Date of Award

1-1-2012

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Othman Ghribi

Abstract

This dissertation work focused on retinal modifications that are relevant to Age-related macular degeneration (AMD) in cholesterol-fed rabbit model of Alzheimer's disease (AD), as AD and AMD share common features. It is unknown whether cholesterol-fed rabbit model of AD displays any AMD features in retina. Previous research showed 27-hydroxycholesterol (27-OHC) involvement in AD like pathology in organotypic hippocampal slices of rabbit brain and human SHSY-5Y neuroblastoma cells. The extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are not known. Various studies suggested estrogen's (E2) role in AMD development. 27-OHC is a ligand for estrogen receptor (ER) and liver X receptor (LXR). 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) are also implicated in AMD development. 25-OHC and 7-KC were shown to be ligands of ER and LXR in various cell types. It is unknown whether 27-OHC, 25-OHC and 7-KC influence ER and LXR transcriptional activity in ARPE-19 cells, a spontaneously arising human RPE cell line with normal karyology.

ARPE-19 cells and cholesterol-fed rabbit eyes were used for the study. Paraffin embedded eye cross sections were used for immunohistochemistry. Cholesterol was quantified by cholesterol/cholesteryl ester quantification kit. Oxysterols in the rabbit retinas were measured by mass spectrometry. Western blotting for detecting proteins, CytoTox-ONE homogenous membrane integrity

assay for measuring lactate dehydrogenase from cells, ELISA (Enzyme-linked immunosorbent assay) for quantifying amyloid beta 1-42 and 1-40, tumor necrosis factor alpha, DCFH-DA (2',7'-dichlorfluorescein-diacetate) assay for measuring reactive oxygen species (ROS), amplex red hydrogen peroxide / peroxidase assay for quantifying hydrogen peroxide and peroxidase activity, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) assay for mitochondrial membrane potential detection, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay for apoptotic cell detection, GSH-Glo assay for glutathione (GSH) quantification, calcium imaging, immunocytochemistry, immunohistochemistry and H&E (hematoxylin and eosin) staining, transfection and dual-luciferase reporter assays were used for this work.

This study showed retinal modifications that are relevant to AMD in cholesterol-fed rabbits. Increased abeta levels, decreased apoptosis regulator Bcl-2 levels, increased apoptosis regulator BAX and growth arrest and DNA damage-inducible protein GADD153 proteins, apoptotic cells, and increased generation of ROS were found in retinas from cholesterol-fed rabbit retinas. Furthermore, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. Oxysterol levels in retinas from cholesterol-fed rabbits were increased. 27-OHC increased abeta peptide production, increased caspase 12 and GADD153, reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, and increased levels of the Nuclear factor NF-kappa-B p65 subunit and Heme oxygenase 1. Additionally, 27-OHC caused GSH depletion, ROS generation, inflammation and apoptotic-mediated cell death in ARPE-19 cells. ARPE-19 cells express ERalpha, ERbeta, LXRalpha and LXRbeta. ER and LXR are transcriptionally active. ER agonist E2 protected cells from 25-OHC and 27-OHC induced cytotoxicity. E2 counteracted 27-OHC and 25-OHC induced mitochondrial membrane potential decline.

Taken together, data demonstrate that cholesterol-enriched diets induce pathological hallmarks suggestive of AMD in rabbit retinas. The cholesterol diet substantially increased concentrations of the major oxysterols, accumulation of which is toxic to retinal cells. Incubation of cells with oxysterols also reproduced the majors effects observed in vivo with the diet rich in cholesterol. These results strongly suggest that hypercholesterolemia and subsequent increase in oxysterol formation may contribute to the pathogenesis of AMD. Our data also shows that ER modulation may play an important role in the cholesterol and oxysterol effects. Specifically, ER agonists may provide protection against oxysterol deleterious effects on retinas. As well, reduction of cholesterol plasma levels may prevent excess conversion of cholesterol to oxysterols and precludes the incidence of AMD.

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