GPR50调控磷酸化tau蛋白水平的作用和机制研究

The abnormal aggregation and clearance deficiency of hyperphosphorylated tau (p-tau) is a key step in the pathogenesis of Alzheimer's disease (AD). Upregulation of autophagy promote the degradation of p-tau, while the autophagy system is impaired in middle and late stage of AD patients. We have previously found that GPR50 was decreased in the brain of AD model mice; overexpression of GPR50 which contain multiple LC3 interacting regions (LIR) will activate autophagy and reduce the content of p-tau, but not change the level of tau mRNA. These indicate that GPR50 can up regulate autophagy and promote the degradation of p-tau, yet it is unknown the specific molecular mechanism of how p-tau degraded by GPR50 activated autophagy. Therefore, this project aims to: .1. Regulate the expression of GPR50, determinate the content of LC3 and autophagy related PI3K/Akt pathways. To elucidate the mechanism of autophagy activated by GPR50;.2. Mutant the LIR sequence of GPR50, transfect them into HEK293/tau cells, analysis the relationship between GPR50, p-tau and LC3. To clarify the mechanism of p-tau degraded by GPR50 activated autophagy;.3. Regulate the level of GPR50 in AD model mice, detect autophagy level, AD like pathological and cognitive ability changes. To prove upregulation of GPR50 can effectively remove p-tau, in order to prevent the occurrence of AD..The results of above proposed research will produce new insight to AD pathogenesis, and provide molecular target for drug prevention.

磷酸化tau（p-tau）异常聚集及其清除障碍是AD发病的关键环节。上调自噬可促进p-tau降解，但AD中晚期自噬系统受损。申请人发现：AD小鼠脑内GPR50降低；过表达含多个LC3结合区（LIR）的GPR50激活自噬，同时p-tau减少，tau mRNA不变，提示GPR50可上调自噬并促进p-tau降解。但p-tau经GPR50激活自噬而降解的具体分子机制不明。为此，本项目拟：1.调控GPR50水平，检测LC3含量、PI3K/Akt自噬相关通路，阐明GPR50激活自噬的机制；2. 突变GPR50的LIR序列，转染于HEK293/tau细胞，分析GPR50、LC3、p-tau相互关系，明确p-tau经GPR50降解机制；3.用GPR50干预AD小鼠，检测自噬活性及AD样病理和认知变化，探明上调GPR50可有效清除p-tau，从而防治AD发生。本研究结果将为AD发病机制及治疗提供新思路。

阿尔茨海默病（Alzheimer's disease， AD）的病理机制目前仍不清楚。Tau蛋白的过度磷酸化在AD的病理机制中起着关键作用。但目前研究发现自噬障碍是导致阿尔茨海默病的一个重要原因。因此，理解自噬的分子调控机制对理解阿尔茨海默病的病理机制非常重要。本项目确定了GPR50一方面作为一个新的自噬受体，通过多个LC3 interacting regions (LIRs)与LC3的结合； 另外一方面GPR50通过激活cAMP/PKA/CREB信号通路，调控自噬相关基因的转录，从而调控基础自噬。通过GPR50在自噬中的这种双重调控作用，GPR50调控tau蛋白磷酸化的水平。另外，本项目GPR50发现在APP/PS1小鼠脑内的表达水平下降，提示GPR50水平下降可能是导致AD脑内自噬障碍的一个重要原因。另外，本项目确定了GPR50是是调控磷酸化tau蛋白水平的关键分子。GPR50是可作为未来AD药物研发的一个靶标分子。本项目首次发现GPCR亦可以作为自噬受体，其不同于其它的自噬受体，GPR50亦可以激活非选择性基础自噬。本项目不仅为理解自噬的分子调控机制，亦为理解阿尔茨海默病的病理机制提供了重要的参考数据。考虑到GPCR在药物研发中的优势，本项目亦为未来以GPCR为靶标的AD药物的研发提供了前期基础。