小胶质细胞TLR3/4-TRIF信号转导对DA神经元存活的作用及机制

Inflammation plays an important role in Dopaminergic (DA) Neuron degeneration, which was activated by TLRs. Most of the TLRs are activated via MyD88 dependent pathway to over-activate microglia to induce inflammation that out of control. Moreover, there is another MyD88 independent signaling pathway named TRIF pathway, which are not elucidated in Parkinson's Disease (PD) till now. In the former research job of our group, we found that TRIF deficiency agravates the injury of PD. TLR3/4 via TRIF signaling pathway may regulate the microglial inflammation via MyD88 to protect the DA Neuron from PD injury. In the project, we plan to confirm that PD injury via MyD88 is regualted by TRIF dependent signaling pathway which is a protective mechanism by using tlr3-/-, trif-/- and myd88-/- mice. In addition, we hope to discover that moderate TRIF signaling activation will regulate the MyD88 dependent inflammation, which will provide insight to PD prevention and PD mechanism activated by innate immunity.

过度炎症反应是多巴胺（DA）神经元退行性病变的重要原因，小胶质细胞TLRs介导天然免疫系统激活炎症反应。大部分TLRs经MyD88依赖途经引发小胶质细胞过度激活，产生炎症失控。TLR3和TLR4下游还存在另一种非MyD88依赖的TRIF途经，该途径对帕金森病（PD）发生的作用与机制未明。课题组前期发现敲除TRIF后DA神经元退行性病变加重，TLR3/4经TRIF信号适度激活可能调控小胶质细胞经MyD88的过度炎症反应，对DA神经元起保护作用。本项目拟采用TLR3、TRIF和MyD88敲基因小鼠证实小胶质细胞通过TLR3/TLR4-TRIF轴拮抗MyD88引起的PD炎症效应和神经毒性，阐明TLR3/TLR4-TRIF轴在DA神经元损伤过程中的保护作用及机制，证实适度增强TRIF信号途径可抑制MyD88途径引发的炎症损伤和神经毒作用。研究结果将为揭示天然免疫参与PD发生及PD防治提供理论基础。

将秋水仙碱注入小鼠纹状体内，发现其黒质致密部和纹状体均出现了一系列的病理改变。应用TRIF敲除鼠和WT小鼠，PD建模后WT组小鼠纹状体内DA和DOPAC显著高于同时段trif-/-组，而在黒质致密部二者的差异并不明显。同时，TH染色也发现WT组小鼠纹状体内TH表达相较于trif-/-组更高，而在黒质致密部两者没有明显差异。这也与我们通过HPLC得到的实验结论相吻合。以上实验结果提示：TLR3下游仅有的TRIF依赖信号途径可能对DA神经元及其微环境具有一定的保护作用。因此，我们使用TLR3的特异性配体polyI：C激活TLR3-TRIF信号通路，从而验证TRIF信号途径的过表达是否能够减轻或延缓DA神经元的退变。实验表明，注射polyI：C后，TH表达较对照组明显增高。同时，使用MN9D（多巴胺能神经元）细胞进行离体实验也得到了相似的结论。总之，无论在体或离体实验，已初步证实TLR3-TRIF信号途径对于由秋水仙碱引起的DA神经元退变具有一定的保护作用。