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¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1995 : ¥1,100,000 (Direct Cost : ¥1,100,000)
The lysosomal pathway has been suggested as the site of formation for amyloidogenic amyloid precursor protein (APP) fragment and amyloid beta protein (Abeta). Lysosomes are, however, meeting places in which several streams of intracellular traffic converge. We describe here an attempt to identify APP processing pathway involving lysosomal system by vesicle specific protein
1. Antibodies raised against transport vesicle specific proteins : There are clathrin-coated vesicle, and non-clathrin coated vesicle, designated as coatmer protein-coated vesicle of transport vesicles. We were successful in raising antibodies against synthetic peptides homologous to amino acid sequence of clathrin, and beta-COP.
2. Proteolytic processing of APP in cultured human myocyte treated with/without concanamycin A (Con A) : Con A inhibits selectively endocytosis from cell surface to intracsllular compartments via clathrin-coated vesicles.
1) Histopathological studies of cultured myocytes : Vacuoles appeared ben
eath the plasma membrane in the cultured myocytes 12 hours after Con A treatment, and increase in number until up to 24 hours.
2) Immunopathological studies of APP and Abeta localization : Con A induced vacuoles were labeled with anti-APP,and anti-Abeta antibodies. A number of these vacuoles were also atained with clathrin antibody. Interestingly, beta-COP antibody labeled unique dot-like structures in the cytoplasm without Con A treatment. Cultured myocytes treated with Con A,vacuoles were labeled with beta-COP antibody. But no immunoreactivities were observed with anti-ERGIC 53, specific for endoplasmic reticulum, and with anti-garactosyltransferase, specific protein to Golgi apparatus.
3. Biochemical studies on proteolytic processing of APP : Metabolic labeling of cultured myocytes were performed of cultured myocytes treated with/without Con A.APP and Abeta in the cell lysates were immunoprecipitated with specific antibodies against APP,and Abeta. Subsequently, eluates were separated on electrophoresis, and then subjected to autoragiography. 4 kDa Abeta fragment was detected in cell lysate without Con A treatment, and this 4 kDa Abeta fragment increased their intensity after Con A treatment.
Our data indicate that non-clathrin coated vesicles to acidic compartments, lysosomes, may play an important role in the generation of amyloidogenic fragments and Abeta, in addition to clathrin-coated vesicles.
3)生化学的検討:Con A添加ないし非添加条件で^<35>S-メチオニンを用いてパルスラベルを行い、APP、Aβ抗体で免疫沈降を施しオートラジオグラフィーにて検討した。Con A非添加条件で4kDaのAβが検出されるが、Con A添加によってこの4kDaバンドは減少したが明らかに存在した。