| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Protein Kinase C(PKC), a calcium-activated, phospholipid-dependent protein kinase, is abundant in the brain. PKC regulates neural functions by phosphorylation of proteins such as ion channels, enzymes, and cytoskeletal proteins. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, is known to cause neurodegeneration related to Alzheimer disease. We reported that oxidized diacylglycerol (DAG-OOH) activates PKC as efficiently as PMA does (S. Takekoshi et al. 1995. Biochem. Biophys. Res. Commun., 217, 654-660). Among 7 PKC isoforms present in rat brain, only PKC α and PKC δ were activated by DAG-OOH. We therefore examined the neurodegenerative effects of DAG-OOH on cultured neurons overexpressing PKC α and PKC δ.
Neuronal cells established from 18-day rat fetus cerebral cortex (PN cells) were employed. We used an adenovirus vector system which allows expression of PKC α and PKC δ gene at a high level. These cultured neurons treated by streptolysin O to let unoxidized DAG and D
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AG-OOH penetrate into PN cells. Those treated cells were observed by 1) phase contrast microscopy, 2) electron microscopy (EM), and 3) immunoblot analysis of phosphorylated Raf, mitogen-activated protein kinase (MAPK; ERK), MAPK/ERK kinase (MEK) and microtubule-associated protein tau.
Within 12h of exposure to DAG-OOH, the PKC δ-overexpressed PN cells exhibited neuritic thinning and characteristic beading, while these changes were not observed in the PKC α-overexpressed PN cells. Both routine EM and tubulin immunohistochemistry revealed the microtubule (MT) disassembly in the "beaded" lesions. This MT damage may be caused by the stagnation of the neurosecretory vesicles in the "beaded" lesions. Next we investigated the effect of DAG-OOH treatment on MAP kinase pathway in the PKC δ-overexpressed PN cells. PD98059, a MEK specific inhibitor, prevented the DAG-OOH-induced neurodegeneration. In addition, DAG-OOH induced a time dependent increase in the phosphorylation of Raf, MEK and ERK. MAP kinase phosphorylates the tau on their microtubule-binding domain, causing their dissociation from microtubules. Immunoblot analysis using phospho-specific tau antibody showed that DAG-OOH treatment induced the tau phosphorylation at threonine-181, serine-202, threonine-205. Phosphorylation of tau may cause MT disassembly in "beaded" lesions because MT disassembly interferes with microtubules stabilizing capacity of microtubules. Those findings may indicate that the DAG-OOH-induced activation of PKC δ is responsible for the activation of MAP kinase pathway and tau phosphorylation, resulting in the neurodegeneration and cell death. Less
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