Elevated vulnerability to oxidative stress‐induced cell death and activation of caspase‐3 by the Swedish amyloid precursor protein mutation

Abstract

The Swedish double mutation (KM670/671NL) of amyloid precursor protein (APPsw) is associated with early-onset familial Alzheimer's disease (FAD) and results in from three- to sixfold increased beta-amyloid production. The goal of the present study was to elucidate the effects of APPsw on mechanisms of apoptotic cell death. Therefore, PC12 cells were stably transfected with human APPsw. Here we report that the vulnerability of APPsw-bearing PC12 cells to undergo apoptotic cell death was significantly enhanced after exposure to hydrogen peroxide compared to human wild-type APP-bearing cells, empty vector-transfected cells, and parent untransfected cells. In addition, we have analyzed the potential influence of several mechanisms that can interfere with the execution of the apoptotic cell death program: the inhibition of cell death by the use of caspase inhibitors and the reduction of oxidative stress by the use of (+/-)-alpha-tocopherol (vitamin E). Interestingly, oxidative stress-induced cell death was significantly attenuated in APPsw PC12 cells by pretreatment with caspase-3 inhibitors but not with caspase-1 inhibitors. In parallel, caspase-3 activity was markedly elevated in APPsw PC12 after stimulation with hydrogen peroxide for 6 hr, whereas caspase-1 activity was unaltered. In addition, oxidative stress-induced cell death could be reduced after pretreatment of APPsw cells with (+/-)-alpha-tocopherol. The protective potency of (+/-)-alpha-tocopherol was even greater than that of caspase-3 inhibitors. Our findings further emphasize the role of mutations in the amyloid precursor protein in apoptotic cell death and may provide the fundamental basis for further efforts to elucidate the underlying processes caused by FAD-related mutations.

Amyloid β-induced Changes in Nitric Oxide Production and Mitochondrial Activity Lead to Apoptosis

“…Cell Culture and Transfection-PC12 cells and HEK cells were transfected with DNA constructs harboring human mutant APP (APPsw, K670M/N671L) gene, the APPwt gene, inserted downstream of a cytomegalovirus promoter using the FUGENE 6 technique (Roche Diagnostics) (37). The transfected cells APPwt PC12, APPsw PC12, and control PC12 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 5% heat-inactivated horse serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 400 g/ml G418 at 37°C in a humidified incubator containing 5% CO 2 .…”

“…Quantification of Apoptosis by Flow Cytometry-Apoptosis was determined by propidium iodide staining and fluorescence-activated cell sorter analysis as described previously (37). PC12 cells and HEK cells were lysed in buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 g/ml propidium iodide.…”

“…APPsw PC12 cells secreted low A␤ levels, reflecting the physiological situation in vivo during cellular metabolism (7), whereas human APP was equally expressed in APPwt and APPsw PC12 cells and was increased 2-fold compared with the endogenous APP expression of control PC12 cells (37). Of note, HEK cells bearing the APPsw mutation exhibited an ϳ20-fold increased A␤ secretion compared with APPsw-bearing PC12 cells (Table I), a scenario that might occur in familial AD and that could explain the more drastic effects in some assay designs.…”

Regulation of gene expression by the amyloid precursor protein: inhibition of the JNK/c-Jun pathway

“…[16][17][18] Cell lines were cultivated in DMEM supplemented with 5% heat-inactivated fetal calf serum, 10% horse serum, 100 U/ml penicillin and 100 mg/ml streptomycin.…”

“…cell lines, PC12 APP M5 and PC12 APP N10, overexpressing equal amounts of the human brain-specific isoform APP695, [16][17][18] which lacks the the Kunitz-type protease inhibitor (KPI) domain. 1 Empty vector-transfected PC12 cells (PC12neo O1) served as control.…”