二氢杨梅素和丹酚酸B通过调节自噬过程参与α-突触核蛋白清除的分子机制研究

Alpha-synuclein aggregation plays a crucial role in pathogenesis in Parkinson disease. The lysosome-autophagy pathway is not only responsible for α-synuclein degradation but is also involved in the intracellular aggregation process. Modulation of activities of lysosome-autophagy pathway has been proven a useful approach to interfere with protein aggregation. We recently found that Dihydromyricetin and Salvianolic acid B, small molecules derived from herbal medicine, could facilitate the clearance of α-synuclein by autophagy. However, the molecular interplay among intracellular pathways, α-synuclein aggregation, release, and response of these chemical compounds remains unknown.. In this proposal, based on a culture cell model of α-synuclein aggregation and a transgenic mouse model of PD, we aim to study the mechanisms underlying α-synuclein accumulation and intracellular clearance by a series of interdisciplinary technologies, especially including super-resolution fluorescence imaging. We propose to precisely locate α-synuclein aggregates and a variety of membrane proteins with some associated regions in lysosome-autophagy pathway and related intracellular pathways in different cell models. We will not just detect the distribution of α-synuclein and autophagic markers in different brain regions of BAC-GFP-α-synuclein transgenic mice in response to the treatment of small chemical molecules and reconcile the involved signaling pathway, but also including the effects on the conformation and fibrous aggregation of α-synuclein by incubating with small chemical molecules. The fulfillment of this project will shed light on cellular autophagy at the single-molecule level to reveal the molecular mechanism of scavenging of misfolded proteins and develop some new effective targets of drugs for intervention to PD and related neurodegenerative diseases.

研究表明帕金森病（PD）发病的主要原因是由于α-突触核蛋白（α-syn）在细胞内的错误折叠和异常聚集形成路易小体。自噬/溶酶体系统是细胞内错误折叠蛋白质的主要降解途径。在前期研究中我们发现两种药物化合物-二氢杨梅素和丹酚酸B对于胞内α-syn的自噬清除有促进作用，而此前关于它们对PD的治疗效果未见报道。为了阐明体内α-syn的降解途径并探讨药物对α-syn自噬性清除的调控机制，本项目拟在α-syn聚集体细胞模型和GFP-α-syn转基因小鼠中分析药物处理与α-syn自噬性降解过程中关键蛋白表达水平变化的相关性，并探讨药物通过对mTOR信号通路的调节从而调控自噬过程的机制，以及药物在体外对α-syn空间构象与纤维状聚集的影响。同时结合超分辨成像结果分析α-syn及其聚集体的降解与自噬、主动运输及膜内吞过程中相关蛋白的关系。本项目将对PD早期预防和治疗中药物新靶点的开发提供理论基础。

细胞自噬过程在维持细胞的稳态和蛋白质降解过程中发挥重要作用。研究发现二氢杨梅素和丹酚酸B两种药物处理后能够一定程度上抑制细胞内α突触核蛋白（α-synuclein, α-syn）聚集体的形成；并且药物处理能够使两种相关受体蛋白LAMP1和LAMP2A表达水平上调，表明二氢杨梅素和丹酚酸B与溶酶体自噬通路的调控存在相关性。对α-syn转基因小鼠进行给药处理后，发现动物体内自噬-溶酶体途径的相关蛋白LAMP1和LAMP2A的表达水平上调，与mTOR通路相关的调节蛋白活性下降，同时α-syn的表达水平下调，进一步说明这两种药物分子对于α-syn的自噬降解具有调节作用。研究证明二氢杨梅素和丹酚酸B处理后转基因小鼠脑组织中的神经炎症水平有所减轻。另一方面，利用单壁碳纳米管结合丹酚酸B后对α-syn转基因小鼠给药处理来研究体内靶向给药途径。结果表明丹酚酸B能与碳纳米管结合并经鼻饲进入脑内，对小鼠体内α-syn的聚集有抑制效果。给药后小鼠体内α-syn表达水平有显著下降，自噬途径相关调节蛋白的表达水平上调。同时碳纳米管载药体在体内表现为低毒性，是一种比较安全的给药体系。本项目研究成果将对帕金森症的早期预防和治疗过程中药物新靶点的开发提供理论基础，并有望对碳纳米管在神经科学和生物医学研究领域的开发和应用提供理论参考。