胞外泌性α-synuclein oligomer-TLR2/4介导的炎性机制对帕金森氏病的病理作用

Parkinson's Disease (PD) is the second most common neurodegenerative disorder following Alzheimer's Disease (AD),and is characterized by a disturbance of the central dopaminergic system. Several lines of studies have shown that a-synuclein displays neurotoxicity to dopaminergic neurons; however, the precise machenisms are unknown. Our preliminary study shows that a-synuclein pronouncedly decreases in 6-OHDA-lesioned PC 12 cells, the in vitro PD model, but its oligomer and toll like receptor 4 are profoundly upregulated. Our preliminary study also shows that a-synuclein seems to co-locolize with toll like receptor 2. Thus We aim to investigate in 6-OHDA-induced PC12 how exocytotic oligomer a-synuclein displays deleterious effects in dopaminergic neurons, and its correlations with toll like receptor 2/4 and how the oligomer activates toll like receptor 2/4. We would like to verify that the possible downstream inflammatory signaling pathway MyD88-Juk-AP1/p38MAPK-NFkB may cause damage in PC12. In addition, we would like to explore how the exocytotic oligomer a-synuclein activates microglia and what the association of inflammatory responses with toll like receptor 2/4 and PC12 is. Through this study, we aim to explore the pathological mechanisms how exocytotic oligomer a-synuclein-toll like receptor 2/4 influences the progression of PD.

帕金森氏病（PD）是一种多巴胺能神经元退化性疾病,研究显示a-synuclein对多巴胺神经元有明显毒性作用,其机制不清。我们前期工作显示a-synuclein在PD细胞模型中明显减低，其oligomer与TLR4含量明显增加,共聚焦预试验指出a-synuclein与TLR2受体似有共定位。我们将在PD模型中明确胞外分泌的a-synuclein oligomer对多巴胺神经元损害及其与TLR2/4的共表达, 并观察a-synuclein oligomer对TLR2/4的激活,证实其下游炎性通路MyD88-Juk-AP1/p38MAPK-NFkB激活对多巴胺神经元的损害,同时探讨胞外分泌a-synuclein oligomer与TLR2/4在小胶质细胞激活产生的炎性反应中与多巴胺神经元损伤间的相关性，以阐释胞外分泌a-synuclein oligomer-TLR2/4在PD发展中的病理机制。

帕金森氏病（PD）是一种多巴胺能神经元退化性疾病,有研究显示 a-synuclein对多巴胺神经元有明显毒性作用,而 TLR2/4 可能作为 a-synuclein的中间受体从而对神经炎症起着至关重要的作用。在我们之前的研究中指出， a-synuclein 与 TLR2受体似有共定位，然而，其介导的病理机制尚未明确，这引起了学者们的高度重视。本项目则旨在探讨胞外性分泌性 a-synuclein oligomer-TLR2/4 介导的多巴胺能神经元死亡机制在PD病理发展过程的作用。本研究通过MPP+处理BV2细胞的PD模型发现，TLR4/NF-κB在神经炎症反应中具有重要的作用。此外，本研究通过观察Cystatin C等生物指标在 SNCA A53T mutant小鼠及Pink1敲除小鼠不同区域中的水平，发现SNCA A53T 突变与神经元内p-a-synuclein沉积增多有关；而实验中两种小鼠Cys C 均升高，Nurr1与VEGF下降，提示Nurr1与VEGF下降，以及Cystatin C 升高可能在疾病早期已发挥作用；本研究也发现Cystatin C反馈性增高与自噬水平调节有关。基于对帕金森病患者与健康人的横断面研究，我们发现PD病人外周血中CYS C明显增高，并与疾病的严重性相关。运用western blotting和免疫荧光技术等进一步检测CYS C对血管生成和多巴胺能神经元存活的影响，我们发现CYS C 通过VEGF-KDR-PKCa/ERK1/2-Nurr1信号通路促进血管生成和多巴胺能神经元存活，从而对帕金森病神经血管单元进行保护。同时，我们在前期研究基础上进一步深入研究发现，Cystatin C过表达抑制α-synuclein寡聚和沉积，从而起到神经保护作用。在PD细胞模型中，过表达 Cystatin C可上调VEGF表达，促进体内和体外的血管生成。基于对帕金森病痴呆（PDD）患者、血管性帕金森综合征伴痴呆（VPD）患者与健康对照者进行横断面研究发现，TFF3、胆碱酯酶活性和Hcy与疾病严重程度显著相关，且可作为鉴别PDD/VPD病人与健康对照者的有效指标。基于本研究在a-synuclein，TLR2/4，CYS C等方面的发现，我们能进一步加深对PD及相关神经退行性疾病的发病机制的认识，也为早中期帕金森病的诊断与治疗提供良好指导意义。