|Budget Amount *help
¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 1993 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1992 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Intracellular trafficking and processing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) was studied.
1.Antibodies against specific protein in intracellular compartments : We were successful in raising specific antibodies against intracellular compartment specific protein, ERGIC53, galactosyltransferase, cathepsin D.
2.APP trafficking in cultured cells : APP and Abeta immunoreactivities were observed in cell surface, and vesicular structures in cytoplasm. Western blot analyzes showed 50,20 kDa amyloidogenic fragments, and 4 kDa, apparently Abeta, in microsomal fraction.
3.Effect of brefeldin A and monensin on APP processing : Brefeldin A and monensin are able to inhibit protein trafficking from endoplasmic reticulum (ER) to Golgi apparatus, and from Golgi apparatus to another cellular compartments. Under brefeldin A treatment, numerous vacuoles appeared in the cytoplasm of the cultured cells, and the vacuoles were immunolabeled with anti-ERGIC53. With monensin treatment, vacuoles appeared in perinuclear cytoplasm, and were stained with anti-APP and anti-Golgi antibodies. Western blot analysis showed reduced intensity of Abeta 4 kDa band.
4.APP trafficking in Hela cells transfected with wild type APP770, and Swedish mutant cDNA : Wild type APP770 and Swedish mutant APP were overexpressed in cultured HeLa cells. APP immunoreactivities appeared to increase in ER,cell surface, and vesicles in the cytoplasm of these transfected cells. APP mRNA and Swedish type mRNA were determined with Northern ELISA,and turned out to increase about 3 to 6 fold compared to plasmid controls.
Our data provide new evidence indicating that APP may process to generate Abeta either in ER,or lysosomes, or both compartments may be involved.