The Study On The Role Of Amyloid Precursor Protein In Capcitative Calcium Entry

Alzheimer's disease (AD) is a progressive dementia affecting a large proportion of aging people. AD is characterized by two hallmark brain lesions, i.e. extracellular deposits of amyloidβ(Aβ) and intracellular neurofibrillary tangles. Aβis derived from the processing of Amyloid precursor protein (APP) byβsecretase andγsecretase.In the present study, we focused on the study of the effect of APP and its proteolytic fragments on capacitative calcium entry (CCE). Following are the main content of the thesis:(1) We compared the CCE in N2a cells stably expressing human APP695 (APPwt) with its wild type counterparts, and found that CCE was depressed in APPwt cells. Experiments using CCD and confocal laser scanning microscope got the same results. We can drawed the conclusion that APP depresses CCE. APP knock-out mouse embryo fibroblast cells have an increased CCE compared to its wild type controls, which suggests that mouse APP has the same CCE depression effect as human APP695.(2) Our results showed that sAPPs do not affect CCE.βsecretase inhibitor couldn't change CCE in APPwt cells, whileγsecretase inhibitor depress CCE in APPwt cells. It's suggested that C-terminal of APP may involved in APP caused CCE depression. We further demonstrated the role of C-terminal of APP in CCE modulation in N2a cells overexpressing C-terminal fragments of APP. Further experiments suggested that the interaction of APP and G protein may contribute to the APP caued CCE depression.(3) In contrast to APPwt cells, N2a cells stably expressing Swedish mutant APP (APPswe) has a potentiated CCE compared to wild type N2a cells. Anti-Aβantibody can depress CCE in both APPswe and APPwt cells, which suggested that Aβcan potentiate CCE. This conclusion can be also demonstrated inγsecretase inhibitor treatment experiment. It has been reported that Aβforms calcium permeable channels on the cell membrane. We treated the cells with an Aβchannel blocker, tromethamine. The results showed that tromethamine can significantly depress Aβcaused CCE potentiation.(4) We further discuss the potential role of tromethamine in AD therapy. Low concentration of tromethamine can not affect the cell viability. Tromethamine could depress cell ROS, and that may be the way through which it affects cell apoptosis and protects the cells from Aβtoxity. In vivo experiments showed that tromethamine could increase the level of phosphoralate erk protein. The mechanisms need further study.