|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1997 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1996 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Recent advance of Alzheimer research revealed the importance of senile plaque amyloids consisting of Abeta as the earliest event of Alzhiemer's disease before appearance of neurofibrillary tangles and neuronal cell loss. Furthermore, genetical study found 4 causal genes linked to familiar Alzheimer's disease, that are, mutant betaAPP,Apolipoprotein E4, S182 (presenilin-1 : PS-1) and E5-1 (presenilin-2 : PS-2). In this study, we planned 1) gene analysis of 2 familiar AD cases ; 2)cloning of wild type of PS-1 and constructing mutant type PS-1 and PS-2 ; 3) making specific antibodies to PS-1 and PS-2 ; 4)establishment of expressing cell lines ; 5)detection of proteolytic fragments and measurement of amount of Abeta secretion ; 6) immunostain and immunoblot analysis of the AD and control brains ; and 7)analysis of transgenic mice expressing mutant PS-1.
Extensive senile plaques and amyloid angiopathy were recognized in PS-1 A260V and PS-1 A285V families. Wild type PS-1 and PS-2, mutant PS-1
(DELTAM146L,DELTAA246E,DELTAL286V,DELTAExon9, DELTAL392V,DELTAC410Y) and mutant PS-2 (DELTAN141I,DELTAM239V) were cloned. Antilbodies to N-terminus of PS-1 (MTELPAPLSYFONAQMSEDNHLS,HSN-2) and C-terminus of PS-1(LVQPFMDQLAFHQFYI,HS-C) were characterized. Transient and stable cell lines expressing PS-1 showed that 44/40 KD full length of PS-1,26/25 KD N-terminal fragments (NTFs) and C-terminal fragments (CTFs) in both wild and mutant cell lines. No significant difference was pbserved in these PS-1 and their fragments. Doubletransfection cell lines expressing betaAPPDELTANL and mutant PS-1 secreted increased amounts of Abeta1-40 and Abeta1-42. In the AD and control brains, 26/25 KD NTFs and 17/16KD CTFs were recognized. Full length PS-1 was not observed. Transgenic mice expressing DELTAM146L and DELTAC410Y showed the some molecule size PS-1 and their fragmentsin the brains. Although HSN-2 and HS-C labeled neurons and processes in the control brains, neurofibrillary tangles and curly fibers were extensively labeled by HS-C suggesting close relationship with PS-1 and cytoskeletal abnormalities in the AD brains. Less