Molecular Basis of rCGG-Mediated Neurodegeneration

项目摘要： 脆性X相关性震颤/共济失调综合征（FXTAS）是一种成人发病的神经退行性疾病 影响脆性X染色体前突变等位基因（55-200 CGG重复）的携带者 FMR 1基因。FXTAS的常见特征包括进行性意向性震颤、步态 共济失调帕金森综合征和认知能力下降FXTAS的神经病理学特征包括 泛素阳性的核内包涵体遍布整个大脑和浦肯野神经元的显着脱落， 小脑本项目的长期目标是了解FXTAS的分子发病机制 并为FXTAS开发有效的治疗干预措施。在分子水平上，FMR 1 CGG 与对照个体相比，前突变携带者表现出FMR 1 mRNA的2至8倍增加。 含有长（~100）CGG三联体的突变体mRNA的表达已被证明在细胞中是有毒的。 动物模型。目前，数据支持两种非相互排斥的分子发病机制 FXTAS的机制：1）RNA功能获得，其中rCGG重复结合蛋白（RBP） 通过长的rCGG重复序列的隔离而变得功能受限，以及2）重复- 相关的非AUG（RAN）翻译，其中通过CGG（或反义CCG）翻译 重复序列导致产生有毒的同源多肽，如FMRpolyG，其反过来干扰 具有多种细胞功能。我们之前的工作已经鉴定了两种已知的RNA结合蛋白， Pur β 1和hnRNP A2/B1，作为受rCGG表达影响的RBP。我们发现， 任何一种蛋白的表达都可以调节rCGG介导的毒性，支持RNA介导的 FXTAS的螯合模型。确定两种机制对FXTAS的贡献 我们已经产生了表达hnRNPA 2/B1并抑制hnRNPA 2/B1的转基因小鼠系， rCGG重复介导的毒性。与此同时，我们进行了全基因组测序和全球范围内的 代谢谱分析方法与果蝇遗传筛选相结合，以确定潜在的额外 FXTAS的遗传修饰剂。我们发现PSMB 5和鞘脂代谢途径 可调节rCGG重复毒性。在本提案中，我们计划进一步检验以下假设： FXTAS是由RBP不适当结合引起的RNA代谢异常引起的 与FMR 1前突变等位基因产生的RNA，以及确定这些额外的 候选修饰剂可以调节FXTAS发病机制。成功完成这些研究 这将大大促进我们对FXTAS分子发病机制的理解。标识 FXTAS中涉及的基因和途径将为未来的药理学研究提供有价值的靶点。 旨在开发治疗药物的研究。

Project Summary: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects the carriers of premutation alleles (55–200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Common features of FXTAS include progressive intention tremor, gait ataxia, Parkinsonism, and cognitive decline. The neuropathological hallmarks of FXTAS include ubiquitin-positive intranuclear inclusions throughout brain and marked dropout of Purkinje neurons in cerebellum. The long-term goal of this project is to understand the molecular pathogenesis of FXTAS and develop effective therapeutic interventions for FXTAS. At the molecular level, FMR1 CGG premutation carriers exhibit a 2 to 8-fold increase in FMR1 mRNA compared to control individuals. Expression of mutant mRNAs containing long (~100) CGG triplets has been shown to be toxic in cell and animal models. Currently, data support two non-mutually exclusive molecular pathogenesis mechanisms for FXTAS: 1) RNA gain-of-function, in which rCGG repeat-binding proteins (RBPs) become functionally limited through sequestration by lengthy rCGG repeats, and 2) Repeat- associated non-AUG (RAN) translation, whereby translation through the CGG (or antisense CCG) repeats leads to the production of toxic homo-polypeptides, such as FMRpolyG, which in turn interfere with a variety of cellular functions. Our previous work has identified two known RNA-binding proteins, Pur  and hnRNP A2/B1, as RBPs affected by expression of rCGG. We showed that increased expression of either protein could modulate rCGG-mediated toxicity, supporting the RNA-mediated sequestration model of FXTAS. To determine the contributions of both mechanisms to FXTAS pathogenesis, we have generated transgenic lines of mice that express hnRNP A2/B1 and suppress rCGG repeat-mediated toxicity. In parallel, we have taken both whole genome sequencing and global metabolic profiling approaches combined with fly genetic screens to identify potential additional genetic modifiers of FXTAS. We have found that PSMB5 and the sphingolipid metabolic pathway could modulate rCGG repeat toxicity. In this proposal, we plan to further test the hypothesis that FXTAS results from abnormal RNA metabolism stemming from inappropriate association of RBPs with the RNA produced by FMR1 premutation alleles, as well as determine whether these additional candidate modifiers can modulate FXTAS pathogenesis. Successful completion of these studies should significantly advance our understanding the molecular pathogenesis of FXTAS. Identifications of genes and pathways involved in FXTAS will provide valuable targets for future pharmacological research aimed at developing drugs for therapy.