Canine Retina Has a Primate Fovea-Like Bouquet of Cone Photoreceptors Which Is Affected by Inherited Macular Degenerations

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations

Retinal localization of proteins of the TNF superfamily and CASP8 in study models.

<p>Immunolabeling of normal, rcd1, xlpra2, and erd retinas with antibodies against TNF superfamily ligands (TNFA, CD40LG, TNFSF8); TNF superfamily receptors (TNFRSF1A, TNFRSF9; TNF superfamily regulator TRADD; and the initiator caspase CASP8. TNFA was localized to the inner retina in Müller cells (Figure S2) at all ages. In mutants, labeling was more intense and present in RPE and remaining cone inner segments. CD40LG and TNFSF8 expressions were comparable to normals, although levels were higher in mutants, particularly at 16 wks in rcd1. Both proteins were found in the inner retina, CD40LG mainly in the GCL and Müller cells (see Figure S2), while TNFSF8 was more widely distributed and co-localized with Müller, horizontal, and ON bipolar cells (see Figure S2). TNFRSF1A localized mainly to the OPL in horizontal cells (see Figure S2). The staining intensity was higher in mutants than in normals, particularly in erd at 7.7 wks and rcd1 at 7 and 16 wks. TNFRSF9 and TRADD labeling was comparable in normal and mutants, although both were slightly more intense in the latter. They both were localized to the OPL and INL, where they co-localized with horizontal cells and TNFRSF9 also with amacrine cells (see Figure S2). TNFRSF9 was present in the majority of INL cell nuclei, while TRADD expression was limited to fewer cells. TRADD labeling was also present transiently in PRs at 7 wks. CASP8 mainly localized to the OPL in horizontal cells (see Figure S2), and the labeling intensity was higher in mutants compared to normals. Scale bar: 20 μm; RPE = retinal pigment epithelium, PR = photoreceptors, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer.</p

Retinal localization of pro-survival proteins in study models.

<p>Immunolabeling of normal, rcd1, xlpra2, and erd retinas at 14.3 to 16 wks with antibodies against pro-survival proteins STAT3, NTF3, and XIAP. Both STAT3 and NTF3 were primarily localized to the inner retina, from the INL to NFL, although the labeled cells differed. Mutant retinas exhibited higher labeling intensities and STAT3, but not NTF3, and showed intense PR inner segment labeling that was absent in normals. STAT3 also co-localized with Müller cells (see Figure S2). Mutant and normal retinas showed similar XIAP labeling pattern, although the intensity was reduced in mutants. XIAP was found in the PR layer, including the OPL-INL interface, as well as the RPE. PR-labeling was restricted to IS that in terms of numbers and shape appeared to be cones. An asterisk indicates that the RPE was missing. Scale bar: 20 μm; RPE = retinal pigment epithelium, PR = photoreceptors, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer.</p

Protein quantification by western blot in retinas of study models.

<p>Protein expression of TNF superfamily ligands (TNFA, CD40LG, TNFSF8), TNF superfamily receptors (TNFRSF1A, TNFRSF9), TNF superfamily regulator TRADD, initiator caspase CASP8, and pro-survival molecules STAT3, NTF3, and XIAP were analyzed at different ages of normal, rcd1, xlpra2, and erd retinas. Up-regulation of TNFA and CD40LG was found in mutants at all ages, particularly at 7-8.3 and 16 wks. TNFSF8 expression was markedly higher in xlpra2 at 7 wks and rcd1 at 16 wks. TNFRSF1A was marginally increased in mutants at all ages, while TNFRSF9, TRADD, and active CASP8 were up-regulated at 7-8.3 and 16 wks. Active CASP8 was also increased in rcd1 at 5 wks. Both STAT3 and NTF3 were up-regulated in mutants at all ages, whereas XIAP expression decreased in mutants after 7 wks, and was particularly low in rcd1 at 7 wks. Either ACTB or GAPDH were used as loading controls. White spaces indicate that the gel was cut. Approximate molecular size markers are indicated. The quantification of the bands illustrated in the Figure is reported in Table S2.</p

Time course of cell death in study models.

<p><b>A</b>) TUNEL-positive PR cells as a function of age (wks) in the superior retinal meridian of normal, rcd1, xlpra2, and erd-mutants. Mean ± SD of 3 measurements taken from the superior retinal meridian of each dog are reported. <b>B</b>) ONL thickness as number of rows of PR nuclei in normal, rcd1, xlpra2, and erd-mutants as a function of age (wks). Mean ± SD of 3 measurements taken from the central and midperipheral regions of the superior retinal meridian of each dog. xlpra2 data are slightly modified from [19] and erd from [20].</p

RNA expression changes of selected pro-death and pro-survival genes in study models.

