|Budget Amount *help
¥12,300,000 (Direct Cost : ¥12,300,000)
Fiscal Year 1999 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Fiscal Year 1998 : ¥8,000,000 (Direct Cost : ¥8,000,000)
The cellular mechanisms underlying production of amyloid β protein (A β), remain key but open issue. An endosomal/lysosomal pathway has been suggested as a site, whereby A β is cleaved from amyloid precursor protein (APP). Previous studies from our laboratory demonstrated that APP, Aβ , and amyloid-associated proteins are involved in the pathogenesis of experimental myopathy induced by chloroquine (CQN) in rats, which is potent agents affecting acidic compartments, and endosomal/lysosomal pathway. Recent immunocytochemical studies showed that presenilin-1 (PS-1) is involved in protein trafficking, including APP. This evidence prompted us to study APP and PS-I processing in cultured myocytes under CQN.
Microvacuoles were recognized in the perinuclear region of cultured myocytes after CQN treatment. These vacuoles were stained with anti APP and PS-I. Double labeling with anti APP and PS-1showed that APP, and PS-I were co-localized in the vacuoles. Northern ELISA demonstrated upregulated m
RNA level of APP, but mRNA of PS-I were unchanged during CQN treatment. About 12 kDa amyloidogenic band was detactable by Western blot analyses using various anti-APP and anti-Aβ antibodies in mitochondrial and microsomal fraction of CQN-treated cells. About 4 kDa band, presumably Aβwas detectable in mitochondrial fraction of both control and CQN-treated cells. 27 and 47 kDa PS-I fragments were detectable in lysate, mitochondrial and microsomal fractions obtained from CQN untreated myocytes with anti-PS-I, N-terminal, and full-length PS band, respectively.
Under Brefeldin, Monensin, and Concanamycin treatment, immunopathological, biochemical, molecular biological studies were performed on cultured myocytes. Our data indicate that the endosomal/lysosomal pathway play an important role in generating amyloidogenic fragments and Aβ from full-length APP and that β-coatmer protein, non-clathrin coated vesicles pathway might 'be involved in APP trafficking to CQN sensitive endosomes.
In conclusion, the experimental cultured myocyte systems with or without CQN treatment provide an excellent model to shed light on the cellular mechanism by which Aβ is produced from APP in terms of the pathogenesis of AD. Less