Date of Award

January 2018

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Janey L. Wiggs

Second Advisor

Ji Liu

Abstract

Early-onset glaucoma has a strong genetic contribution, but only 20% of affected patients have mutations in known disease-causing genes. Whole exome sequencing of 27 early-onset glaucoma families found three families with ARHGAP33 mutations, suggesting ARHGAP33 as a promising candidate. This study explores the functional impact of ARHGAP33 mutations by using morpholino-mediated knockdown of the ARHGAP33 ortholog in zebrafish embryos. Since missense alleles are hypothesized to disrupt protein function, zebrafish morphants are expected to have underdeveloped eyes and reduced visual function.

Embryos at the 1-8 cell stage were injected with 2.3 nL of 0.3 mM ARHGAP33 translation-blocking (TB), 0.3 mM ARHGAP33 splice-site blocking (SS), or 0.3 mM standard control (STD) morpholinos, and one group was left uninjected. Zebrafish were stained with acetylated-tubulin antibody and imaged under confocal microscopy to measure optic nerve width at 3 days post fertilization (dpf). At 5 dpf, zebrafish were embedded in glycol methanlacrylate acrylic resin, sectioned coronally, and stained with hematoxylin and eosin to count retinal ganglion cells. Gross eye area measurements and visual function using the Visual Motor Response (VMR) system were also conducted at 5 dpf.

Zebrafish in the TB and SS groups had significantly smaller eye areas (72,001 m2 7,451 m2 and 74,498 m2 7,481m2, respectively) compared to fish in the STD group (82,801 m2 7,646 m2, p < 0.001). They also had significantly decreased eye/head area ratios (0.24 0.02 and 0.25 0.02, respectively) than STD fish (0.28 0.02, p < 0.001). In addition, the optic nerve widths of TB and SS fish were significantly thinner (7.22 m 1.06 m and 7.32 m 1.19 m) than those in the STD group (8.85 m 1.17 m, p < 0.009), and they had reduced retinal ganglion cell layer counts (152 11.5 and 155.6 8.1) compared to STD fish (181.1 9.8, p < 0.005). Lastly, both TB and SS fish had reduction in activity level in response to light change compared to STD fish in VMR testing, indicating reduced visual function.

The ARHGAP33 TB and SS morpholinos both had significant negative effects on eye development and visual function. ARHGAP33 knockout mice have been shown to express less Tropomyosin receptor kinase B (TrkB), which is needed for retinal ganglion cell survival. Together, these findings suggest a possible role for ARHGAP33 in glaucoma-mediated neurodegeneration.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/25/2100

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