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¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 2005 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2004 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 2003 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Deposition of amyloid β protein (Aβ) in the brain is an invariant neuropathological feature of Alzheimer's disease (AD). Aβ is generated by two cleavages of amyloid precursor protein (APP). The initial cleavage by BACE is followed by γ-secretase cleavage of the C-terminal APP fragment. Once formed, Aβ is mainly degraded small regulatory peptides by neprilysin (NEP). The generative process of AD is linked to a shift in the balance between Aβ production and degradation. We investigated the possible functional significance of BACE and NEP in cultured myocyte systems under chloroquine (CQN) as an experimental model to study APP processing for Aβ production.
1.Human myocyte (CCL136) was transfected with mutation APPcDNA. The APPmRNA level reached the maximum after 12 hours CQN treatment. Microvacuoles were appeared in perinuclear area 12 hours after CQN treatment, and increased in number until up to 24 hours. Red granular materials around these vacuoles, and inside the vacuoles were stained
2.Perinuclear and heterogeneous materials surrounding vacuoles containing stained granular bodies reacted with anti Aβ antibody. Double labeling with anti Aβ and anti BACE antibodies, demonstrated that Aβ and BACE were co-localized in some of the vacuoles.
3.4,12,20,28,60,70 kDa bands were detected using anti Aβ antibody, 12,35,40,70 kDa bands were detected using anti BACE antibody, 70,80 kDa bands were detected using anti NEP antibody by Western blotting. 4 kDa band, apparently Aβ, became remarkable after CQN treatment.
4.BACEmRNA levels were reduced 50% in the siRNA treated myocyte cells relative to control. Double immunostaining of BACEmRNA knockdown myocyte with anti Aβ and anti BACE antibodies, immunoreactivities were co-localized in some of the microvacuoles, but apparently decreased in number
5.NEPmRNA vector was tranfected into myocyte using Lipofectamine. The recombinant myocyte which increased of NEPmRNA level is not provided at present.
CQN myocyte system is a good experimental model to study the balance between the generation and degradation of Aβ appears altered. Less