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

Vitamin 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 (Aß) 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 Aß. Mitochondrial function was shown to be affected by the increased level of Aß and may induce cell death. Here, we aimed to compare the effects of ATF and GTF on their ability to reduce Aß 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 Aß 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

DNA damage and protein oxidation associated with ageing correlate with cognitive dysfunction in a Malaysian population.

Ageing is associated with increased oxidative stress accompanied by cognitive decline. The aim of this study was to evaluate oxidative stress biomarkers and their possible relationship with cognitive performances during ageing among the Malay population. Approximately 160 healthy Malay adults aged between 28 and 79 years were recruited around Selangor and Klang Valley. Cognitive function was assessed by Montreal Cognitive Assessment (MoCA), forward digit span (FDS), backward digit span (BDS), digit symbol, Rey Auditory Verbal Learning Test immediate recalled [RAVLT(I)] and delayed recalled [RAVLT(D)], and visual reproduction immediate recalled (VR-I) and delayed recalled (VR-II). DNA damage, plasma protein carbonyl and malondialdehyde (MDA) levels were also determined. Cognitive function test showed significant lower scores of MoCA, BDS, RAVLT(I), RAVLT(D), digit symbol, VR-I, and VR-II in the older age group (60 years old) compared with the 30-, 40-, and 50-year-old group. The extent of DNA damage was sequential with age: 60 > 50 > 40 > 30, whereas protein carbonyl was higher in 40-, 50-, and 60-year-old groups compared with the youngest group (30 years old). However, the MDA level was observed unchanged in all age groups. Approximately 21.88% of the participants had cognitive impairment. Multiple logistic regression analysis revealed that DNA damage and protein carbonyl levels are predictors for cognitive impairment in healthy Malays. In conclusion, cognitive decline occurred in healthy adult Malay population at an early age of 30 years old with corresponding higher DNA damage and protein oxidation.

Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aß). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aß42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aß42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aß42 alone and that more extensive Aß formation, such as accumulation of intraneuronal Aß, Aß plaques, soluble oligomeric Aß (oAß), and insoluble fibrillar Aß (fAß) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aß42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aß2/40 ratio compared to wild-type and single-mutant cells.

Antioxidant modulation in restoring mitochondrial function in neurodegeneration.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the leading causes of disability associated with neurodegeneration worldwide. These diseases are influenced by multiple genetic and environmental factors and share similar mechanisms as both are characterized by accumulation and aggregation of misfolded proteins - amyloid-beta (Aß) in AD and α-synuclein in PD. Over the past decade, increasing evidence has shown that mitochondrial dysfunction and the generation of reactive oxygen species (ROS) are involved in the pathology of these diseases, and the contributions of these defects to the cellular and molecular changes that eventually cause neuronal death have been explored. Using mitochondrial protective agents, such as antioxidants, to combat ROS provides a new strategy for neurodegenerative treatment. In this review, we highlight the potential of multiple types of antioxidants, including vitamins, phytochemicals, fatty acids and minerals, as well as synthetic antioxidants specifically targeting the mitochondria, which can restore mitochondrial function, in the treatment of neurodegenerative disorders at both the pre-clinical and clinical stages by focusing on AD and PD.