<p>The fold change (FC) differences measured by qRT-PCR of selected genes in mutants compared to normals are shown at different ages. Genes belong to different functional categories: <b>A</b>) pro-death members of the extrinsic apoptotic pathway <i>CASP8</i>, <i>TNFA</i>, <i>TNFRSF1A</i>, <i>TRADD</i>; <b>B</b>) pro-survival <i>STAT3</i>, <i>NTF3</i>, <i>XIAP</i>, <i>IL10</i>. An asterisk indicates statistical significance; bars show SD of biological triplicates. Note that values on the Y-axis are not the same for all graphs, due to the highly variable FC differences in gene expression.</p

Protein expression changes of RHO, SAG, and ARR3 in study models.

<p><b>A</b>) Western blot analysis of normal, rcd1, xlpra2, and erd retinas showed decreased levels of the rod-specific protein RHO in the tested mutants at 5, 7, and 16 wks. Decreased levels compared to normals were also observed for SAG, a major protein of the retinal rod outer segments in rcd1 at 5, 7, and 16 wks and in xlpra2 at 16 wks. The quantification of the bands illustrated in the Figure is reported in Table S2. B) Immunolabeling of normal (4, 7, 16 wks), rcd1 (5, 7, 16 wks), xlpra2 (5, 7, 16 wks), and erd (4.3, 8.3, 14.1 wks) retinas with antibodies against RHO, the cone-specific ARR3, and SAG. An asterisk denotes mislocalization of RHO and SAG in the inner retina of mutants. Note that the findings are representative for the entire retina. Scale bar: 20 μm; PR = photoreceptors; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer. </p

RNA expression changes of retinal genes in study models.

<p><b>A</b>) Differentially expressed (DE) retinal genes in rcd1, xlpra2, and erd-mutants compared to normals at 5, 7, and 16 wks. No differences were found at 3 wks. DE genes are listed in alphabetical order, first the up-regulated and then the down-regulated, and are reported with the fold change (FC) differences. Note that in erd at 11.9-14.1 wks a reduced number of genes was tested (see Material and Methods). The complete list of tested genes is in Table S1. n.s. = not statistically significant differences; n.t. = not tested. <b>B</b>) FC differences between rcd1, xlpra2, and erd compared to normals at different ages (3, 5, 7, 16 wks) for <i>RHO</i>, <i>OPN1SW</i>, <i>OPN1LW</i>, <i>SAG</i>, and <i>CNGB3</i>. An asterisk indicates statistical significance, bars show SD of biological triplicates.</p

IRE1-XBP1 pathway in mutant T4R <i>RHO</i> and <i>WT</i> canine retinas 6 hours after light exposure.

<p><b>(A)</b> RT-PCR analysis of <i>XBP1</i> splicing in light exposed (E) compared to shielded (S) T4R <i>RHO</i> and <i>WT</i> retinas. RT-PCR of canine <i>XBP1</i> generated a 289 bp fragment, which represents the unspliced form of canine <i>XBP1</i>. The 263 bp fragment, which represents the spliced form of canine <i>XBP1</i> was not observed except in the tunicamycin treated normal canine fibloblasts (NCF). A retina from a wild-type dog kept under standard ambient kennel illumination (K) was used as a control of basal <i>XBP1</i> expression and splicing. <b>(B)</b> Differential expression of genes <i>XBP1 and ASK1</i> in the retinas of three RHO ^T4R/T4R mutant dogs following light exposure. Three different sets of primers were used to specifically amplify the unspliced (u), spliced (s) and both (total) <i>XBP1</i> transcripts. Displayed are the mean fold change (FC) difference compared to the contralateral shielded retinas. Error bars represent the FC range (FC min to FC max). <b>(C)</b> Immunoblots showing the protein levels of total and phosphorylated forms of XBP1 in light exposed (E) compared to shielded (S) retinas of mutant (<i>RHO</i>^T4R/T4R, and <i>RHO</i>^T4R/+), and <i>WT</i> (+/+) dogs. A single retina from a wild-type dog kept under standard ambient kennel illumination (K) was included as a control of basal levels of XBP1.</p

PERK-elF2α-ATF4 pathway in mutant T4R <i>RHO</i> and <i>WT</i> canine retinas 6 hours after light exposure.

<p><b>(A)</b> Immunoblots showing the protein levels of total and phosphorylated forms of eIF2α in light exposed (E) compared to shielded (S) retinas of mutant (<i>RHO</i>^T4R/T4R, and <i>RHO</i>^T4R/+), and <i>WT</i> (+/+) dogs. A single retina from a <i>WT</i> dog kept under standard ambient kennel illumination (K) was included as a control of basal levels of total and phosphorylated eIF2α. MDCK cells either treated with DMSO or Tunicamycin (Tun) were used as controls of P-eIF2α expression and antibody specificity. <b>(B)</b> Differential expression of gene <i>ATF4</i> in the retinas of three RHO ^T4R/T4R mutant dogs following light exposure. Displayed is the mean fold change (FC) difference compared to the contralateral shielded retinas. Error bars represent the FC range (FC min to FC max).</p