颗粒中来抑制mRNA的翻译。我们建立了一个小鼠模型，CUGBP 1-S302 A敲入小鼠，其仅表达CUGBP 1 REP。我们发现S302 A ki小鼠在6个月大时出现脑萎缩，浦肯野细胞丢失。S302 A ki小鼠的脑萎缩伴随着Ub蛋白在小脑中的积累，表明CUGBP 1 REP抑制UPS。由于CUGBP 1 REP在rCGG ki小鼠的小脑中增加，我们认为rCGG重复序列至少部分地通过升高CUGBP 1 REP来抑制UPS。 基于这些数据，我们提出了一个假设，即rCGG重复通过在早期阶段增加CUGBP 1 REP触发FXTAS病理，并且这种增加导致在疾病晚期阶段抑制UPS，从而导致脑病理。将在表达rCGG重复序列的tet调节细胞系（Aim 1）、rCGG ki、rCGG转基因和S302 A ki小鼠（Aim 2）中检验这一假设。我们将确定CUGBP 1 REP的增加是否先于UPS的抑制和Ub阳性包涵体的形成。识别触发FXTAS进展的早期毒性事件将为开发FXTAS治疗方法提供基础。 公共卫生相关性：脆性X相关震颤/共济失调综合征（FXTAS）是一种影响老年患者的进行性神经系统疾病，其携带脆性X综合征基因突变前范围内的CGG扩增。这种病无药可治。尽管目前的证据表明RNA CGG重复序列具有毒性作用，但rCGG重复序列导致FXTAS脑萎缩的分子机制尚不清楚。我们建议应用基础科学的专业知识，以确定由rCGG重复引起的早期分子异常。本研究的结果将为FXTAS的治疗提供依据。

DESCRIPTION (provided by applicant): Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disease characterized by tremor with ataxia, brain atrophy and cognitive defects. FXTAS is caused by CGG expansions, varying in the length from ~55 to 200 repeats (CGG55-200), in the 5' UTR of the FMR1 gene. The CGG expansions of more than 200 repeats in the same gene cause the Fragile X Syndrome (FXS), an intellectual disability, distinct from FXTAS. Long CGG expansions silence the transcription of the FMR1 gene; while short expansions elevate the transcription of the FMR1. Numerous models show that the FMR1 mRNA with CGG55-200 repeats or RNA CGG55-200 repeats outside of the FMR1 mRNA cause neurodegeneration. The main pathologic feature of FXTAS is the formation of the ubiquitin (Ub)-positive inclusions that contain the proteasome, RNA-binding proteins and proteins associated with stress response. The role of these inclusions in FXTAS pathogenesis is not well understood. We have examined the activity of the ubiquitin-proteasome system (UPS) in the FXTAS mouse model, rCGG ki mice, and found accumulation of the Ub-protein conjugates in the cerebellum in these mice. We also observed that rCGG repeats increase the levels of un-phosphorylated at S302 form of RNA-binding protein, CUGBP1, named CUGBP1REP. One of the known functions of CUGBP1REP is inhibition of translation of mRNAs by recruiting them into Stress Granules. We generated a mouse model, CUGBP1-S302A knock in mice, which express only CUGBP1REP. We found that S302A ki mice develop brain atrophy at 6-months of age with the loss of the Purkinje cells. The brain atrophy in the S302A ki mice is accompanied by the accumulation of the Ub-proteins in cerebellum showing that CUGBP1REP inhibits the UPS. Since CUGBP1REP is increased in the cerebellum of rCGG ki mice, we suggest that the rCGG repeats inhibit the UPS, at least in part, through the elevation of CUGBP1REP. Based on these data, we propose a hypothesis that rCGG repeats trigger FXTAS pathology by the increase of CUGBP1REP at early stages and that this increase leads to inhibition of the UPS at late stages of the disease contributing to brain pathology. This hypothesis will be tested in the tet-regulated cell lines expressing rCGG repeats (Aim 1) and in rCGG ki, rCGG transgenic and in S302A ki mice (Aim 2). We will determine if the increase of CUGBP1REP precedes the inhibition of the UPS and formation of the Ub- positive inclusions. The identification of the early toxic events which trigger progression of FXTAS will provide a basis for the development of the therapeutic approaches for FXTAS. PUBLIC HEALTH RELEVANCE: The Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurological disease affecting elderly patients that carry the CGG expansions in the pre-mutation range in the gene for Fragile X syndrome. There is no cure for this disease. Whereas the current evidence suggests the toxic role of the RNA CGG repeats, the molecular mechanisms by which the rCGG repeats cause brain atrophy in FXTAS are not known. We propose to apply basic science expertise to identify early molecular abnormalities caused by rCGG repeats. The results of this work will provide a basis for the development of the therapy for FXTAS.