Elovl4 5-bp deletion does not accelerate cone photoreceptor degeneration in an all-cone mouse

Abstract

Mutations in the elongation of very long chain fatty acid 4 (ELOVL4) gene cause Stargardt macular dystrophy 3 (STGD3), a rare, juvenile-onset, autosomal dominant form of macular degeneration. Although several mouse models have already been generated to investigate the link between the three identified disease-causing mutations in the ELOVL4 gene, none of these models recapitulates the early-onset cone photoreceptor cell death observed in the macula of STGD3 patients. To address this specifically, we investigated the effect of mutant ELOVL4 in a mouse model with an all-cone retina. Hence, we bred mice carrying the heterozygously mutated Elovl4 gene on the R91W;Nrl-/- all-cone background and analyzed the retinal lipid composition, morphology, and function over the course of 1 year. We observed a reduction of total phosphatidylcholine-containing very long chain-polyunsaturated fatty acids (PC-VLC-PUFAs) by 39% in the R91W;Nrl-/-;Elovl4 mice already at 6 weeks of age with a pronounced decline of the longest forms of PC-VLC-PUFAs. Total levels of shorter-chain fatty acids (< C26) remained unaffected. However, this reduction in PC-VLC-PUFA content in the all-cone retina had no impact on morphology or function and did not accelerate retinal degeneration in the R91W;Nrl-/-;Elovl4 mice. Taken together, mutations in the ELOVL4 gene lead to cone degeneration in humans, whereas mouse models expressing the mutant Elovl4 show predominant rod degeneration. The lack of a phenotype in the all-cone retina expressing the mutant form of the protein supports the view that aberrant function of ELOVL4 is especially detrimental for rods in mice and suggests a more subtle role of VLC-PUFAs for cone maintenance and survival.

Fig 1. Evaluation of Elovl4 expression in the all-cone mice.

(A) mRNA levels of total (left), mutant (middle) and wt (right) Elovl4 analyzed by semiquantitative real-time PCR. Expression was normalized to Actb and expressed relative to 6-week-old R91W;Nrl^-/- mice (left and right panel), or to 6-week-old R91W;Nrl^-/-;Elovl4^mut mice (middle panel). n.d.: not detected. Shown are means ± SD. n = 3. ***: P < 0.001; ****: P < 0.0001. (B) Schematic representation of binding sites for primers used to discriminate between total, wt and mutant Elovl4 transcripts (adapted from Vasireddy et al. [33]). (C) Western blot detection of indicated proteins in R91W;Nrl^-/-;Elovl4^mut (mut), R91W;Nrl^-/- (ctrl) and 129S6 (wt, rod-dominant) mice at 6, 12 and 24 weeks of age as indicated. Shown are representative blots of n = 3.

Fig 2. ELOVL4 distribution in R91W;Nrl^-/-;Elovl4^mut, R91W;Nrl^-/- and C57BL/6J (wt) rod-dominant retina of 12-week-old mice.

Red: ELOVL4; green: PNA (cone PR segments); blue: DAPI. PRS: photoreceptor segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 μm. Shown are representative images of n = 3.

Fig 3. Retinal FA composition.

(A) Percentage of the sum of PC-VLC-PUFAs in the total PC pool at indicated ages. (B) All individually identified VLC-PUFAs of the PC pool at indicated ages. The glycerolipids are composed of a PC headgroup, a DHA (22:6) and a VLC-PUFA (e.g.: PC-58:12 (22:6/36:6)) (C) Ratio of n6 to n3 FA in total lipids at indicated ages. Shown are means ± SD (n = 4; *: P < 0.05; ****: P < 0.0001).

Fig 4. Analysis of retinal morphology.

(A) Representative micrographs of retinas from R91W;Nrl^-/-;Elovl4^mut and R91W;Nrl^-/- mice at indicated ages. (B) Cone nuclei count in the central retina. RPE: Retinal pigment epithelium, other abbreviations as in Fig 2. Red bars: R91W;Nrl^-/-;Elovl4^mut mice; blue bars: R91W;Nrl^-/-. Scale bar: 20 μm. Shown are means ± SD (n = 3).

Fig 5. Light-adapted ERG to test retinal function.

(A) ERG traces of early (6 w), intermediate (24 w) and late (52 w) time points. (B) Photopic b-wave amplitudes plotted against the logarithm of light intensities in R91W;Nrl^-/-;Elovl4^mut and control R91W;Nrl^-/- mice at indicated ages. Shown are means ± SD (6 weeks, n = 5; 12 weeks, n = 6; 24 weeks, n = 6; 38 weeks, n = 4; and 52 weeks, n = 3).