CGG repeat associated translation in Fragile X gene function and disease

项目摘要 在FMR 1基因的5' UTR中CGG重复的中间扩增（55-200个重复之间）是FMR 1基因的突变的基础。 一种与年龄相关的神经退行性疾病，脆性X综合征和脆性X相关震颤/共济失调 综合征（FXTAS）。虽然导致FXS的完全突变（>200个重复扩增）通常导致FXS， FMR 1表达的甲基化和沉默，FXTAS患者表达正常或升高量的FMR 1 mRNA，但FMRP减少。大的转录CGG重复序列作为RNA或通过触发 重复相关的非AUG启动（RAN）翻译，允许产生有毒的均聚物 导致神经退化的肽。因此，FXTAS的有效疗法需要 同时阻断CGG RAN并增强FMRP的产生。以前的数据提供了证据，RAN 翻译抑制下游FMRP的翻译，但也可能在调节mGluR-LTD中起关键作用， 突触可塑性的一种形式这一发现显示在正常重复大小除了扩大 条件在这个提议中，我将研究RAN翻译在调节FMRP合成中的天然作用 基础和刺激内源性FMR 1位点的代谢型谷氨酸受体后， 患者干细胞衍生的神经元。这项工作将确定阻止RAN翻译的潜力， 同时减少有毒RAN产物和增强FMRP的方法。另外，我将直接测试这个 利用选择性靶向RAN起始位点（RAN）的反义寡核苷酸的治疗机制 ASOs）在FMR 1转录本上。在报告系统和人类细胞系中，这些ASO抑制RAN 在正常和扩增的重复大小的翻译。RAN ASO还增强了内源性FMRP，降低了内源性FMRP。 毒性RAN蛋白，FMRpolyG，和来自患有神经系统疾病的患者的神经元中增强的神经元存活。 未甲基化的完全突变（UFM），其中FMR 1被有效地转录，但翻译不良。在这个项目中， 我将用RAN ASO治疗FXTAS患者干细胞衍生的神经元，并评估ASO介导的改善 神经元存活、钙信号传导和神经突生长的疾病特异性缺陷。所有这些 实验将证明CGG RAN在调节FMRP合成中的天然功能， 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，本建议书中描述的培训将为我未来的翻译研究做好准备， 神经系统疾病的建模。这个奖项下的活动将培养我在未来的研究蓬勃发展 培养下一代的科学家。

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.