鱼藤酮影响溶酶体膜通透性引发α-突触核蛋白自噬性降解障碍导致帕金森症的机制探讨

Parkinson's disease (PD) is one of the neurodegenerative disorders characterized by the selective loss of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc). It has been found that pesticides contribute to pathogenesis of degeneration of nigrostriatal DA neurons in animal models. As a broad-spectrum pesticide, rotenone, a typical environmental toxin, belongs to the family of isoflavones naturally found in the roots and stems of several plants. Rotenone is widely used in rodent model systems to elicit a parkinsonian syndrome with selective degeneration of DA neurons and the formation of Lewy bodies, cytoplasmic inclusions composed predominantly of alpha-synuclein, which is the pathological hallmark of PD. Excessive accumulation of misfolded alpha-synuclein, which may be less degraded, has been implicated in the cause of the disorder. Rotenone can inhibit the transfer of electrons from iron-sulfur (Fe-S) center to ubiquinone which increases reactive oxygen species (ROS) production and ROS results in lysosomal membrane permeabilization (LMP). We have found that environmental neurotoxin rotenone may contribute to the stalling of autophagy, which may be due to the impaired lysosomal quantity and integrity, and damage the DA neurons. Now the aim is to prove rotenone-induced LMP results in the impaired lysosomal membrane while cathepsins are released from lysosome into the cytosol. Therefore, autophagic flux could be impaired which induces the accumulation of alpha-synuclein. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, can modulate lysosomal proteostasis. Autophagy inducers may ameliorate toxicity of rotenone by nuclear translocation of TFEB which upregulates LAMP2 protein level and accelerates alpha-synuclein degredation.

帕金森病（PD）是一种以中脑黑质多巴胺（DA）神经元缺损为主要病理基础的慢性进展性神经退行性疾病，α-突触核蛋白（alpha-synuclein）的折叠异常和降解减少可能是其在细胞内聚集，导致DA神经元损伤的机制之一。鱼藤酮作为PD环境致病因素之一，我们预实验发现可能造成溶酶体功能缺陷导致自噬流障碍，引发alpha-synuclein蓄积，损伤DA神经元。本项目试图证明：①鱼藤酮通过损伤线粒体，上调活性氧（ROS）水平，引起溶酶体膜通透性增高，使溶酶体蛋白水解酶外漏；②溶酶体功能受损导致自噬流阻滞，alpha-synuclein聚集体增多；③自噬激动剂通过提高转录因子EB蛋白表达量和（或）核转录水平，增加溶酶体膜蛋白LAMP2蛋白合成量，加速自噬流进程，清除自噬底物alpha-synuclein和损伤的线粒体起到神经保护作用。本项目研究将为阐明PD致病机制中自噬流障碍提供理论和实验依据。

环境毒素鱼藤酮能够引发α-突触核蛋白（α-synuclein）在中脑黑质区多巴胺（DA）神经元中蓄积、DA神经元退行性病变，其分子机制尚不完全明确。鱼藤酮皮下注射Lewis大鼠，免疫组织化学、免疫印迹法及行为学检测中脑DA神经元损伤情况，免疫荧光法检测DA神经元内α-synuclein聚集体，电镜观察自噬体数量，PCR法检测α-synuclein的mRNA水平，免疫印迹法检测α-synuclein以及自噬标记物 LC3-II，Beclin 1和p62等蛋白水平，免疫荧光法双标TH/α-synuclein，TH/LAMP2，TH/cathepsin D。鱼藤酮体外干预PC12细胞，检测α-synuclein、LC3-II、 Beclin 1、p62等蛋白水平。使用自噬阻滞剂Bafilomycin A1判断鱼藤酮干预后自噬流通畅与否。免疫荧光法双标LAMP2/cathepsin D；提纯溶酶体后进行孵育，收取上清液后检测cathepsin D漏出；N-乙酰-β-D氨基葡萄糖苷酶检测试剂盒进一步明确溶酶体膜通透性改变情况。验证自噬激动剂海藻糖对环境毒素鱼藤酮导致的神经细胞毒性的保护作用，并且检测海藻糖使用后鱼藤酮体内外帕金森症模型转录因子EB（TFEB）、LC3-II和LAMP2蛋白表达量，免疫荧光法检测外源性 GFP-LC3 脂质体转染后点状聚集体变化情况和LysoTracker观察溶酶体荧光强度，质核分离及免疫荧光法观察自噬激动剂海藻糖使用后TFEB蛋白入核现象。环境毒素鱼藤酮导致Lewis大鼠中脑DA神经元丢失，伴随大量α-synuclein蓄积，同时自噬体数量显著增多，cathepsin D从溶酶体内漏出到胞浆中。鱼藤酮能够明显上调PC12细胞α-synuclein、LC3-II、Beclin 1、p62等蛋白水平，提示自噬流阻滞。鱼藤酮导致PC12细胞溶酶体膜通透性增高，使其中水解酶大量释放到胞浆中，这种损伤作用作用可以被活性氧清除剂tiron所缓解。自噬激动剂海藻糖能够引发TFEB发生核转位，恢复溶酶体功能，加速自噬体的清除，降解α-synuclein；海藻糖对鱼藤酮体内外模型具有保护作用。我们发现溶酶体损伤是环境毒素鱼藤酮导致帕金森症的机制之一，保护溶酶体功能将成为抗帕金森症药物研发的重要靶点。