FXTAS: Mechanisms and Modifiers

摘要 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，影响 脆性X智力低下1(FMR1)具有预突变等位基因(55~200个CGG重复)的个体。普普通通 FXTAS的特征包括进行性意向震颤、步态共济失调、帕金森症和认知能力下降。 外显率与年龄有关，到80岁时，男性携带者的外显率达到~75%。高达~15%的女性患有 预突变也显示出FXTAS的症状。FXTAS的神经病理特征包括泛素- 脑部核内包涵体呈阳性，小脑内浦肯野神经元明显缺失。 在分子水平上，FMR1前突变等位基因的FMR1mRNA和表达增加了2到8倍 在细胞和动物模型中，含有长(~100)个CGG三联体的突变的mRNAs已被证明是有毒的。 目前的数据支持FXTAS的两种非互斥的分子致病机制：1)RNA获得- Of-Function，其中扩展的CGG在RNA(RCGG)中的表达干扰了RNA结合的子集 蛋白质(RBP)，通过隔离在功能上限制其可用性，以及2)重复相关的非AUG (RAN)翻译，通过rCGG(和/或反义RCCG)重复的翻译导致 有毒同源多肽的生产，其中最丰富的是FMRPoly甘氨酸(FMRPolyG)，依次 干扰细胞功能。我们和其他人已经开发并使用了多种小鼠模型来研究 这些机制。纳尔逊、托德、艾伦和金团队之前使用模型生物(苍蝇、老鼠)所做的工作 细胞模型已经发现了几种受rCGGs表达影响的限制性商业惯例。其中包括Purα、hnRNP A2/B1、DROSHA/Dgcr8和TDP43。增加这些蛋白的表达可以调节rCGG介导的 模型系统的毒性，支持FXTAS的RNA介导的封存模型。此外，范围 翻译产物在患者包涵体和小鼠模型中发现，似乎也具有毒性 无数的研究。在确定RAN翻译和RBP封存的贡献的研究中 FXTAS发病机制的研究，我们已经建立了表达hnRNP A2/B1的转基因小鼠系 在不改变FMRPolyG阳性包涵体的情况下抑制rCGG重复序列介导的毒性。平行 埃默里脆性X中心的工作使用了前突变携带者的全基因组序列(WGS)分析 FXTAS起病早或晚，结合苍蝇遗传筛查确定其他遗传修饰物， 影响FXTAS发病年龄。了解这些基因中变异的作用可能会提示 治疗靶点。在这一应用中，我们建议确认和扩展遗传修饰因子的识别 通过序列分析和分析埃默里利用人类遗传学确定的FXTAS潜在修饰物 贝勒、埃默里和密歇根的模型系统研究使用了额外的苍蝇、细胞和老鼠模型。在……里面 通过与项目1和项目3的协调，我们期望提高对导致FXTAS和 其他脆性X相关疾病，如脆性X相关原发卵巢功能不全(FXPOI)。

SUMMARY Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects individuals with premutation alleles (55–~200 CGG repeats) in fragile X mental retardation 1 (FMR1). Common features of FXTAS include progressive intention tremor, gait ataxia, Parkinsonism, and cognitive decline. Penetrance is age-dependent and reaches ~75% in male carriers by age 80. Up to ~15% of women with premutations also show symptoms of FXTAS. The neuropathological hallmarks of FXTAS include ubiquitin- positive intranuclear inclusions throughout the brain and marked dropout of Purkinje neurons in the cerebellum. At the molecular level, FMR1 premutation alleles exhibit a 2 to 8-fold increase in FMR1 mRNA and expression of mutant mRNAs containing long (~100) CGG triplets has been shown to be toxic in cell and animal models. Current data support two non-mutually exclusive molecular pathogenesis mechanisms for FXTAS: 1) RNA gain- of-function, in which the expression of expanded CGGs in RNA (rCGG) interferes with a subset of RNA-binding proteins (RBPs), functionally limiting their availability through sequestration, and 2) Repeat-associated non-AUG (RAN) translation, whereby translation through the rCGG (and/or antisense rCCG) repeats leads to the production of toxic homo-polypeptides, the most abundant of which is FMRpolyGlycine (FMRpolyG), that in turn interfere with cellular functions. Multiple mouse models have been developed and used by us and others to study these mechanisms. Previous work by the Nelson, Todd, Allen and Jin groups using model organisms (flies, mice) and cell models has identified several RBPs affected by expression of rCGGs. Among these are Pur α, hnRNP A2/B1, DROSHA/DGCR8, and TDP43. Increasing expression of these proteins can modulate rCGG-mediated toxicity in model systems, supporting the RNA-mediated sequestration model of FXTAS. In addition, RAN translation products are found in patient inclusions and mouse models and appear to also confer toxicity in numerous studies. In studies to determine the contributions of both the RAN translation and RBP sequestration mechanisms to FXTAS pathogenesis, we have generated transgenic lines of mice that express hnRNP A2/B1 and suppression of rCGG repeat-mediated toxicity without alteration of FMRpolyG positive inclusions. Parallel efforts at the Emory Fragile X Center used whole genome sequence (WGS) analysis of premutation carriers with early or late onset of FXTAS, combined with fly genetic screens to identify additional genetic modifiers that influence age of onset of FXTAS. Understanding the role of variation in these genes could suggest candidate therapeutic targets. In this application, we propose to confirm and extend identification of genetic modifiers through sequence analysis and analyze potential modifiers of FXTAS identified at Emory using human genetics and model system studies at Baylor, Emory and Michigan using additional fly, cell and mouse models. In coordination with Projects 1 and 3, we expect to improve understanding of mechanisms that lead to FXTAS and other Fragile X-associated Disorders, such as Fragile X associated Primary Ovarian Insufficiency (FXPOI).