All ETDs from UAB

Advisory Committee Chair

Alecia K Gross

Advisory Committee Members

Timothy W Kraft

Thomas T Norton

Steven J Pittler

Bradley K Yoder

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) School of Optometry

Abstract

Retinitis pigmentosa is one of the most common inherited blinding disorders, affecting 1 in 4000 individuals world-wide. Approximately 30% of retinitis pigmentosa cases are dominantly inherited and approximately 30% of these dominant mutations are found in rhodopsin. Most rhodopsin mutations result in misfolded protein aggregates and subsequent apoptosis while others have been shown to result in apoptosis associated with mislocalization, diminished/enhanced activity, or faulty protein-protein interactions. This work serves to test for mechanisms by which the naturally-occurring Ter349Glu rhodopsin mutation causes rod cell death. Ter349Glu rhodopsin was tested for the ability to bind the ligand 11-cis retinal, photoactivate, and activate the G-protein transducin in vitro. The effects of Ter349Glu rhodopsin on protein localization and retinal inflammation was assayed both in vitro and in vivo using transfected cells, transgenic Xenopus laevis, and a knock-in mouse all expressing the Ter349Glu rhodopsin mutant. The impact on retinal structure, retinal vasculature and photoreceptor ultrastructure by Ter349Glu rhodopsin was examined in the knock-in mouse. I found Ter349Glu rhodopsin absorbs maximally at 500 nanometers and behaves similarly to wild-type rhodopsin in in vitro G-protein activation assays. In mouse kidney epithelium cells, mislocalization occurred at high levels of expression while normal localization occurred at low levels. Transgenic Xenopus laevis expressing Ter349Glu rhodopsin also exhibited both proper localization to the rod outer segment and improper localization to the plasma membrane of the inner segment and synaptic region. The Ter349Glu rhodopsin knock-in mouse displayed expedited, early-onset degeneration in homozygotes with a loss of proper outer segment development and labeling for markers of inflammation including activated macrophages and proteins involved in the Janus kinase/signal transducer and activator of transcription pathway. Ter349Glu rhodopsin heterozygotes displayed a delayed degenerative phenotype with slower apoptosis, a dispersed loss of proper disc formation and orientation, mislocalization and endoplasmic reticulum retention, and signal transducer and activator of transcription 3 activation. In all, this work shows the severe phenotype observed in human patients could likely be attributed to a number of mechanisms including improper disc/outer segment morphogenesis, possible endoplasmic reticulum stress associated with aggregation, as well as retinal inflammation.

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Optometry Commons

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