外泌体通过递送miRNAs介导神经元变性在肌萎缩侧索硬化中的分子机制

肌萎缩侧索硬化（ALS）是一种严重致死性神经变性疾病，其上下运动神经元损伤的机制不清。申请者前期发现ALS患者血浆外泌体导致了NSC34细胞变性，进一步在ALS患者与健康对照外泌体中发现差异表达的miRNA，并在ALS患者IPSCs来源的运动神经元得到验证，且运动神经元分泌的外泌体可以导致正常神经元ALS相关的病理蛋白表达增加，结合既往的研究推测血浆外泌体miRNA异常与ALS发病相关。本研究拟通过外泌体功能实验、细胞生物行为学改变及芯片表达谱筛查等，在细胞水平探讨外泌体miRNA导致运动神经元选择性损伤的机制；在转基因ALS小鼠中干扰miRNA表达后观察对其表型的影响；最后结合外泌体miRNA在ALS患者和IPSCs运动神经元的表达变化，探讨外泌体miRNA在ALS发生发展中的预警机制。本项目对阐明外泌体miRNA在ALS发病中的作用及探索ALS早期诊断和治疗策略具有重要意义。

Amyotrophic lateral sclerosis (ALS）is a fatal neurodegenerative disease which pathological mechanism is largely unknown. Accumulative evidences suggest exosome has emerged as a new mechanism involved in ALS pathogenesis, although the accurate pathogenic mechanism of ALS remains unclear. miRNA, a non-coding RNAs, which is a kind of main components in exosome, were suggested to play an important role in the development of ALS, however, there were systematically no studies on the mechanisms that dysregulated miRNA in exosome cause ALS. In our previous study, we found the normal proliferation and apoptosis of NSC34 cell were affected after treating with plasma exosome from ALS patients, in addition, accumulations of the TDP-43 in the cytoplasm of NSC34 cells was observed after treating with plasma exosome from SALS and C9orf72-ALS patients, but not in NSC34 cell with plasma exosome from SOD1-ALS or HCs. Then, we investigated the exosomal miRNAs expression profiles by affymetrix miRNA 4.0 microarray in SALS, C9orf72-ALS and health controls, and found 25 dysregulated expression miRNAs between SALS and 27 dysregulated expression miRNAs between C9orf72-ALS and HCs. Among them, three miRNAs were validated by RT-PCR in plasma exosome from 30 SALS patients and exosome from IPSCs-derived motor neuron of ALS patients. In addition, after treating with exosome from motor neuron of ALS patients, healthy motor neuron showed higher expression of ALS pathogenic related protein, such as TDP43 and p62. Combination the results of previous studies, we speculate exosomal miRNA involved in ALS pathogenesis. In the present project, we plan to fully explore the role and pathogenesis of exosomal miRNA in amyotrophic lateral sclerosis by cell and animal experiments. In cell model, firstly, we investigate the function of exosome which mediate the information exchange among targeted cells by exosomal miRNA; then the biological behavior of NSC34 cell and motor neuron will be tested by interfering the expression of exosomal miRNA; Combination the data from bioinformatics analysis and mRNA expression from microarray, we’ll analyze the molecular mechanism of miRNA involved in neurodegeneration of ALS. In animal model, the dysregulated expression miRNA and location in cellular and subcellular level will be tested in C9orf72-ALS transgenic and wild mouse, then the phenotype of C9orf72-ALS transgenic and wild mouse will be assessed after interfering the dysregulated miRNA expressing by CRISPR-Cas9. In addition, the exosomal miRNA level and clinical information from baseline and follow up will be investigated in anther 60 ALS patients, combination the data of exosomal miRNA level from motor neuron in different stages, we expect to find the relationship between miRNA expression and the development and prognosis of ALS. Overall, the results of the present project will provide novel insights into the mechanisms of exosomal miRNA contribute to ALS, and identify genes and pathways as potential targets for further development of the therapeutic reagents.