MANF通过改善溶酶体功能清除α-synuclein的神经保护机制研究

Parkinson's disease (PD) is a neurodegenerative disease characterized by abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons of substantia nigra. Our previous studies have confirmed that mesencephalic astrocyte-derived neurotrophic factor (MANF) could protect dopaminergic neurons in both rats, mice and Caenorhabditis elegans PD models. We further found that MANF could promote the clearance of α-syn, but the mechanisms remain unclear. RNA-seq analysis revealed that MANF could promote the expression of lysosomal-associated genes and transcription factors. As to the modulation of lysosomal function by transcription factors, we hypothesize that MANF may promote α-syn clearance by regulating transcription factors to improve lysosomal function. This study will explore the role of lysosomal function in the clearance of α-syn by MANF using α-syn-regulated expression cell line and PD mouse model combined with qRT-PCR and co-immunoprecipitation techniques. Then, we filter target transcription factors using luciferase reporter assay and subsequent validate their functions based on gene knockout methods. Finally, the binding sites of transcription factors are detected by ChIP-seq. This study will clarify the mechanism of MANF clearing α-syn by improving lysosomal function, which provide new theoretical and experimental basis for the intervention of α-syn-induced neurotoxicity.

帕金森病（PD）是以黑质多巴胺能神经元内α-突触核蛋白（α-syn）异常聚集为特征的神经退行性疾病。课题组前期研究证实中脑星形胶质细胞源性神经营养因子（MANF）在PD大/小鼠及转基因线虫模型中均具有神经保护作用，进一步发现MANF可促进α-syn清除，但其机制不明。表达谱分析显示MANF可促进溶酶体相关基因及转录因子的表达。基于转录因子对溶酶体功能的调节作用，我们推测MANF可通过调控转录因子改善溶酶体功能促进α-syn的清除。本研究拟利用α-syn调控表达细胞株及PD小鼠模型结合qRT-PCR和免疫共沉淀技术探索溶酶体功能在MANF清除α-syn中的作用；利用荧光素酶报告基因检测筛选目标转录因子并通过基因敲除进行功能验证；最后利用ChIP-seq对转录因子的结合位点进行检测，阐明MANF改善溶酶体功能促进α-syn清除的作用机制。为建立以α-syn神经毒性为靶标的干预手段提供新思路。

α-突触核蛋白（SNCA）的异常聚集及其诱导的炎症反应是帕金森病(PD)的核心病理特征。我们前期研究发现，中脑星形胶质细胞源性神经营养因子(MANF)可清除SNCA，保护多巴胺能神经元，但其机制不明。在本研究中，通过构建鱼藤酮诱导的PD小鼠模型，证实AAV8-MANF可抑制PD模型小鼠黑质区SNCA的积聚，保护多巴胺能神经元，改善模型动物的运动功能。通过建立SNCA调控表达细胞模型，发现分子伴侣介导的自噬(CMA)和巨自噬均参与了MANF对野生型SNCA的清除。当CMA途径受损时，MANF可通过调节转录因子Nrf2提前激活巨自噬参与对SNCA的清除。此外，通过建立6-OHDA诱导的大鼠模型，发现AAV8-MANF可抑制黑质区小胶质细胞的浸润；进一步利用LPS诱导的炎症细胞模型证实MANF可通过调节Nrf2抑制炎症反应。本研究揭示了MANF清除SNCA的具体机制，有望为建立以SNCA神经毒性为靶标的干预手段提供新思路。