Date of Award

2015

Embargo Period

8-1-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Neuroscience

College

College of Graduate Studies

First Advisor

Bärbel Rohrer

Second Advisor

DeAnna Adkins

Third Advisor

Carl Atkinson

Fourth Advisor

Gautam Ghatnekar

Abstract

Age-related macular degeneration (AMD) is a multifactorial disease and is regarded as the predominant cause of central vision loss in the elderly in industrialized countries. A critical target tissue in AMD is the retinal pigment epithelium (RPE), which together with Bruch’s membrane forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction in AMD might result from attenuation and disruption of intercellular tight junctions. Zonula occludens-1 (ZO-1) is a major structural protein of intercellular junctions. A connexin-based peptide mimetic, αCT-1 (Alpha Connexin carboxy-Terminal 1), was developed which competitively inhibits ZO-1 interaction with its binding partners. We hypothesized that targeting ZO-1 signaling using αCT-1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions. Choroidal neovascularization (CNV) was induced using laser-photocoagulation; RPE-cell barrier loss was triggered by bright light exposure (3000 lux for 3 hours). Both models lead to VEGF- dependent loss of cell junctions. The αCT-1 peptide was delivered via daily eyedrops. CNV lesion sizes were determined using optical coherence tomography (OCT). RPE flatmounts were stained for cell-junction proteins ZO-1 and occludin. Cell profiler software was used to examine the RPE tiling pattern. In vitro experiments using ARPE-19 cells showed that αCT-1 stabilizes intercellular tight junctions. αCT1 treatment reduced CNV development and fluid leakage, and damage was correlated with disruption in cellular integrity of the surrounding RPE cells. Light-damage significantly disrupted RPE cell morphology, which was prevented by αCT-1 pre-treatment. In vitro experiments using ARPE-19 cell monolayers suggest that αCT-1 stabilizes intercellular tight junctions. Taken together, stabilization of cellular junctions with αCT-1 was effective in ameliorating RPE dysfunction in AMD models of photo-coagulation-induced CNV and bright-light exposure RPE-cell barrier loss. Future research will include additional investigation into the peptide’s mechanism of action.

Rights

All rights reserved. Copyright is held by the author.

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