ABSTRACT

Establishing mitochondrial cholesterol enrichment as a fundamental causal mechanism underlying the dysfunctional clearance of neurotoxic substances by microglia would help pave the way toward the discovery of potential new therapeutics for Alzheimer’s disease (AD). The dissertation is divided into four chapters with the corresponding objectives:1. The first chapter is a literature review describing the mechanisms by which cholesterol has been implicated in influencing microglia phenotype and function, and the implications for a role in contributing to the pathophysiology of AD. We describe both what is known and the research gaps that exist and should be explored for the discovery of novel therapies for AD 2. The second chapter is an investigation on the effects of a 36h fast on HDL particle size distribution by transmission electron microscopy (TEM), and ability to efflux cholesterol from activated microglia. Importantly, HDL from 36h fasted individuals effluxed cholesteryl esters from immortalized human microglia (HMC3) loaded with cholesterol + amyloid beta oligomers (AβO) 10-fold more effectively than HDL from postprandial individuals, and microglia treated with cholesterol+AβO had reduced apolipoprotein E (ApoE) secretion, which was attenuated more effectively by HDL from 36h fasted individuals than HDL from postprandial individuals. 3. In the third chapter we use a multi-stimulant approach to test the usefulness of the HMC3 microglia cell line in duplicating critical aspects of the dysfunctional microglia phenotype and to better mimic the multi-factorial nature of AD, which is associated with multiple comorbidities, including diabetes and cardiovascular disease. HMC3 microglia were treated with cholesterol (Chol), AβO, lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol+AβO+fructose+LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol+AβO+ fructose+LPS increased mitochondrial Chol content. Microglia treated with combinations containing cholesterol+AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol+AβO+fructose+LPS having the strongest effect. Combination treatment with Chol+AβO+fructose+LPS also induced APOE and TNF-alpha expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events.