Cerebellar gene expression analysis of Gb5-deficient mice

Gb5 is a divergent, evolutionarily-conserved, member of the heterotrimeric G protein b subunit family that is expressed principally in brain and neuronal tissue. Among Gb isoforms, Gb5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-g like (GGL) domains. Previous studies employing Gb5 knockout mice have shown that Gb5 is an essential stabilizer of GGL domain-containing RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. The purpose of this study is to better understand the functions of Gb5 in the brain outside the visual system by employing molecular biology, immunohistochemistry and confocal imaging technologies. We show here that mice lacking Gb5 have a markedly abnormal neurologic phenotype that includes neurobehavioral developmental delay, wide-based gait, motor learning and coordination deficiencies, and hyperactivity. Using immunohistochemical analysis and a green fluorescent reporter of Purkinje cell maturation we show that the phenotype of Gb5-deficient mice includes, in part, delayed development of the cerebellar cortex, an abnormality that likely contributes to the neurobehavioral phenotype. Multiple neuronally-expressed genes are dysregulated in cerebellum of Gb5 KO mice. Brain tissues from WT and KO with three biological replications of mice were collected, frozen in liquid nitrogen, and stored at -70°CGb5是异三聚体G蛋白β亚基家族（heterotrimeric G protein β subunit family）中一类趋异且进化保守的成员，主要在大脑及神经组织中表达。在所有Gβ亚型中，Gb5的独特之处在于其可与含有G蛋白γ样（G protein-g like, GGL）结构域的G蛋白信号调节蛋白（regulator of G protein signaling, RGS）R7亚家族成员发生异二聚化。既往针对Gb5基因敲除小鼠（knockout mice）的研究表明，Gb5是含GGL结构域的RGS蛋白的必需稳定因子，并可调控视网膜光转导过程的失活以及视网膜双极细胞的正常生理功能。本研究旨在通过分子生物学、免疫组织化学（immunohistochemistry）及共聚焦成像（confocal imaging）技术，深入阐明视觉系统外Gb5在大脑中的功能。本研究发现，缺失Gb5的小鼠呈现显著异常的神经表型，具体包括神经行为发育迟缓、宽基底步态、运动学习与协调功能缺陷以及活动过度。通过免疫组织化学分析及浦肯野细胞（Purkinje cell）成熟的绿色荧光报告基因（green fluorescent reporter）检测，我们证实Gb5缺陷小鼠的表型部分源于小脑皮层（cerebellar cortex）发育延迟，该异常可能是其神经行为表型的致病基础。在Gb5基因敲除（knockout, KO）小鼠的小脑组织中，多种神经表达基因存在表达失调情况。本研究收集了野生型（wild type, WT）与KO小鼠的脑组织样本，每组设置3次生物学重复（biological replications），将样本置于液氮中速冻后于-70℃保存。