Depletion of Very Long Chain Polyunsaturated Fatty Acids Causes Functional and Ultrastructural Changes in Photoreceptors Independent of Mutant ELOVL4 Expression

Abstract

Purpose: The fatty acid elongase-4 (ELOVL4) synthesizes retinal very long chain polyunsaturated fatty acids (VLCPUFAs). Autosomal dominant Stargardt macular dystrophy (STGD3) mutant ELOVL4 is misrouted to rod outer segments of Xenopus laevis. We hypothesize that expression of the mutant ELOVL4 affects retinal VLCPUFAs and alters photoreceptor structure and function leading to retinal degeneration in STGD3 and that expression of a single copy of the mutant ELOVL4 in the absence of WT ELOVL4 will exacerbate the rate of retinal dysfunction.

Methods: We generated conditional Elovl4 Knock-In mice (cKI) that express only the mutant ELOVL4 but not wild-type ELOVL4 in photoreceptor cells by crossing Cre+/Elovl4flox/flox with floxed-Elovl4 mice to generate four genotypes: Cre+/Elovl4flox/stgd3 (cKI), Cre-/Elovl4flox/stgd3 (KI), Cre+/Elovl4flox/wt (Het) and Cre-/Elovl4flox/wt (WT). Retinal function was determined at 2 years of age by scotopic and photopic electroretinography (ERG). The ERG responses of rod photoreceptors were modeled based on the biochemistry of phototransduction and retinal ultrastructure was evaluated using transmission electron microscopy (TEM).

Results: cKI mice expressing a single copy of mutant ELOVL4 have reduced retinal ELOVL4, and the sum of retinal VLCPUFAs was depleted to 0.58% compared to 7.4% in WT. At 2 years of age, the dominant negative effect of the mutant ELOVL4 on retinal VLCPUFA biosynthesis was evident as the sum of VLCPUFA in KI was 2.7% compared to 5.7% in Hets. Functionally, there was an age-dependent decline in rod but not cone photoreceptor function in the cKI mice, which had significantly reduced a- and b-wave amplitude responses when compared to WT. The scotopic b-wave amplitudes were more significantly affected in the cKI compared to WT mice. Retinal outer nuclear layer thickness was reduced in the cKI compared to WT mice, but was not different from KI mice. At the TEM level, the cKI mice appear to have thicker sub-RPE deposits, and more lysosomes and lipofuscin compared to WT mice. Non-digested lysosomes and putative outer segment phago-lysosomes were detected embedded in the basal deposit in the cKI mice.

Conclusions: Significant depletion of retinal VLCPUFA causes an age-related functional decline and RPE ultra-structural changes in cKI mice which are not exacerbated by expression of mutant ELOVL4.