肌萎缩性脊髓侧索硬化症相关蛋白TDP-43对细胞自噬的调控作用及致病机理

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTD) and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional RNA-binding protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this project, our preliminary data indicate that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in autophagy-lysosome pathway. We will use a combination of molecular and cellular biology, biochemical and pathological approaches to further investigate the mechanism of TDP-43-mTORC1-TFEB mediated autophagy and explore the role of this regulation in disease pathogenesis using cultured cells and transgenic animal models. This study will be helpful for us to further understanding the basic molecular mechanism underlying ALS, and could lead to novel therapeutic interventions of ALS.

肌萎缩性脊髓侧索硬化症（ALS）是一种进行性发展的致死性神经退行性疾病，伴随有脑与脊髓中运动神经元的特异性损伤。TAR DNA结合蛋白（TDP-43）被认为是ALS、额颞叶痴呆和其它神经退行性疾病发病中的重要参与者。尽管研究发现TDP-43作为RNA结合蛋白可调控RNA成熟过程并鉴定出一些底物RNA，但TDP-43具体通过何种调控靶点以何种方式参与神经退变仍不明确。申请人通过前期的研究工作发现TDP-43可以通过TFEB这一重要的细胞自噬和溶酶体发生调控因子调控细胞自噬-溶酶体基因表达水平，且这一过程依赖于TFENB上游信号mTORC1中的核心组分raptor。本课题拟用分子细胞生物学，生物化学以及病理学研究手段在培养细胞和转基因动物模型中深入研究TDP-43调控细胞自噬的机制，并探索这种调控在ALS疾病发生中的作用。本研究有助于进一步从分子水平了解ALS发病核心机理，并为治疗提供新思路。

在此项目的研究过程中，我们发现在细胞和果蝇模型中TDP-43的缺失可以导致自噬体和溶酶体生成的显著增加。同时，自噬主要的转录因子TFEB也呈现核移位并进一步增加自噬-溶酶体系统基因的表达。更深入的研究显示TDP-43是通过raptor影响mTORC1信号通路，进一步影响自噬通路的。另一方面，TDP-43的缺失可以阻断自噬体-溶酶体的融合，这种影响是通过Dynactin 1的负调控实现的。因此我们得出结论，在细胞中TDP-43功能缺失将会导致不成熟自噬体和溶酶体的异常聚集，最终破坏整个自噬-溶酶体系统。更加有趣和重要的是，在TDP-43敲除的果蝇模型中，mTORC1抑制剂雷帕霉素可以加速神经退行性疾病的进程， mTORC1激动剂PA则可以改善神经退行性表型。这些动物模型中的结果也支持了我们的观点，即TDP-43介导的神经退行性疾病是通过调控mTORC1信号通路进而影响细胞自噬，且在自噬系统紊乱的情况下一味促进自噬体生成反而加速神经损伤。本项目获得的结果为我们更好的了解神经退行性疾病以及寻找未来药物靶点提供了新思路。

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