GPR52调控变异HTT蛋白水平的验证与机制研究

Huntington's disease (HD) is the third most prevalent neurodegenerative disorder with no cure. HD is an autosomal dominant monogenetic disorder caused by an expanded CAG repeat in exon 1 of the HTT gene, resulting in the expression of an mHTT protein with a polyglutamine repeat containing greater than ~36Q. Although it is reported that wild-type HTT (wtHTT) has neuroprotective functions; the gain of toxic function of mHTT, rather than loss of activity of wtHTT, is the major cause of the disease. Research groups have reported that expression of mHTT protein causes neuronal death, dementia, and motor dysfunction via a variety of cellular processes such as transcriptional dysregulation, imbalances in axonal transport, endocytosis/exocytosis, and calcium homeostasis. While each of these processes is critical to survival, it is unclear which one(s) underlie the etiology from mHTT expression to neuronal toxicity. Given the complex nature of HTT biology following its synthesis, we postulated that targeting HTT protein level at a point upstream of the pathological phenotypes would be the best way to mitigate any possible toxic phenotypes. There is evidence that, by lowering mHTT, the associated toxic phenotypes may be inhibited or reversed.Therefore, lowering mHTT level may provide an effective approach to treat HD by ameliorating all the downstream toxic effects, and identifying genes and pathways that regulate mHTT level is of great interest. To achieve this goal, we have performed genome-wide RNAi screenings and identified GPR52 as a promising target in suppressing mHTT protein level. Preliminary validation showed that knock-down of GPR52 decreased mHTT protein level and rescued relavant disease phenotypes.In this project, we will further validate the potential role of GPR52 in HD and mHTT clearance, and reveal its mechanism, especially the potential involvement of G protein relavant pathways.

亨廷顿病是一种具代表性的神经退行性及多聚谷氨酰胺疾病，由基因HTT的CAG重复区域的变异引起。直接降低变异HTT蛋白水平理论上可以抑制其下游各种机制产生的毒性，并在各种疾病模型中被证实可以显著抑制疾病相关表型。寻找影响变异HTT蛋白水平的基因或信号通路，对选择适合小分子作用的潜在靶位点，开发针对此类疾病新药有着重要意义。我们前期工作通过高通量筛选发现敲低GPR52这个G蛋白偶联受体可以降低变异HTT蛋白水平，在细胞及动物模型中初步验证其对变异HTT蛋白水平的调控。本项目将利用基因操控、信号通路的小分子调控、细胞及动物疾病表型分析等方法，在神经元及动物模型中对GPR52对变异HTT蛋白水平的调控机理深入研究，进一步确认GPR52的作用并揭示其降低变异HTT的机制，特别是与G蛋白的信号通路的关联。本项目的成功实施将对基于调控GPR52的抗亨廷顿病药物提供细胞生物学层面的理论支持。

亨廷顿病主要由HTT基因突变产生的突变HTT蛋白的细胞毒性导致。尽管变异HTT蛋白广泛表达，亨廷顿病中的神经退行主要发生在大脑纹状体，特别是其中表达多巴胺受体二型（D2）的中间多棘神经元（medium spiny neuron）是最早死亡的一种。项目负责人实验室通过遗传学筛选发现富集于D2型中间多棘神经元的孤儿G蛋白偶联受体GPR52可以有效稳定纹状体的突变HTT蛋白，而非大脑皮层的突变HTT蛋白。因此，GPR52可能通过特异性地稳定纹状体神经元的突变HTT蛋白导致纹状体神经元对变异HTT蛋白更敏感，从而使其在亨廷顿病中更容易死亡。类似的机制也可能存在于其他神经退行性疾病。. 项目负责人课题组进一步揭示了GPR52调控变异HTT蛋白稳定性的机制。此调控过程依赖于GPR52的G蛋白偶联受体活性和下游的第二信使cAMP，但是并不依赖于经典的cAMP感应蛋白PKA，而是依赖于小G蛋白介导的变异HTT的胞内运输。有趣的是，GPR52是一个单外显子基因，完全位于另外一个基因RABGAP1l的一个内含子中，且方向一致。进一步研究发现，RABGAP1l可以通过其对小G蛋白RAB39B的阻断，对抗GPR52的作用，为HTT蛋白提供相互平衡的调控。这是首次发现一个基因和它内含子所包含的基因在功能上直接相互联系。. GPR52的发现不仅为疾病神经元死亡的区域特异性提供了可能解释，而且也为疾病治疗提供了可能的药靶。GPR52的敲低或敲除，显著拯救了人类干细胞分化亨廷顿病神经元模型的神经元萎缩及死亡，以及在体果蝇遗传学动物模型的疾病表型。G蛋白偶联受体是目前最主要的一类药物靶点，因此GPR52的制药潜力巨大。后续研究将围绕针对GPR52的药物研发以及哺乳动物模型的验证展开。..项目相关通讯作者论文已发表于eLife、Nature Chemical Biology等期刊。