The role of S182 and E5-1 on AB secretion in Alzheimer's Disease.

阿尔茨海默病研究的最新进展揭示了由Abeta组成的老年斑淀粉样蛋白作为阿尔茨海默病在出现神经原纤维缠结和神经细胞丢失之前的最早事件的重要性。此外，遗传学研究还发现了4个与常见阿尔茨海默病相关的致病基因，即突变的β-APP、载脂蛋白E4、S182(早老素-1：PS-1)和E5-1(早老素-2：PS-2)。在本研究中，我们计划1)对2例常见AD病例进行基因分析；2)克隆PS-1野生型并构建突变型PS-1和PS-2；3)制备针对PS-1和PS-2的特异性抗体；4)建立表达细胞系；5)检测蛋白水解物片段和测定Aβ分泌量；6)对AD和对照脑进行免疫染色和免疫印迹分析；7)分析表达突变型PS-1的转基因小鼠，发现PS-1A260V和PS-1A285V家族存在广泛的老年斑和淀粉样血管病变。野生型PS-1和PS-2，突变型PS-1…克隆了DELTAM146L、DELTAA246E、DELTAL286V、DELTAExon9、DELTAL392V、DELTAC410Y和突变体PS-2(DELTAN141I、DELTAM239V)。对PS-1的N端抗体(MTELPAPLSYFONAQMSEDNHLS，HSN-2)和PS-1的C端抗体(LVQPFMDQLAFHQFYI，HS-C)进行了鉴定。瞬时和稳定表达PS-1的细胞系表明，野生型和突变型PS-1、26/25kD的N-末端片段(NTF)和C-末端片段(CTF)的全长为44/40kD。PS-1及其片段的差异无统计学意义。表达APPDELTANL和突变型PS-1的双感染细胞株分泌Abeta1-40和Abeta1-42的量增加。在AD组和对照组脑中识别出26/25kD的NTF和17/16KD的CTF。未观察到全长PS-1。表达DELTAM146L和DELTAC410Y的转基因小鼠在脑内显示了一定分子尺寸的PS-1及其碎片。虽然HSN-2和HS-C标记了对照脑中的神经元和突起，但HS-C广泛标记了神经原纤维缠结和卷曲的纤维，提示与PS-1和AD脑内细胞骨架的异常密切相关。较少

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 … More (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