Author ORCID Identifier
Date Available
2-10-2023
Year of Publication
2023
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Physiology
First Advisor
Dr. Lance A. Johnson
Abstract
Excess lipid droplet (LD) accumulation is associated with several pathological states, including neurodegenerative disorders such as Alzheimer’s disease (AD). However, the mechanism(s) by which changes in LD composition and dynamics may contribute to the pathophysiology of AD remains unclear. Apolipoprotein E (ApoE) is a droplet-related protein with a common variant (ApoE4) that confers the largest increase in genetic risk for late-onset AD. Interestingly, ApoE4 is associated with both increased neuroinflammation and excess LD accumulation. This dissertation work seeks to quantitatively profile the lipid and protein composition of LDs between the ‘neutral’ ApoE3 and ‘risk’ ApoE4 isoforms, in order to gain insight into potential LD-driven contributions to AD pathogenesis.
Targeted replacement mice expressing human ApoE3 or ApoE4 were injected with saline (control) or LPS (inflammatory stimulus) and after 24 hours, hepatic lipid droplets were isolated and droplet proteomes and lipidomes were analyzed. Quantitative proteomics showed that LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for proteins involved in lipid ß-oxidation. Lipidomics revealed a shift in the distribution of glycerophospholipids in E4 LDs with an increase in multiple phosphatidylcholine (PC) species. There was also substantial overlap between LD proteins and AD-proteomes of human whole brain tissue. To translate these findings to the brain, primary microglia from the same strain of mice were exposed to exogenous lipid, inflammatory stimulation, necroptotic N2A cells (nN2A), or a combination of treatments to evaluate lipid droplet accumulation and impact on cell function. Microglia from ApoE4 mice accumulated more LDs at baseline, with exogenous OA, LPS stimulation, and nN2As as a percentage of E3 control across multiple experiments. E4 microglia also secreted significantly more cytokines (TNF, IL-1β, IL-10) than E3 microglia in the control, oleic acid, and nN2A treatment conditions. Interestingly, droplet inhibitors for ACAT and DGAT both decreased droplet accumulation in cells, but did not ameliorate the cytokine response. Finally, we have established a biobank of APOE genotyped peripheral blood mononuclear cells (PBMCs) from research participants. These easily accessible immune cells will serve as a highly translational model to understand LD dynamics as it relates to ApoE and AD risk.
In summary, E4 cells accumulate more LDs compared to E3 under all conditions tested, while the proteomic profile of E4 LDs support the hypothesis that E4 expression increases inflammation under basal conditions. This increased LD formation in non-aged, non-diseased E4 cells may suggest preclinical dysfunction associated with the highest risk APOE genotype, and a better understanding of LD dynamics within these cells and their functional implications may provide novel targets to improve E4-related outcomes.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2023.031
Funding Information
This work was supported by the National Institutes of Health (Johnson - 1R01AG060056, R01AG062550, and R01AG080589; Morganti - R01AG070830 and RF1NS118558; Devanney - F31AG076282; Golden - T32AG057461), the Cure Alzheimer’s Fund, and the Alzheimer’s Association.
Recommended Citation
Friday, Cassi, "The Influence of APOE Genotype on Lipid Droplet Dynamics" (2023). Theses and Dissertations--Physiology. 61.
https://uknowledge.uky.edu/physiology_etds/61
