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Uzoamaka Nwagbo, Paul S Bernstein; Elovl4b Ablation in Zebrafish Leads to Loss of Ocular C30 to C34 Very-Long-Chain Polyunsaturated Fatty Acids. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2486 – F0193.
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Very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are a distinct class of lipids with chain lengths greater than 24 carbons found in the retina and a few other tissues in vertebrates. ELOVL4 is a member of the ELOVL elongase family responsible for the rate-limiting step of VLC-PUFA biosynthesis. Patients with autosomal dominant Stargardt-3 disease, a juvenile form of macular degeneration, have genetic mutations in ELOVL4 leading to the formation of a non-catalytically active protein and loss of retinal VLC-PUFAs. Interestingly, low retinal VLC-PUFAs are also characteristic of age-related macular degeneration. Moreover, retinas isolated from patients with age-related macular degeneration are reported to have low omega-3 (n-3) to omega-6 (n-6) fatty acid ratios compared to healthy controls.The retinal pathology caused by VLC-PUFA depletion has historically been difficult to study because homozygous Elovl4 mutations cause skin and neurological disorders in humans and neonatal lethality in mice. Mice with homozygous Elovl4 mutations are susceptible to catastrophic drying from losing their protective skin barrier. Thus, we created and examined a zebrafish model of Elovl4 deficiency and haploinsufficiency.
We created a deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. F0 mosaic mutant zebrafish were screened and outcrossed with wild-type fish over two generations to generate a stable line. At ~4 months post-fertilization, their eyes were isolated, total fatty acids extracted, and the quantity of n-3 and n-6 VLC-PUFAs was determined through gas chromatography and mass spectrometry.
We found that homozygous Elovl4b mutant zebrafish eyes had no detectable C30 to C34 VLC-PUFAs in their eyes, in contrast to age-matched wild-type controls. Heterozygous fish with one functional copy of Elovl4b had intermediate lipid profiles.
Our data indicate that the loss of Elovl4b in zebrafish alters ocular biochemistry in a way that is comparable to macular degeneration. We expect diminished ERG amplitudes and visual function in the homozygous and heterozygous fish and altered retinal histology. We plan to correct such abnormalities by feeding synthetic VLC-PUFAs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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