Functional analysis of protein kinase C(PKC), a molecule involved in plasticity, development and cell death in central nervous system, using gene-manipulated mice.

1)利用tet调控系统建立了脑区特异性、可诱导表达GammaPKC-GFP的转基因小鼠。在这些小鼠中，GammaPKC-GFP在浦肯野细胞中大量表达。我们制备了这些小鼠的小脑切片，并试图观察GammaPKC-GFP在活体浦肯野细胞中的移位。平行纤维的跨突触刺激触发了pkc-gfp沿树突的移位。这种易位从细胞的外周传播到胞体，表明神经活动可以控制GammaPKC的易位。这一现象为发展从蛋白质水平监测神经活动的方法提供了新的视角。2)最近有报道称遗传性脊髓小脑性共济失调14型(SCA14)是由GammaPKC错义突变引起的。为了阐明SCA14致病的分子机制，我们将SCA14突变引入人GammaPKC基因，并与GFP融合，在CHO细胞中表达突变型GammaPKC-GFP。在表达细胞中可见突变型GammaPKC的聚集。此外，GammaPKC-GFP在其受体介导的转位过程中经常聚集。这些结果表明突变的GammaPKC的聚集形成参与了SCA14的发病。3)我们对来自不明原因的日本SCA患者的基因组DNA进行了基于PCR的筛选。我们发现了新的日本人SCA14家族，并证明了SCA14在日本人中是非常罕见的。

1)We developed brain region specific and inducible transgenic mice expressing gammaPKC-GFP using tet-regulated system. In these mice, gammaPKC-GFP was abundantly expressed in Purkinje cells. We prepared the cerebellar slices from these mice and attempted to observe gammaPKC-GFP translocation in living Purkinje cells. Trans-synaptic stimulation of parallel fiber triggered gammaPKC-GFP translocation along the dendritic shafts. This translocation propagated from peripheral to soma of the cells, indicating that neural activity can control gammaPKC translocation. This phenomenon provides the novel insight for developing the methods to monitor the neural activity in a protein level.2)Recently it was reported that the hereditary spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations of gammaPKC. To elucidate the molecular mechanism underlying the SCA14 pathogenesis, we introduced SCA14 mutations into human gammaPKC cDNA, fused it with GFP cDNA and expressed mutant gammaPKC-GFP in CHO cells. The aggregation of mutant gammaPKC was obviously seen in the expressing cells. Moreover, gammaPKC-GFP was frequently aggregated during the process of its receptor-mediated translocation. These results suggest that the aggregate formation of mutant gammaPKC is involved in the SCA14 pathogenesis.3)We performed PCR-based screening of genomic DNA prepared from undefined Japanese SCA patients. We found new Japanese SCA14 family and proved that SCA14 is very rare in Japanese SCA population.