TFEB调控的自噬—溶酶体通路在FUS突变的肌萎缩侧索硬化的作用及其机制研究

The pathogenesis of amyotrophic lateral sclerosis (ALS) is unclear. We previously found that FUS mutations are the most common mutation in Chinese ALS patients, patients carrying different mutations in FUS presented with different phenotypes varying in severity, which is correlated with abnormal aggregations of mutated FUS protein in the cytoplasm. Abnormal aggregation of mutated FUS protein in the cytoplasm is directly related with the pathogenesis of ALS, transcription factor EB (TFEB) can positively regulate autophagy-lysosome pathway (ALP) and thus enhance degradation of protein in cells, but the role of TFEB in ALS is unclear. In this study, we analyze, in cellular model and animal model of ALS, the nuclear localization of TFEB and extend of dysfunction of ALP in different disease stages, as well as the correlation of nuclear localization of TFEB, extend of dysfunction of ALP caused by different FUS mutations and severity of phenotype of these mutations; we also down-regulate or up-regulate TFEB, or increase the activity of TFEB, analyzing the effect and mechanism of TFEB on phenotype of ALS by regulating clearance of FUS mutations; furthermore, we explore the interaction and associated structure domain between FUS protein and TFEB. This study will disclose that FUS mutations cause neurodegeneration due to decreased degradation and resulting aggregation in the cytoplasm, which is caused by interacting with TFEB and inhibiting nuclear translocation of TFEB. This study will not only furnish a sound theoretical basis for the hypothesis of abnormal protein aggregation in the pathogenesis of ALS, but also provide new treatment target for ALS.

肌萎缩侧索硬化（ALS）的发病机制尚不明确。我们前期研究发现FUS突变是中国ALS患者最常见的基因突变，不同FUS突变的临床表型严重程度不同，并与其在细胞质的异常聚积程度呈正相关。FUS突变蛋白在细胞质的异常聚积与ALS发病直接相关，转录因子EB（TFEB）可正调控自噬-溶酶体通路（ALP），促进细胞内蛋白降解，但在ALS的作用未明。本项目在ALS细胞和动物模型中分析不同疾病阶段ALP和TFEB核转位特点，不同FUS突变对ALP和TFEB核转位的影响与其临床表型严重程度的相关性；并下调或上调TFEB表达、增加TFEB活性，探讨TFEB调节FUS蛋白降解影响ALS表型的机制；进而确认FUS与TFEB的相互作用及作用位点，从而揭示FUS突变与TFEB相互作用抑制其核转位致突变蛋白清除障碍发生聚积并引起神经元变性的机制。本项目可完善ALS“异常蛋白聚积学说”，并提供TFEB神经保护的新证据。

本项目临床部分研究发现中国和欧美ALS患者基因突变构成存在显著差异，中国ALS患者最常见的基因突变是SOD1突变和FUS突变，FUS基因突变患者具有特定的临床表型，发病年龄轻，绝大多数青年起病甚至可以表现为少年型ALS，疾病进展迅速、生存期短。由于FUS突变蛋白在细胞质的异常聚积与ALS的发病直接相关，ALS的细胞模型发现FUS突变可抑制自噬体的形成和自噬溶酶体的合成，因此我们假设FUS 蛋白与TFEB存在相互作用，FUS突变时由于入核转运障碍细胞质中FUS突变蛋白增多并与TFEB相互作用，抑制TFEB的核转位而影响其功能，导致TFEB调控的自噬溶酶体通路障碍使FUS突变蛋白降解减少，出现 FUS 突变蛋白在神经元细胞质中聚积并引起ALS的神经元变性。. 本项目主要研究内容包括三部分：FUS突变对TFEB核转位和自噬溶酶体通路的影响；TFEB活性或表达改变时对FUS蛋白降解及ALS表型的影响；FUS蛋白与TFEB 相互作用及作用位点的确定。. 本项目研究表明FUS突变ALS存在自噬和线粒体自噬通路的障碍，而且与FUS突变临床表型严重程度相关。正常情况下TFEB存在于细胞核和细胞质中，以细胞核为主，FUS突变时TFEB可从细胞核转位到细胞质中，在细胞质和细胞核中与突变的FUS蛋白共聚集；饥饿处理（应激）时TFEB被激活，可刺激更多的TFEB易位到细胞核中，由于FUS突变与TFEB在细胞质中结合，影响TFEB的作用，使之不能进行相应饥饿反应，转移到细胞核中。使用mTOR抑制剂CCI-779可使TFEB活性增强，LC3-II/I表达上调，BNIP3、PINK1表达下调，TFEB活性增强后增加了FUS-R521C突变的神经元毒性。尽管FUS蛋白与TFEB存在共聚集，但二者间无直接相互作用。高通量转录组测序发现三种FUS突变型与野生型相比上调基因只有1个基因，下调基因为7种基因。此外本研究还使用Cas9基因编辑技术构建FUS-R521C突变和FUS-P525L突变的斑马鱼模型。后续我们将继续对高通量转录组测序发现的基因进行验证，并使用FUS突变斑马鱼模型进行进一步的研究。本项目说明TFEB调控的自噬溶酶体通路障碍在FUS突变ALS发病中起着重要的作用，丰富了ALS“异常蛋白聚积学说”的理论依据，为探讨TFEB作为ALS治疗靶点提供基础。

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