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¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Proteolytic proccessing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) in lysosomal pathway, and an effect of chloroquine, a potent lysosomotropic agent, on the processing were studied using variety of cultured cells.
1.Antibodies raised against specific protein to intracellular compartments under lysosomal pathway : Lysosomal pathway consists of endosome, primary, secondary lysosomes, and transport vesicles. There are specific proteins to these intracellular compartments, transferrin receptor for endosome, rab 7 for primary lysosome, and cathepsin D for secondary lysosome. Antibodies against synthetic peptides homologous to amino acid sequence of these proteins were successfully established.
2.Proteolytic processing of APP to A beta in cultured human cells under Chloroquine treatment : HUT78, derived from human T cell, U937, derived from human monocyte, and HeLa cell were cultured.
1) Proteolytic processing of APP to A beta in cultured human cells without Chl
(1) Immunohistochemical studies : Antibodies against intracellular compartment specific protein stained unique intracellular structures respectively. Transferrin receptor antibody labeled relatively large vesicles, and rab 7 antibody immunostained numerous small vesicles. Cathepsin D antibody labeled intracellular granular materials. APP and Abeta (Abeta17-28) antibodies demonstrated cell surface dot-like structures and cytoplasmic fine granular materials.
(2) Biochemical studies of APP processing : Many amyloidogenic and non-amyloidogenic APP fragments were detectable in cell fractionate. Western blot analyzes showed that 4 kDa Abeta fragment, was observed in mitochondrial and cytosolic fractions.
2) Proteolytic processing of APP to Abeta in cultured human cells under Chloroquine treatment : Chloroquine induced vacuoles in cytoplasm of the cultured cells after 12 hours. Vacuoles increased in number until up to 24 hours.
(1) Immunohistochemical studies : A number of these vacuoles were stained with transferrin receptor antibody, rab 7 antibody, and were also labeled with both APP and Abeta antibodies.
(2) Biochemical studies of APP processing : 4 kDa Abeta fragment became visualized more intensely after chloroquine treatment in mitochondrial and cytosolic fractions.
Our data provides further evidence for participation of acidic compartments, for example, lysosomal pathway, in the generation of amyloidogenic processing proteolytic cleaved APP products and Abeta.