突变亨廷顿蛋白抑制突触小泡蛋白2C表达的分子机制研究

Huntington's disease (HD) is a neurodegenerative disorder caused by mutation of the gene for huntingtin (Htt). There is defect in neuronal transmission in the brains of HD patients and transgenic mouse models. Mutant Htt could affect the expression of a series of synaptic proteins through gene dysregulation, causing dysfunction of synaptic transmission. Synaptic protein 2 (SV2) is a class of glycoprotein involved in regulation of synaptic transmission, isoform C of which is selectively expressed in the HD pathology-selective regions, such as basal ganglia. Our preliminary work has found that transcription of SV2C gene is downregulated in the striatum of HD transgenic mouse brains. Bioinformatic analysis has showed that repressor element silencing transcription factor (REST) could be an inhibitory transcription factor of SV2C gene. It is likely that REST regulates expression of SV2C viainteracting with a sequence in the promoter of SV2C gene, which is similar to the sequence of repressor element 1(RE1). Our preliminary data also showed that the alteration of REST expression caused the change in transcription of SV2C gene. In the present project, we will firstly try to demonstrate that REST is an inhibitory transcription factor of SV2C gene, and then analyze the role of REST and RE1-like sequence in mutant Htt inhibiting transcription of SV2C gene, probing for the underlying molecular mechanism of mutant Htt inhibiting SV2C expression via REST. Hopefully, the project could provide new evidence for revealing the mechanism of synaptic transmission impairment by mutant Htt and for finding new target of treating HD.

亨廷顿病(HD)由亨廷顿蛋白(Htt)基因突变所致，HD患者和转基因动物脑内有明显的突触功能障碍，突变Htt通过对基因转录的异常调控影响多种突触蛋白的表达与突触传递障碍有关。突触小泡蛋白2(SV2)是一类对突触传递具有调节作用的糖蛋白，其C亚型SV2C选择性表达于基底神经节等HD病变脑区。我们在HD转基因小鼠纹状体等脑区发现SV2C基因转录明显降低；生物信息学分析显示，阻遏子元件沉默转录因子(REST)可能是SV2C基因的转录抑制因子，可能通过SV2C基因启动子上与阻遏子元件1(RE1)序列相似的RE1样序列互作调控SV2C表达；我们的初步实验也显示，改变REST表达水平可改变SV2C的转录表达。本项目将在证明REST是SV2C基因的转录抑制因子基础上，分析REST和RE1样序列在突变Htt抑制SV2C表达的分子机制，为揭示突变Htt损害突触传递功能的机制和发现治疗HD的新靶点提供依据。

亨廷顿病(Huntington’s disease, HD)是一种常染色体显性遗传的神经变性疾病。HD早期症状的出现与突触功能的异常有关。HD患者和HD转基因动物脑内有明显的突触功能障碍，突变Htt通过对基因转录的异常调控影响多种突触蛋白的表达。突触小泡蛋白2（Synaptic vesicle protein 2, SV2）是神经元突触小泡和内分泌细胞分泌颗粒上的一种跨膜糖蛋白，具有SV2A、SV2B 和 SV2C三个亚型，突变Htt是否通过对SV2的表达和/或翻译后修饰的影响或与其发生异常相互作可能而引起HD未见报道。本项目对 SV2表达的影响及其机制和病理学意义进行了研究。结果显示，突变Htt显著抑制选择性表达于HD累及脑区的SV2中的C亚型(SV2C)的转录表达,而对A、B两种亚型(SV2A和SV2B)的转录表达无明显影响；阻遏子元件沉默转录因子(REST)通过与SV2C基因启动子中阻遏子元件1(RE1)相互作用而抑制SV2C转录活性；突变Htt能促进REST核转位及与SV2C启动子的结合，沉默REST显著可阻断突变Htt对SV2C转录表达的抑制；组蛋白去乙酰化酶1/2（HDAC1/2）选择性抑制剂丁酸钠能减弱突变Htt对SV2C转录和表达的下调。由此表明，突变Htt 可通过促进REST从细胞质到细胞核的转位而增强REST与SV2C启动子中RE1的结合，进而抑制SV2C基因的转录和表达；突变Htt对SV2C表达的抑制至少部分是由突变Htt通过组蛋白去乙酰化而增强REST作用所致。此外，本研究还观察到SV2C参与神经细胞内突触小泡运输，过表达SV2C和丁酸钠处理能显著减弱突变Htt对突触小泡运输的抑制，表明突变Htt可通过促进HDAC1/2的活性而抑制SV2C表达，进而抑制突触小泡的运输。因此，HD中选择性病理学变化可能与突变Htt通过选择性抑制SV2C表达而抑制突触小泡的运输，进而引起突触功能障碍有关，对SV2C基因的转录调控可作为探索治疗HD方法的靶点。