The toxicity of the RNA CGG repeats in FXTAS

描述（由申请人提供）：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，以震颤伴共济失调、脑萎缩和认知缺陷为特征。FXTAS是由FMR1基因5' UTR中的CGG扩增引起的，长度从~55到200重复（CGG55-200）不等。同一基因中超过200个重复的CGG扩增导致脆性X综合征（FXS），这是一种不同于FXTAS的智力残疾。长CGG扩增使FMR1基因的转录沉默；而短时间的扩增会提高FMR1的转录水平。许多模型表明，具有CGG55-200重复序列的FMR1 mRNA或FMR1 mRNA外的RNA CGG55-200重复序列导致神经退行性变。FXTAS的主要病理特征是形成泛素（Ub）阳性包涵体，内含蛋白酶体、rna结合蛋白和与应激反应相关的蛋白。这些包涵体在FXTAS发病机制中的作用尚不清楚。我们在FXTAS小鼠模型rCGG - ki小鼠中检测了泛素-蛋白酶体系统（UPS）的活性，发现这些小鼠的小脑中有ub蛋白偶联物的积累。我们还观察到rCGG重复序列增加了S302形式的rna结合蛋白CUGBP1（命名为CUGBP1REP）的未磷酸化水平。CUGBP1REP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。我们建立了小鼠模型CUGBP1-S302A敲入小鼠，该敲入小鼠只表达CUGBP1REP。我们发现S302A ki小鼠在6个月大时出现脑萎缩，浦肯野细胞丢失。S302A ki小鼠的脑萎缩伴随着小脑中ub蛋白的积累，表明CUGBP1REP抑制UPS。由于CUGBP1REP在rCGG小鼠小脑中升高，我们认为rCGG重复序列至少在一定程度上通过CUGBP1REP的升高抑制了UPS。基于这些数据，我们提出了一种假设，即rCGG重复序列在早期通过CUGBP1REP的增加触发FXTAS病理，并且这种增加导致疾病晚期UPS的抑制，从而导致脑病理。这一假设将在表达rCGG重复序列的受tet调控的细胞系（目的1）、rCGG ki、rCGG转基因和S302A ki小鼠（目的2）中得到验证。我们将确定CUGBP1REP的增加是否先于UPS的抑制和Ub阳性包涵体的形成。识别引发FXTAS进展的早期毒性事件将为FXTAS治疗方法的发展提供基础。

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.