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

<div><p>Mutations in the elongation of very long chain fatty acid 4 (<i>ELOVL4</i>) 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 <i>ELOVL4</i> 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 <i>Elovl4</i> gene on the <i>R91W;Nrl</i>^<i>-/-</i> 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 <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i> 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 <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i> mice. Taken together, mutations in the <i>ELOVL4</i> gene lead to cone degeneration in humans, whereas mouse models expressing the mutant <i>Elovl4</i> 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.</p></div

Evaluation of <i>Elovl4</i> expression in the all-cone mice.

<p><b>(A)</b> mRNA levels of total (left), mutant (middle) and <i>wt</i> (right) <i>Elovl4</i> analyzed by semiquantitative real-time PCR. Expression was normalized to <i>Actb</i> and expressed relative to 6-week-old <i>R91W;Nrl</i>^<i>-/-</i> mice (left and right panel), or to 6-week-old <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i>^<i>mut</i> mice (middle panel). n.d.: not detected. Shown are means ± SD. n = 3. ***: P < 0.001; ****: P < 0.0001. <b>(B)</b> Schematic representation of binding sites for primers used to discriminate between total, <i>wt</i> and mutant <i>Elovl4</i> transcripts (adapted from <i>Vasireddy et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190514#pone.0190514.ref033" target="_blank">33</a>]). <b>(C)</b> Western blot detection of indicated proteins in <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i>^<i>mut</i> (mut), <i>R91W;Nrl</i>^<i>-/-</i> (ctrl) and <i>129S6</i> (<i>wt</i>, rod-dominant) mice at 6, 12 and 24 weeks of age as indicated. Shown are representative blots of n = 3.</p

Analysis of retinal morphology.

<p><b>(A)</b> Representative micrographs of retinas from <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i>^<i>mut</i> and <i>R91W;Nrl</i>^<i>-/-</i> mice at indicated ages. <b>(B)</b> Cone nuclei count in the central retina. RPE: Retinal pigment epithelium, other abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190514#pone.0190514.g002" target="_blank">Fig 2</a>. Red bars: <i>R91W;Nrl</i>^<i>-/-</i><i>;Elovl4</i>^<i>mut</i> mice; blue bars: <i>R91W;Nrl</i>^<i>-/-</i>. Scale bar: 20 μm. Shown are means ± SD (n = 3).</p

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

<p>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.</p

Primers for qPCR.

<p>Primers for qPCR.</p