|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2003 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 2002 : ¥2,200,000 (Direct Cost : ¥2,200,000)
To understand molecular mechanisms of the proteolytic processing of APP (generation of Aβ) by presenilin-dependent γ-secretase, the relationship between γ-and γ' (ε)-cleavages of APP was investigated.
1. Amino acid substitutions in the vicinity of γ'-cleavage sites of APP altered γ'-cleavage sites as well as γ-cleavage sites, resulting in altered proportions of Aβ42 production.
2. Effects of familial Alzheimer's disease-associated mutations of APP and presenilins on γ-and γ'-cleavage sites were investigated. All the mutations examined here altered γ-cleavage sites and increased the proportion of Aβ42 production. Those mutations altered also γ'-cleavage sites and caused an increase in the proportion of CTFγ49-99 production. Although a potential link between the production of Aβ42 and CTPγ49-99 was observed, the proportion of Aβ4O and Aβ42 did not faithftully reflect that of CTFγ50-99 and 49-99.
These results indicate an intimate interrelation between γ-and y '-cleavages.
3. To evaluate the hypothesis that y '-cleavage precedes γ-cleavage, we sought to find an intermediate product, Aβ species longer than Aβ42/43. Using newly developed urea gel systems and an N-end-specific Aβ monoclonal antibody, the longer Aβs, Aβ1-43, Aβ1-45, Aβ1-46, and Aβ1-48, were identified unambiguously within the various cell types. The precise analysis of those longer Aβs suggests that multiple γ-secretase-dependent cleavage sites are present between γ-and 'γ'-cleavage sites of APP and that APP is cleaved successively at every three residues from the carboxyl-terminus generated by γ'-cleavage.