| Co-Investigator(Kenkyū-buntansha) |
WAKABAYASHI Ichiro Yamagata University, School of Midicine, Deparment of Anatomy and Cell Biology, Professor, 医学部, 教授 (70220829)
SAINO Sachiko Yamagata University, School of Midicine, Deparment of Anatomy and Cell Biology, Associate Professor, 医学部, 講師 (50312559)
HOZUMI Yasukazu Yamagata University, School of Midicine, Deparment of Anatomy and Cell Biology, Assistant Professor, 医学部, 助手 (00372334)
NAKANO Tomoyuki Yamagata University, School of Midicine, Deparment of Anatomy and Cell Biology, Assistant Professor, 医学部, 助手 (00333948)
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| Budget Amount *help |
¥12,500,000 (Direct Cost: ¥12,500,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2003: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Research Abstract |
We have so far isolated and characterized several isozymes of diacylglycerol kinase(DGK) from rat brain cDNA library. Our series of studies revealed the heterogeneity of the enzyme in terms of molecular structure, tissue distribution, enzymatic activity, and the localization of gene expression, and provided functional implications for DGK family. Interestingly, most of the isozymes show abundant expression in distinct patterns in the brain, suggesting the importance of this enzyme in neuronal function. Among these, mRNA for DGKbeta is detected uniquely in restricted regions of the brain, such as the caudate-putamen, accumbens nucleus, olfactory tubercle, olfactory bulb, and hippocampal pyramidal cell layer. In our previous experiments using transfected COS-7 cells, DGKbeta is shown to be distributed throughout the cytoplasm in a filamentous pattern. However, detailed subcellular localization of this isozyme remains unclear in neurons. To address this point, we performed in the present
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study immunohistochemical examination on rat brain with a specific antibody against DGKbeta, together with cDNA-transfection into primary cultured neurons using GFP expression vector. Under the con focal laser scanning microscopy, the immunoreactivity for DGKbeta was detected in neurons of the corresponding brain regions that express the mRNA abundantly and appeared as ring-like structures. To examine the presice localization of DGKbeta, we employed immunoelectron microscopy to localize the immunolabelled structures. Dark immunoreaction products in pre-embedding immunoperoxidase electron microscopy were distributed diffuselly within dendrites and spines in neurons. Furthermore, most of the particulate metal marker in pre-embedding silver-intensified immunogold electron microscopy were deposited in association with the cytoplasmic face of the cell membrane in dendrites and spines in neurons. In the transfected hippocampal neurons in culture, GFP-DGKbeta was predominantly detected as button-like structures in the presumed dendritic processes. These results suggest that DGKbeta may be involved in synaptic transmission at postsynaptic site and signal transduction in neuronal cells.
In addition, it should be also noted that two of the isozymes, DGKzeta and DGKiota, contain a nuclear localization signal, suggesting possible roles of these isozymes in nuclear processes. We have revealed selective translocation of DGKzeta in hippocampal neurons under transient forebrain ischemia. We found that DGKzeta is translocated from the nucleus to the perikaryal cytoplasm in hippocampal pyramidal cells immediately after 20 min of ischemic insult. Previous studies have shown that neuronal death in the hippocampus occurs after 48-72 h reperfusion, but not immediately, under transient ischemia, a phenomenon called "delayed neuronal death." Therefore we speculate that the nucleo-cytoplasmic translocation of DGKzeta in the hippocampal neurons at an early phase of ischemic insult may be one of the triggering events of the delayed neuronal death, but not a result of dying process. Less
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