| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The mechanisms of specific localization of synaptic proteins at synaptic site of central nervous system (CNS) have been unclear. Little is known how the soluble proteins functioning in synapses localize at synapse in particular. Complexin (CPX) is a soluble pre-synaptic regulatory protein, and is thought to regulate exocytosis of neurotransmitters release from pre-synapse. Here we show the initial study to elucidate the localization mechanism of soluble synaptic proteins in synapse by using CPX as a model molecule.
The myc-tagged CPX (mCPX) construct was transfected into rat primary hippocampus neuron with liposome reagent, and the localization of expressed protein was evaluated by the fluorescent intensity analysis of images up taken by laser confocal scanning microscopy. In the dendrite, fluorescence intensities from mCPX and EGFP, a nonspecific soluble protein control, decreased at periphery of the dendrite. In the axon, the mCPX was transferred to periphery of the axon although the
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EGFP showed the same tendency with that at dendrite, indicating that CPX has a specific mechanism to be transferred to the axon.To identify the domain required for the specific transfer to axon, the deletion analysis was conducted. Fluorescent analyses indicated that the deletion of 19 amino acid residues, G71-P89, within the "central core" resulted the loss of the specific axonal localization of CPX derivatives. The EGFP derivative fused with the G71-P89 peptide showed axonal localization, indicating that the G71-P89 region conditions the axonal transport of CPX.
SNAP-25, a member of SNARE proteins, is essential for the secretory vesicle fusion with plasma membrane and phosphorylated at the Ser187 residue by the phorbor-ester stimulation in PC12 cells. The pharmacological study is important to elucidate the function of the protein phosphorylation in cellular function. In addition, to know how the phosphorylatin state in living organ is also important for understanding the biological system. Here we show the mode of the SNAP-25 at Ser187 in rat brain by using anti-phosphorylated SNAP-25 antibody as a probe. Western blotting of rat brain lysates indicated that SNAP-25 is phosphorylated in rat brain. The developmental patterns of expression and the SNAP-25 phosphorylation was not pararell. The SNAP-25 expression is detected from E18 embryonic brain and the expression continues to increase lineally up to P25. The phosphorylation of SNAP-25 cannot be detected until P9 and then the phosphorylation increment continue to P25. Thus the SNAP-25 phosphorylation is regulated developmentally in rat brain. The time course study using the rat hyppocampal culture showed similar phosphorylation pattern of SNAP-25, implying the SNAP-25 phosphorylation is genetically programmed under the interaction between neuronal cells and glial cells. In the investigation of the effect of environmental stimulation that confers the neural plasticity, the SNAP-25 phosphorylation shows small decrement by in vivo long-term-potentiation. The decrement of the SNAP-25 phosphorylation is also observed in the rat brain introduced epilepsy by kainate. These results clearly exhibit that the phosphorylation state of SNAP-25 at Ser187 is altered by developmental stage and environmental change, thus the SNAP-25 phospohorylation takes important roles in higher brain function(s). Less
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