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Epigenetic regulation of interleukin 6 in age-related cognitive decline and Alzheimer's disease

Sawkulycz, Xenia (2018) Epigenetic regulation of interleukin 6 in age-related cognitive decline and Alzheimer's disease. Masters thesis (MSc), Manchester Metropolitan University.

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Abstract

Introduction: Alzheimer’s disease (AD) is a neurodegenerative condition that is becoming a global crisis with no current cure. Neuroinflammation is widely thought to play a role in the pathogenesis of this disorder. Peripheral levels of the cytokine interleukin 6 (IL-6), that has both pro- and anti-inflammatory properties have been linked to dementia and age-related cognitive decline. Polymorphisms in the IL-6 gene have also been associated with neurodegenerative diseases. However, it remains to be determined how IL-6 levels are regulated in AD within brains and how this relates to neuropathology. Aim: The aim of this project was to investigate if and how the IL-6 gene is regulated in the brain during cognitive decline and AD and its relationship to neuropathological markers of AD. Methods: This study investigated IL-6 within brains from the University of Manchester Age and Cognitive performance research Cohort (ACPRC) and the Dyne – Steele study, that have been longitudinally followed over a 20-year period for cognition and characterised in neuropathology stages. Genotyping for the single nucleotide polymorphism (SNP) rs1800795 in the IL-6 locus was performed using KASP and IL-6 gene expression was measured by RT-PCR and normalised using two housekeeping genes (βactin and GAPDH). ELISA was used to determine IL-6 protein levels in brains and bisulphite pyrosequencing was used to quantify methylation levels of CpG sites at the IL-6 promoter. Finally, immunofluorescence was used to compare control and early AD prefrontal cortex brain samples to enable investigation of the distribution of IL-6 and an important regulator, NFkB. Results: Between genotypes, no significant difference was observed in age of death, whole brain weight, post mortem delays and neuropathology stages. IL-6 protein levels were analysed at the different neuropathology stages (BRAAK, THAL and CERAD) with no significance determined between each of the stages in the neuropathological hallmarks. Levels of IL-6 protein and IL-6 mRNA expression were analysed between the different genotypes with no significant differences. IL-6 gene expression and IL-6 total protein levels showed no correlations, even when further stratified by genotype. Immunofluorescence determined that IL-6 is present in the prefrontal cortex of both control and early AD samples and NFkB is activated. Distribution of DNA methylation at 4 CpG sites in the IL-6 promoter were determined. CpG site 2 depends on the rs1800795 SNP, which is only present in the G allele carriers. Interestingly, in the G allele carriers, levels of methylation at CpG sites 3 and 4 were significantly lower and higher, respectively. Levels of DNA methylation between control and AD groups were not significantly different at each of the CpG sites and did not correlate with protein levels. Levels of IL-6 mRNA and DNA methylation stratified by genotype revealed no correlation. Discussion: This study is the first to investigate IL-6 in AD brains and mechanisms underlying its epigenetic and genetic regulation. IL-6 protein levels and mRNA levels are found not to be impacted by the genotype and in disease state. Interestingly, DNA methylation was shown to be altered in the brains by the presence of a SNP, suggesting gene-environment interactions however, this was not influenced by disease state. In summary, the research has shown that IL-6 is present in control and AD brains and that the SNP has an impact on methylation levels between genotype at different CpG sites. However direct linear correlations between methylation, protein and expression levels did not exist. IL-6 is an important mediator of inflammation and further work is needed to understand how this factor is involved in neuroinflammation and regulated in the pathogenesis of AD.

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