The effect of expressing APP and APP carboxyl-terminal fragment in human neuroblastoma IMR-32

• Main Authors: Shih-Jyue Liang, 梁世玨
• Other Authors: Huey-Jen Tsay
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/97952459464715736624

碩士 === 國立陽明大學 === 神經科學研究所 === 93 === Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder in the aged population and is characterized by the presence of senile plaques and neurofibrillary tangles in the brain. The major component of senile plaques is amyloid protein β (Aβ) peptides deposition. Aβ is processed from β-amyloid precursor protein (APP) via β- and γ-secretase. Many researches are focused on Aβ�nneurotoxicity. It is known that many genetic mutants of APP can promote Aβ production. The mechanism in sporadic cases of AD is not fully understood. Oxidative stress is defined as a disturbance in the equilibrium status of free radicals and the antioxidant defense mechanisms. Furthermore, oxidative stress has long been implicated in a number of age-associated disorders, including AD. Calcium homeostasis plays many roles in neuronal cell functions including signaling pathway, synaptic plasticity and apoptosis. Disturbance of intracellular calcium homeostasis has been implicated in the pathogenesis of the AD. Swedish mutant and Flemish mutant of APP in familial AD had intensively studied. These mutations can alter APP processing or increase Aβ production. It had been reported that expression of APP or APP C-terminal fragment (APP-CTF) is toxic to neurons. In early study, conditional expressed APP-CTF by using a tetracycline-responsive promoter system was investigated. Expression of this fusion protein in neuroblastoma SK-N-MC resulted in pronounced cytotoxicity. In present study, human neuroblastoma IMR-32 was used to establish stable lines expressing mutant APP and APP-CTF. Different level of soluble APPα, intracellular APP and Aβ1-40 production were found in these clones. Compared with vector-transfected controls, the APP and APP-CTF mutant clones had smaller cell bodies and clustered together. The spontaneous cell death also frequently happened in APP and APP-CTF mutant clones. Using these clones, I tried to elucidate the effect of APP and APP-CTF in cells. After hydrogen peroxide and thapsigargin treatment, there were no remarkable vulnerability differences between these stable clones. But, compared with vector-transfected clones, expressing mutant APP or APP-CTF in IMR-32 cells had effect on cell growth. In further experiments, we compared the mitochondria activity in these stable clones by MTT reduction assay. The O.D. values from different stable clones didn’t show significant difference in 6, 24 and 48 h after cell seeding. By PI staining and flow cytometry, G2/M phase peak was not clear in C4 and C28 clones. It also seemed more pAPP-CT100fle cells were located in S phase, and further experiments were needed to confirm this observation.