Semester

Fall

Date of Graduation

2020

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Visvanathan Ramamurthy

Committee Member

Max Sokolov

Committee Member

Peter Stoilov

Committee Member

Michael D. Schaller

Committee Member

David M. Smith

Committee Member

Gordon P. Meares

Abstract

Photoreceptor neurons are modified primary cilia with an extended ciliary compartment known as the outer segment (OS). The mechanisms behind the elaboration of photoreceptor cilia, OS morphogenesis, and maintenance remain poorly understood. In this work, we focused on dissecting the role of two ciliary proteins, the small GTPase ADP-ribosylation factor-like GTPase 13B (ARL13B) and Bardet-Biedl Syndrome-8 (BBS8) in the context of photoreceptor biology. Both BBS8 and ARL13B are linked to defects in ciliogenesis (cilia development) and Retinitis Pigmentosa (vision loss). ARL13B is implicated in regulating ciliary length, and BBS8 is part of the Bardet-Biedl Syndrome complex (BBSome); the BBSome is a stable multiprotein complex linked to retinitis pigmentosa, yet its role in photoreceptor neurons remains poorly understood.

To investigate the role of ARL13B and BBS8 in the development and maintenance of ciliated photoreceptor neurons in vivo, we generated pan-retina knockout models of both proteins, with ablation of the protein taking place at embryonic day 9.5. Additionally, to study the role of these proteins in fully mature rod photoreceptors, we utilized a rod photoreceptor-specific tamoxifen-inducible murine model (PDE6g-CreERT2 ). Our studies showed that both ARL13B and BBS8 are needed for photoreceptor viability during postnatal retinal development; ARL13B mutants, however, displayed additional defects in retinal proliferation compared to BBS8. In BBS8 mutants, outer segments and their characteristic membranous discs were able to develop, but they became highly dysmorphic over time. In contrast, outer segments in ARL13B-null retina failed to form, where the OS did form, they resembled highly vesiculated rudiments. The length of the microtubule axoneme was affected in both models of cilia disease. However, BBS8-null axonemes had increased tubulin acetylation. Although tubulin hyperacetylation was not present in ARL13B-null retina, we found intraflagellar transport protein-88 (IFT88), to be highly mislocalized. IFT88 is an IFT-B complex protein crucial for the transport of tubulin dimers to the growing axoneme.

In conclusion, in this study, we sought to perform a careful analysis of the different phenotypic profiles present in both BBS8 and ARL13B mutant models. We were able to show apparent deviations in photoreceptor cellular processes ranging from early retinogenesis, photoreceptor outer segment development, maintenance, and protein transport. Current studies are now focused on determining what specific protein domains of both ARL13B and BBS8 correlate to different photoreceptor processes and how these domains are affected in diseased states.

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