调控表达MANF治疗帕金森病的作用及机制研究

Mesencephalic-astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor identified in recent years with higher selectivity for dopaminergic neurons and stronger ability for neural regeneration than glial-cell-line-derived neurotrophic factor(GDNF) and brain derived neurotrophic factor(BDNF) in a low concentration. MANF cannot cross the blood brain barrier by itself, so it is very important to achieve consistent and regulated expression of MANF gene in the brain. We have succeeded in controlled and efficient expression of target genes with the adeno-associated viral (AAV) vector. Based on this, we have designed two AAV-MANF vectors which could express MANF either consistently or induced by Rapamycin. Cell type and efficiency of the recombinant AAV-MANFs transfection, as well as the expression and regulation of MANF. Then, protect effect of AAV-MANF on dopaminergic neurons will be evaluated.The protection effect of MANF on PD mice will be eluciated, as well as its effect on the function of mitochondrion, apoptosis-related proteins and up-regulation of α-synuclein, to find out the potential mechanisms and lay down foundation for gene therapy of Parkinson disease.

中脑星形胶质细胞源性神经营养因子(MANF)是多巴胺能神经元新型高选择性神经营养因子，在低浓度时即具有较其它胶质细胞源性和脑源性神经生长因子更强的神经保护及促神经修复作用。MANF不透血脑屏障，因而限制了其在临床上的应用。实现MANF在脑内的可调控表达对于帕金森病的临床治疗具有重要的科学意义。本项目以我们前期构建的可持续表达MANF的AAV-MANF质粒和受雷帕霉素诱导调控的重组AAV-MANF表达系统为基础，以MANF在体内和体外对多巴胺能神经元的保护作用研究为主线，阐明调控表达MANF对帕金森病动物模型的保护效果及其对小胶质细胞、线粒体功能和凋亡相关蛋白的影响，探讨上调MANF表达对α-synuclein异常聚集的影响，为揭示MANF对帕金森病的治疗作用及相关机制奠定基础。

中脑星形胶质细胞源性神经生长因子(MANF) 是多巴胺能神经元新型高选择性神经营养因子,在低浓度时即具有较其他胶质细胞源性和脑源性神经生长因子更强的神经保护作用及促神经修复作用。本项目通过构建可持续表达MANF的AAV-MANF质粒和受雷帕霉素诱导调控的重组AAV-MANF表达系统为基础，以MANF在体内和体外对多巴胺能神经元的保护作用研究为主线，阐明了调控表达MANF对帕金森病动物模型的保护效果及其对多巴胺能神经元的及其SHSY-5Y细胞的保护作用，与上调Grp-78、HSP70表达有关；同时阐明了MANF的自噬调节作用与AMPK/mTOR通路有关；MANF可通过PI3K/AKT/GSK3β通路上调Nrf2的表达及核内转移对抗6-OHDA诱导的SH-SY5Y细胞死亡。本项目设计出雷帕霉素诱导型AAV-MANF载体（AAV-TAR-BiDi-Ci-MANF），通过雷帕霉素的诱导，可对导入脑细胞内MANF基因的表达进行有效调控，从而克服了MANF作为新型多巴胺能神经特异性营养因子，因不能透过血脑屏障而限制其在临床上应用的弊端。