DNA damage, micronutrient and lipid profiles of human APOE ε4 carriers

Abstract

Alzheimer’s disease (AD) is an increasing global health problem that is expected to affect 65.7 million people by 2030. The major susceptibility gene, the Apolipoprotein (APOE) ε4 allele is associated with a 4-fold increased risk for AD if one ε4 allele is present and can advance the age-of-onset of AD by 7-9 years. Carriers of the ε4 allele are also associated with poorer cognitive performance, increased amyloid plaque and neurofibrillary tangle burden and greater levels of neuronal cell death. Previous studies have demonstrated that AD individuals possess elevated levels of genomic instability and an altered lipid and nutritional profile. At present the mechanism by which APOE ε4 may accelerate the onset of AD is unknown but may include genomic instability in AD individuals via promoting the intracellular accumulation of the neurotoxic amyloid beta 42 peptide (Aβ₄₂) or directly altering the homeostasis of important lipids such as Docosahexaenoic acid (DHA) and cholesterol, which are crucial for optimal brain function. To date, there is limited information regarding the genomic instability profile of APOE ε4 carriers and their nutritional status. The primary aims of the thesis were to investigate the following hypothesis: (i) APOE ε4 carriers without cognitive impairment have greater levels of chromosomal DNA damage and cell death as measured in human peripheral lymphocytes compared to non-ε4 carriers. (ii) APOE ε4 carriers without cognitive impairment have an altered lipid and nutritional profile compared to non-ε4 carriers. The thesis comprised of two distinct studies: (i) An in vitro study which investigated whether long term supplementation of two fatty acids present in fish oil, i.e. Docosahexaenoic acid (DHA) and Furan Fatty Acids (FFA) can prevent oxidative stress-induced cell death and DNA damage in an astrocytoma cell lines with or without the APOE ε4 allele. (ii) An in vivo study investigating the primary hypothesis that APOE ε4 carriers that were not cognitively impaired exhibit greater levels of chromosomal DNA damage and an altered lipid and nutritional profile compared to non-ε4 carriers. The Cytokinesis-block micronucleus cytome (CBMN-Cyt) assay was the primary genome instability assay used in both studies, as it is a comprehensive technique that allows chromosomal DNA damage to be measured visually in once divided cells that are recognised by their binucleate appearance. Examples of chromosomal DNA damage scored include micronuclei (MNi, chromosome breakage and/or loss), nuclear buds (NBUDs, DNA amplification and or removal of DNA repair complexes) and nucleoplasmic bridges (NPBs, DNA misrepair and/or telomere end fusions). Previous studies have demonstrated that DHA promotes antioxidant defences in the brain, while FFA is emerging as a potential antioxidant and believed to contribute to the benefits of fish oil. The cytotoxic and genotoxic effects of DHA and FFA was investigated in in vitro cultures of U87MG (APOE ɛ3/ɛ3) and U118MG (APOE ɛ2/ɛ4) astrocytoma cell lines with and without a hydrogen peroxide (H₂O₂, 100 μM) challenge. The APOE ε4 cell line was found to be more sensitive to the cytostatic (P < 0.001), cytotoxic (i.e., apoptosis, P < 0.001) and DNA damaging effects (i.e. MNi, P < 0.001; NPBs, P < 0.001 and NBUDs, P < 0.01) of H₂O₂ when compared to the non-ε4 cell line. DHA at 100 μg/mL significantly affected cytostasis (P < 0.05) and increased DNA damage in the form of NPBs and MNi (P < 0.05) in both cell lines, whereas it decreased necrosis (P = 0.0251) in the non-ε4 cell line. FFA had no effect on chromosomal DNA damage in both cell lines investigated. Findings from the in vivo study suggest that APOE ε4 carriers do not experience significantly different rates of cytostasis, cytotoxicity and/or chromosomal DNA damage (MNi, NPBs and NBUDs) as measured by the CBMNCyt assay, when compared to non-APOE ε4 carriers after correcting for age and gender. However, there was a trend for increased NPBs and NBUDs in homozygous ε4 carriers compared to non-carriers. Analysis of plasma nutrients and lipids showed no significant differences between ε4 and non-ε4 carriers with the exception of Phosphorus (P = 0.042), total plasma cholesterol (P < 0.0001) and LDL-cholesterol (P < 0.0001) which were higher in APOE ε4 carriers. This study was the first to characterise the CBMN-Cyt assay chromosomal DNA damage profile of APOE ε4 carriers. Although no statistically significant differences for any of the cytome biomarkers in our cohort was reported, the limited trend for increased NPBs (r = 0.118, P = 0.06) suggests the possibility of the loss of systemic genome integrity as measured in surrogate tissues such as lymphocytes but may not adequately explain why APOE ε4 carriers are at higher risk of developing AD. The results of this study are not generalisable to other cohorts with poor lifestyle habits or to non-healthy or older APOE ε4 carriers in which the effects of APOE ε4 may be more evident (i.e. smokers and obese individuals). In addition, whilst there were no substantial differences in chromosomal DNA damage biomarkers in the form of MNi, NPBs and/or NBUDs in peripheral lymphocytes, DNA damage studies using a large cohort to increase statistical power and other biomarkers such as the buccal micronucleus cytome assay, telomere length and integrity, comet assay and γH2AX would allow a more complete characterisation of the DNA damage profile of APOE ε4 carriers and a more definitive assessment of the role of DNA damage in APOE ε4-related pathology.