Study on the physiological function of synuclein family

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1) It was found that a-synudem is consiturtively phosphoiylated in both cells HEK 293 and FC12 which had been previously transfected with α-synudein gene. In both cell lines phosphorylalion was highly sensitive to phosphatases, since okedaic acid markedly stabilized phosphate incorporation. We have identified a major phosphorylation site at serine 129 which is phosphoiylated by casein kinases. It may be indicated that the function of α-synuciein is regulated by phosphorylation/dephosphorylation reaction.2) β-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that β-synuclein protein was expressed within the cytoplasm and nudeus. Furthermore, β-synuclein immunoreadivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues.3) We have prqared cDNA library fmm the cerebrum of reptiles (blue green snake) and amphibian (bullfrog) to clone cDNA of synudein family. Domain I of human β-synuclein … More was used as a prove. Ten positive clones have been obtained from the eDNA library of blue green snake. The sequence analysis is underadvance,4) Using yeast two-hybrid system, we have identified 14-3-30 and metallothionein2A as β-synudein-binding protein. It has been shown that both proteins interact with β-synuclein in rat brain lysate, indicating that those interactions between β-synuclein and 14-3-30 and metallothionein2A occure even under the physiological conditions. Interestingly, it was also shown that β-synuclein bound to metallothionein3.5) Transgenic mice using Cre-loxp system were produced to clarify the physiological function of α- and β-synucleins. In this experiment expression of synudeins is region specific in tissue since it is regulated by promotor used. We produced transgenic mice of two category, Cre-donor and Cre-acceptor mice. CaMkll promotor-Cre (hippocampus), GFAP promotor-Cre (astrocytes), Pgk2 promotor-Cre (testes), CAG promoter-Cre (whole body), Keratin14 piomotor-Cre (skin) transgenic mice were produced as Cre-dpnor, and loxP-neo-loxP (LNL)-cz-synudein and LNL-α-synuclein mice were as Cie-acceptor. Less