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

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

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

Primers for qPCR.

<p>Primers for qPCR.</p

PBN and certain PBNDs inhibited rhodopsin regeneration.

<p>Rats were treated with either PBN or certain PBNDs before 2h of light adaptation followed by 2.5h of dark adaptation after which the animals were euthanized and retinas harvested for rhodopsin assay. Saline treated retina recovered ~ 75% rhodopsin in 2.5h dark. PBN and its derivatives, however, blocked rhodopsin regeneration significantly. LA, light adapted; DA, dark adapted. [* = <i>p</i><0.01; ** = <i>p</i><0.001; <i>n</i> = 4–6]</p

PBND tested for RPE-65 enzyme inhibition efficiency.

<p>The structure of PBN and the derivatives of PBN that are used in various <i>in vitro</i> and <i>in vivo</i> assays are shown in Fig 1A–1D. Concentration-dependent inhibition of RPE65 and generation of 11-<i>cis</i>-retinol in <i>in vitro</i> microsome assays were performed with representative images shown in Fig 1E, 4-F-PBN; Fig 1F, 4-CF₃-PBN; and Fig 1G, 4-CH₃-PBN. A table of the IC₅₀ values of the compounds tested for their inhibition efficiency for RPE 65, (Fig 1J), show various inhibition rates for which we determined which PBN-derivatives looked the most promising to continue our analysis.</p

Histological analysis of rat retina confirms protection from light damage.

<p>Vehicle or Saline treated (Vehicle LD) rats lost most of their photoreceptors from the central retina. Effect is more pronounced in the superior retina. NLD is non-light-damage control. Fig 4A shows the ONL thickness across the retina in the vertical meridian. PBN treatment (PBN LD) retinas retained ~90% of photoreceptor cells. 4-CF₃-PBN is comparable to PBN in protection, whereas 4-F-PBN appears better than PBN. 4-CH₃-PBN also protects the retina significantly, although its effect is slightly less than PBN. Fig 4B shows central retinal outer retinal layer (ONL) thickness in both inferior and superior retina. [** = <i>p</i><0.001; <i>n</i> = 8–10]</p

Systemically administered PBN and PBND protect rat retina from light damage.

<p>Electroretinographic (ERG) responses were recorded for a series of flash stimuli intensities at 0.04, 4, 200, 400, and 2000 cd.sec/m². ERG responses presented here are from the intensities of 4 and 400 cd.sec/m². The dim flash (4 cd.sec/m²) stimulates the rod photoreceptor cells and the bright flash (400 cd.sec/m²) stimulates both rod and cone photoreceptors. Therefore, the blue bars represent only rod responses and the red bars represent mixed response from both rod and cone photoreceptors. Fig 3A shows A-wave responses, and Fig 3B shows B-wave responses. NLD represents non-light-damaged group and LD represents light-damaged groups. [## = <i>p</i><0.001: NLD vs. LD saline-treated; * = <i>p</i><0.01 and ** = <i>p</i><0.001: LD saline vs. PBNDs; <i>n</i> = 4–6]</p

PBN and 4-CH₃-PBN topically administered in baboon eyes slows rate of rhodopsin regeneration.

<p>Baboon eyes were treated topically with drug or vehicle every 15 minutes over a 2h period before eyes were harvested and dark adapted 2h before retinal harvest. 10% PBN showed a roughly 50% higher inhibition rate compared to 5% CH₃-PBN suggesting both drugs to be effective. [** = <i>p</i><0.001; <i>n</i> = 6–8 eyes]</p