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

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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.