S6K表达下调对脆性X相关震颤/共济失调综合征转基因果蝇的作用及机制研究

Fragile X associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegene- rative disorder , has been recognized mainly among many male premutation carriers,and an RNA gain-of-function mechanism has been suggested as the culprit in FXTAS. Transcriptional dysfunction of the fragile X mental retardation 1 (FMR1) gene containing a highly polymorphic CGG repeat in the 5'-untranslated region increases the levels of FMR1 mRNA，but the translation of the FMR1 mRNA is influenced by expand CGG repeats that are prone to forming hairpin secondary construct. On the other hand, there are protein complexes physically interacting with CGG repeats, all these proteins are RNA-binding proteins, and have been shown to function in transcription, mRNA trafficking, splicing and translation.Ubiquitin-positive intranuclear inclusions contanining FMR1 mRNA and protein complexes have also been found in the neurons of definitive FXTAS patients. During the Development Phase S6K has emerged as a crucial effector of mTOR signaling contributing to the regulation of cell growth, proliferation by controling the transcription, translation and inhabiting autophagy，but in no longer split cells. hyperactivity S6K not conducive to cell repair.. In the present study, we will first definite whether the neurodegeneration of FXTAS transgenic drosophila model can be inhibited by down-regulated expression of S6K through many different methods including FISH/IF,RNA interfere ，then determinate whether there are close relationships between inhibited neurodegeneration and arrested transcription, inhibition of sequestration of RNA binding proteins by CCG repeats of FMR1 mRNA, or accelerated FMR1 mRNA aggregates elimination. Present study will offer a new clue for developing therapy of FXTAS.

脆性X相关震颤/共济失调综合征(FXTAS)是一种因FMR1基因 CGG重复前突变（premutation）所致的主要累及男性的迟发性神经变性疾病。FMR1 mRNA因转录失调而异常增多，其5'端CGG重复序列形成的发夹式二级结构不但使其翻译受阻，还大量募集与RNA剪接、转运有关的RNA结合蛋白，形成FXTAS特征性的核内包涵体。在发育期，S6K是重要的细胞生长调控因子，具有促进mRNA转录、蛋白翻译，抑制自噬对细胞器的吞噬等作用，但在不再分化的细胞，高活性的S6K却不利于细胞修复。在本课题中，我们拟通过细胞和FXTAS转基因果蝇模型，应用FISH/IF、RNA干扰等方法，明确S6K表达下调是否能通过抑制FMR1基因转录，减少FMR1 mRNA对RNA结合蛋白的募集，或促进自噬对FMR1 mRNA聚集物的清除，而对FXTAS转基因果蝇具有神经保护作用，为探索FXTAS的治疗提供新的思路。

在本研究中我们在前期成功构建了含有90个CGG重复的FMR1 mRNA在果蝇眼睛表达的GMR-GAL4/UAS系统FXTAS转基因果蝇模型基础上，构建了FMR1 mRNA在运动神经元表达的NRV2-GAL4/UAS系统FXTAS转基因果蝇模型，并发现S6K表达下调抑制了其翅膀异常，改善其果蝇飞行能力。RT-PCR分析证明S6K表达下调通过调控转录、翻译减少了FMR1 mRNA表达量；同时我们也发现敲除自噬相关基因显著抑制了S6K表达下调对FXTAS转基因果蝇模型的保护作用。这些研究结果表明S6K表达下调一方面通过转录调控，一方面通过自噬机制抑制了FXTAS转基因果蝇模型，改善了其运动能力。本研究结果为探索FXTAS疾病治疗提供一种了新的尝试。