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Production of amyloid beta protein (Abeta), which characteristically deposits in Alzheimer's disease (AD) affected brain, by proteolytic processing of amyloid precursor protein (APP) is a key issue in the pathogenesis of AD.We describe here a novel experimental model in vitro, in which Abeta is cleaved from APP in cultured human myocyte after chloroquine (CQN) treatment.
1. Pathological studies on cultured of human myocyte with/without CQN treatment.
1) Histopathological studies of cultured myocytes : Vacuoles appeared in perinuclear area of the cultured myocytes 12 hours after CQN treatment, and increase in number until up to 24 hours. Modified Gomori method stained red granular materials around these vacuoles, and inside the vacuoles.
2) Immunopathological studies of APP and Abeta localization : CQN induced vacuoles were stained with anti-APP,and anti-Abeta antibodies. A number of these vacuoles were also labeled with antibodies against specific protein to intracellular compartments, ra
b 7 and cathepsin D for lysosomes, only a few of these vacuoles were stained with anti-garactosyltransferase. But no immunoreactivities were observed with anti-ERGIC 53, specific for endoplasmic reticulum.
2. Biochemical studies on proteolytic processing of APP : Western blot analyzes demonstrated there were many amyloidogenic or non-amyloidogenic APP fragments in cell fractions obtained from cultured myocytes with CQN treatment. 4 kDa fragment, apparently Abeta, become remarkable after CQN treatment.
3. Molecular biological studies of APP gene expression : mRNA of APP gene was determined sequentially by Northern ELISA.The mRNA began to increase 3 hours after CQN treatment, and showed a peak 9 hours after CQN treatment, 2.5-fold increase compared to mRNA of myocyte without CQN treatment.
This novel experimental model provides in vitro model to study mechanism underlying proteolytic process of APP into Abeta production. Our data indicate that acidic compartments, lysosomal pathway, may play an important role in the generation of amyloidogenic fragments and Abeta.
本実験系はAPPからAβ生成機構を解明する際に有用な新しいin vitroのモデルとなることが示された。また、リソソーム経路などの、いわゆる酸性コンパートメントがAPPの分解過程に関与しAβの生成に重要な役割を演じていることを支持する新しい証左を提供するものと考えられる。 Less