CGG repeat associated translation in Fragile X gene function and disease

项目摘要 FMR1基因5‘非编码区CGG重复序列的中间扩展(55-200个重复序列)是基础 年龄相关性神经退行性疾病、脆性X综合征和脆性X相关震颤/共济失调 综合征(FXTAS)。而导致FXS的完全突变(&gt；200重复扩展)通常会导致 FMR1表达的甲基化和沉默，FXTAS患者表达正常或升高的FMR1 但降低了FMRP。大量转录的CGG重复序列作为RNA或通过触发 重复相关的非8月启动(RAN)翻译，允许生产有毒的均聚体 导致神经退化的多肽。因此，治疗FXTAS的有效方法需要 同时阻断CGG RAN，提高FMRP的产量。之前的数据提供了证据，表明 翻译抑制下游FMRP的翻译，但也可能在调节mGluR-Ltd.中发挥关键作用， 突触可塑性的一种形式。这一发现在正常重复大小下显示，除了扩展的 条件。在这个提案中，我将研究RAN翻译在调节FMRP合成中的天然作用 在基础和在刺激内源FMR1基因的代谢性谷氨酸受体时 患者干细胞来源的神经元。这项工作将确定阻止RAN转换作为 方法在减少有毒RAN产物的同时，提高FMRP。此外，我将直接测试这一点 利用选择性靶向RAN起始点(RAN)的反义寡核苷酸的治疗机制 ASOS)在FMR1的转录记录上。在报告系统和人类细胞系中，这些ASO抑制范围 以正常和扩展重复大小进行翻译。RAN ASOS还提高了内源性FMRP，降低了 毒性RAN蛋白、FMRPolyG和增强的神经元存活 非甲基化全突变(UFM)，其中FMR1被有效转录，但翻译水平较低。在这个项目中， 我将用RAN ASO治疗FXTAS患者干细胞来源的神经元，并评估ASO介导的改善 神经元存活、钙信号和轴突生长方面的疾病特异性缺陷。加在一起，这些 实验将证明CGG RAN在调节FMRP合成和 证明靶向RAN翻译具有纠正多种疾病相关特征的潜力 脆性X相关疾病。 此外，这份提案中描述的培训将为我未来从事翻译研究和 神经系统疾病的模型。该奖项下的活动将训练我在未来的研究中茁壮成长 让我为培养下一代科学家做好准备。

Project Summary Intermediate expansion of a CGG repeat in the 5’ UTR of the FMR1 gene (between 55-200 repeats) underlies an age-related neurodegenerative disorder, Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). While full mutations (>200 repeat expansions) resulting in FXS typically lead to methylation and silencing of FMR1 expression, FXTAS patients express normal or elevated amounts of FMR1 mRNA but have reduced FMRP. Large transcribed CGG repeats are potentially toxic as RNA or by triggering Repeat associated non-AUG initiated (RAN) translation, allowing for production of toxic homopolymeric peptides that contribute to neurodegeneration. Therefore, effective therapies for FXTAS need to simultaneously block CGG RAN and enhance production of FMRP. Previous data provides evidence that RAN translation inhibits translation of downstream FMRP, but may also play a critical role in regulating mGluR-LTD, a form of synaptic plasticity. This finding was shown at normal repeat sizes in addition to the expanded condition. In this proposal, I will investigate the native role of RAN translation in regulating FMRP synthesis both basally and upon stimulation of the metabotropic glutamate receptor at the endogenous FMR1 locus in patient stem cell-derived neurons. This work will determine the potential of blocking RAN translation as a method to simultaneously reduce toxic RAN products and enhance FMRP. Additionally, I will directly test this therapeutic mechanism utilizing an antisense oligonucleotide that selectively targets RAN initiation sites (RAN ASOs) on the FMR1 transcript. In reporter systems and human cell lines, these ASOs suppress RAN translation at both normal and expanded repeat sizes. RAN ASOs also enhanced endogenous FMRP, reduced toxic RAN protein, FMRpolyG, and enhanced neuronal survival in neurons derived from a patient with an unmethylated full mutation (UFM), in which FMR1 is efficiently transcribed, but poorly translated. In this project, I will treat FXTAS patient stem cell-derived neurons with RAN ASOs and evaluate ASO-mediated amelioration of disease-specific deficits in neuronal survival, calcium signaling, and neurite outgrowth. Together, these experiments would demonstrate a native function for CGG RAN in regulating FMRP synthesis and demonstrate that targeting RAN translation has the potential to correct multiple disease relevant features in Fragile X-associated disorders. Furthermore, the training described in this proposal will prepare me for a future in translational research and modeling of neurological disease. The activities under this award will train me to thrive in a future research career and prepare me to train the next generation of scientists.