Data_Sheet_3_Disposition of Proteins and Lipids in Synaptic Membrane Compartments Is Altered in Q175/Q7 Huntington’s Disease Mouse Striatum.xls

Dysfunction at synapses is thought to be an early change contributing to cognitive, psychiatric and motor disturbances in Huntington’s disease (HD). In neurons, mutant Huntingtin collects in aggregates and distributes to the same sites as wild-type Huntingtin including on membranes and in synapses. In this study, we investigated the biochemical integrity of synapses in HD mouse striatum. We performed subcellular fractionation of striatal tissue from 2 and 6-month old knock-in Q175/Q7 HD and Q7/Q7 mice. Compared to striata of Q7/Q7 mice, proteins including GLUT3, Na+/K+ ATPase, NMDAR 2b, PSD95, and VGLUT1 had altered distribution in Q175/Q7 HD striata of 6-month old mice but not 2-month old mice. These proteins are found on plasma membranes and pre- and postsynaptic membranes supporting hypotheses that functional changes at synapses contribute to cognitive and behavioral symptoms of HD. Lipidomic analysis of mouse fractions indicated that compared to those of wild-type, fractions 1 and 2 of 6 months Q175/Q7 HD had altered levels of two species of PIP2, a phospholipid involved in synaptic signaling, increased levels of cholesterol ester and decreased cardiolipin species. At 2 months, increased levels of species of acylcarnitine, phosphatidic acid and sphingomyelin were measured. EM analysis showed that the contents of fractions 1 and 2 of Q7/Q7 and Q175/Q7 HD striata had a mix of isolated synaptic vesicles, vesicle filled axon terminals singly or in clusters, and ER and endosome-like membranes. However, those of Q175/Q7 striata contained significantly fewer and larger clumps of particles compared to those of Q7/Q7. Human HD postmortem putamen showed differences from control putamen in subcellular distribution of two proteins (Calnexin and GLUT3). Our biochemical, lipidomic and EM analysis show that the presence of the HD mutation conferred age dependent disruption of localization of synaptic proteins and lipids important for synaptic function. Our data demonstrate concrete biochemical changes suggesting altered integrity of synaptic compartments in HD mice that may mirror changes in HD patients and presage cognitive and psychiatric changes that occur in premanifest HD.

突触功能异常被认为是引发亨廷顿病（Huntington’s disease, HD）患者认知、精神及运动障碍的早期病理变化之一。在神经元中，突变型亨廷顿蛋白会聚集形成包涵体，并与野生型亨廷顿蛋白分布于相同的位点，包括细胞膜及突触部位。本研究针对亨廷顿病模型小鼠的纹状体突触的生化完整性展开了探究。我们对2月龄及6月龄的敲入型Q175/Q7 HD小鼠与Q7/Q7小鼠的纹状体组织进行了亚细胞分级分离。与Q7/Q7小鼠的纹状体相比，6月龄Q175/Q7 HD小鼠纹状体中，葡萄糖转运体3（GLUT3）、钠钾ATP酶（Na+/K+ ATPase）、N-甲基-D-天冬氨酸受体2b（NMDAR 2b）、突触后致密蛋白95（PSD95）及囊泡谷氨酸转运体1（VGLUT1）等蛋白的分布发生了改变，而2月龄小鼠未出现此类变化。上述蛋白均分布于细胞膜、突触前膜及突触后膜，这一结果支持了“突触功能异常是亨廷顿病患者认知与行为症状的致病诱因”这一假说。对小鼠分级组分的脂质组学分析显示，与野生型小鼠相比，6月龄Q175/Q7 HD小鼠的组分1与组分2中，两种参与突触信号传导的磷脂酰肌醇二磷酸（PIP2）的含量发生了改变，胆固醇酯水平升高，而心磷脂组分的含量则有所下降。在2月龄小鼠中，则检测到酰基肉碱、磷脂酸及鞘磷脂的组分水平升高。电镜（EM）分析显示，Q7/Q7与Q175/Q7 HD小鼠纹状体的组分1及组分2中，均存在游离突触囊泡、单个或成簇分布的囊泡填充轴突终末，以及内质网（ER）与内体样膜结构。但与Q7/Q7小鼠相比，Q175/Q7 HD小鼠纹状体组分中的颗粒团块数量显著减少，且体积更大。对人类亨廷顿病死者的壳核组织进行分析后发现，钙连蛋白（Calnexin）与GLUT3这两种蛋白的亚细胞分布与对照组壳核存在差异。我们的生化分析、脂质组学分析及电镜分析结果均表明，亨廷顿病突变会导致与突触功能密切相关的突触蛋白及脂质的定位出现年龄依赖性紊乱。本研究数据证实了具体的生化改变，提示亨廷顿病模型小鼠的突触区室完整性受损，这一变化或可模拟人类亨廷顿病患者的病理改变，并预示临床前亨廷顿病患者即将出现的认知与精神症状。