Molecular Basis of rCGG-Mediated Neurodegeneration

项目总结： 脆性X相关震颤/共济失调综合征(FXTAS)是一种成人起病的神经退行性疾病 这会影响脆性X精神疾病的前突变等位基因(55-200个CGG重复)的携带者 迟滞1(FMR1)基因。FXTAS的共同特征包括进行性意向震颤、步态 共济失调、帕金森症和认知衰退。FXTAS的神经病理特征包括 脑内泛素阳性的核内包涵体和浦肯野神经元的显著丢失 小脑。这个项目的长期目标是了解FXTAS的分子发病机制 并为FXTAS制定有效的治疗干预措施。在分子水平上，FMR1 CGG 与对照组相比，前突变携带者FMR1mRNA的表达增加了2到8倍。 含有长(~100)CGG三联体的突变mRNAs的表达在细胞中被证明是有毒的 和动物模型。目前，数据支持两种非互斥的分子发病机制 FXTAS的机制：1)RNA功能增益，其中rCGG重复结合蛋白(RBPs) 通过长时间rCGG重复的隔离而变得功能受限，以及2)重复- 相关的非8月(RAN)翻译，由此通过CGG(或反义CCG)翻译 重复序列会导致有毒同源多肽的产生，如FMRPolyG，这反过来又会干扰 具有多种细胞功能。我们之前的工作已经确定了两个已知的RNA结合蛋白， Pur和hnRNPA2/B1，作为受rCGG表达影响的限制性商业惯例。我们发现增加了 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的封存模型。确定这两种机制对FXTAS的贡献 发病机制，我们已经建立了表达hnRNP A2/B1的转基因小鼠并抑制了 RCGG重复序列介导的毒性。与此同时，我们同时进行了全基因组测序和全球 代谢谱方法与苍蝇基因筛查相结合以识别潜在的 FXTA的遗传修饰物。我们已经发现PSMB5和鞘磷脂代谢途径 可调节rCGG重复毒性。在这项提案中，我们计划进一步检验以下假设 FXTAS是由于限制性商业惯例的不适当关联引起的RNA代谢异常 用FMR1产生的RNA预突变等位基因，以及确定这些额外的 候选修饰物可以调节FXTAS的发病机制。成功完成这些研究 将极大地促进我们对FXTAS分子发病机制的认识。身份识别 FXTAS涉及的基因和途径的研究将为未来的药理学提供有价值的靶点 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.