Comparative Effects of Alpha- and Gamma-Tocopherol on Mitochondrial Functions in Alzheimer’s Disease In Vitro Model

Abstract

AbstractVitamin E acts as an antioxidant and reduces the level of reactive oxygen species (ROS) in Alzheimer’s disease (AD). Alpha-tocopherol (ATF) is the most widely studied form of vitamin E besides gamma-tocopherol (GTF) which also shows beneficial effects in AD. The levels of amyloid-beta (Aβ) and amyloid precursor protein (APP) increased in the brains of AD patients, and mutations in the APP gene are known to enhance the production of Aβ. Mitochondrial function was shown to be affected by the increased level of Aβ and may induce cell death. Here, we aimed to compare the effects of ATF and GTF on their ability to reduce Aβ level, modulate mitochondrial function and reduce the apoptosis marker in SH-SY5Y cells stably transfected with the wild-type or mutant form of the APP gene. The Aβ level was measured by ELISA, the mitochondrial ROS and ATP level were quantified by fluorescence and luciferase assay respectively whereas the complex V enzyme activity was measured by spectrophotometry. The expressions of genes involved in the regulation of mitochondrial membrane permeability such as voltage dependent anion channel (VDAC1), adenine nucleotide translocase (ANT), and cyclophilin D (CYPD) were determined by quantitative real-time polymerase chain reaction (qRT-PCR), while the expressions of cyclophilin D (CypD), cytochrome c, Bcl2 associated X (BAX), B cell lymphoma-2 (Bcl-2), and pro-caspase-3 were determined by western blot. Our results showed that mitochondrial ROS level was elevated accompanied by decreased ATP level and complex V enzyme activity in SH-SY5Y cells expressing the mutant APP gene (p < 0.05). Treatment with both ATF and GTF reduced the mitochondrial ROS level with maximum reduction was observed in the cells treated with high concentrations of ATF and GTF (p < 0.05). However, only GTF at 80 µM significantly increase the ATP level and complex V enzyme activity (p < 0.05). VDAC1 and CYPD were downregulated and CypD protein was significantly overexpressed in cells transfected with the wild-type (WT) and mutant APP gene (p < 0.05). Cytochrome c release, the ratio of BAX/Bcl-2, and pro-caspase-3 expression increased in cells expressing mutated APP gene (p < 0.05). The expression of CypD and pro-caspase 3 protein, and the ratio of BAX/Bcl-2 were increased in the following order; SH-SY5Y-APP-WT < SH-SY5Y-APP Swe <SH-SY5Y-APP Swe/Ind. Treatment with both ATF and GTF reduced the release of cytochrome c and the ratio of BAX/Bcl-2. However, only GTF significantly reduced the expression of CypD and pro-caspase-3, suggestive of its unique role in AD. In conclusion, GTF has an effect that was not shown by ATF and thus suggest its potential role in the development of therapeutic agents for AD.