2 results
2095 Drug screening and hit identification for night blindness with zebrafish
- Logan Ganzen, Yuk Fai Leung
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- Journal:
- Journal of Clinical and Translational Science / Volume 2 / Issue S1 / June 2018
- Published online by Cambridge University Press:
- 21 November 2018, p. 11
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OBJECTIVES/SPECIFIC AIMS: Retinitis pigmentosa (RP), also known as night blindness, is an incurable disease which affects ~1 in 4000 individuals globally. Since there are no effective treatment options for RP, the goal of this project is to identify novel drug treatments that can prevent or slow the disease progression. To this end, we optimized a behavioral assay, visual-motor-response (VMR) assay, to investigate rod function (Ganzen et al., ARVO, 2017; Ganzen et al., IJMS, 2017). This was done utilizing a transgenic zebrafish RP model expressing human rhodopsin with the Q344X mutation. In this study, we used this model to perform a proof-of-concept screen for drugs which may improve the vision of the larvae. METHODS/STUDY POPULATION: To screen for beneficial drugs, the SCREEN-WELL® REDOX library was chosen for screening. This library was selected to identify a compound that may alleviate any excessive oxidative stress in the diseased retina. The Q344X zebrafish line suffers from significant rod degeneration by 7 days postfertilization (dpf) and displayed deficits in VMR under scotopic conditions (Ganzen et al., ARVO, 2017). The Q344X larvae were drug treated beginning at 5 dpf at 10 μM. Compounds that were toxic at this concentration were retested at 1 μM. The 5 dpf stage was chosen as most of the rods are intact, and these concentrations were chosen to optimize the drug effect based on similar studies. Hits were identified by assays that provided a robust and reproducible enhancement in the Q344X VMR. The retinae of any drug hits were dissected from larvae crossed with a rod EGFP reporter line and whole-mounted to analyze rod survival via fluorescence. To determine if drug effects were exerted through the retina, eyeless chokh mutant zebrafish were exposed to the drug and tested with the same assay. RESULTS/ANTICIPATED RESULTS: Of the 84 compounds tested, we identified 1 drug that ameliorated the VMR of the Q344X scotopic VMR. Eyeless chokh mutant zebrafish larvae did not exhibit the same VMR when treated with the same drug. Histological analysis suggested increased rod survival in the drug-treated retina of Q344X mutants. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate that the vision of the Q344X zebrafish was improved via this beneficial drug treatment. Since eyeless chokh larvae did not respond to the same treatment, the drug likely mediated its positive effects through the Q344X retina, likely by improving rod survival. Together, our results have identified a beneficial drug that may treat RP.
2217: A transgenic retinitis pigmentosa zebrafish model for drug discovery
- Logan Ganzen, Chi Pui Pang, Mingzhi Zhang, Motokazu Tsujikawa, Yuk Fai Leung
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- Journal:
- Journal of Clinical and Translational Science / Volume 1 / Issue S1 / September 2017
- Published online by Cambridge University Press:
- 10 May 2018, pp. 3-4
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OBJECTIVES/SPECIFIC AIMS: Retinitis pigmentosa (RP) is a hereditary retinal degeneration disease that affects ~1 in 4000 individuals globally, and there are currently no effective treatment options available. In order to identify potential drug treatments, we optimized our existing a behavioral assay around a transgenic zebrafish carrying a truncated human rhodopsin transgene [Tg(rho:Hsa.RH1_Q344X)]. This line was also crossed with the Tg(-3.7rho:EGFP) reporter for rod visualization. The Q344X larvae experiences significant rod photoreceptor death by 7 days postfertilization (dpf) (Nakao et al., 2012). METHODS/STUDY POPULATION: To assess the vision of the Q344X zebrafish, the VMR assay was run under a dim-light condition based on recorded rod b-waves in larval fish (Moyano et al., 2013) and the minimum cone activation threshold in mice (Cachafeiro et al., 2010). Specifically, Q344X and control larvae at 7 dpf were placed into a 96-well plate and acclimated to a dim-light source (1.802e-05 μ W/cm2 at 500 nm) for 1 hour. The VMR was tracked and quantified during light offset. The total distance traveled was averaged and analyzed at 1 second poststimulus. Retinas were dissected from Q344X and control larvae and whole-mounted to validate the rod degeneration in the Q344X model. RESULTS/ANTICIPATED RESULTS: We found that the Q344X larvae displayed an attenuated VMR (0.121±0.041 cm) to the dim-light offset as compared with the control larvae (0.2751±0.038 cm) (two-sample t-test; p-value=4.619e-14, n=19). Analysis of whole-mounted retinae indicated significant rod degeneration at 7 dpf compared with controls (control: 87 rods/retina, Q344X: 9.3 rods/retina, Welch two-sample t-test, p-value=1.4e4). It is unlikely that the cones of the zebrafish contributed to this VMR since the light intensity of the assay was below the cone detection threshold of mice. As the only apparent difference between the 2 groups of larvae is significant rod degeneration, it can be concluded that the behavioral phenotype was a result of the degeneration. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that the attenuated Q344X VMR is a result of the rod photoreceptor death. This behavioral phenotype can be taken advantage of to develop a drug screening assay. Future steps will screen chemical libraries to identify compounds that ameliorate the rod degeneration. Compounds that prevent degeneration are expected to result in a significant increase in locomotion in response to the dim visual stimulus.