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

Comparative analysis of differentially expressed (DE) genes by qRT-PCR in study models: Pro-inflammatory immune response group.

<p>DE genes (p<0.05 and FC≥+/-2) between rcd1, xlpra2, erd and xlpra1 mutants compared to normal at different ages.</p

Functional cell-cell interactions in retina responsible for stimulating and suppressing inflammation.

<p>Schematic representation of glia-photoreceptor interactions in the retina. The connections are based on reported cellular interactions (for description, please see text). Microglia, the resident immune cells, are responsible for initiating an inflammatory response. Microglia activation in the degenerating retina is triggered by different DAMPs. Although some trigger molecules which activate microglia are predicted to be released from injured cells, e.g. ATP, other molecules have not yet been identified. Activated microglia are capable of acquiring diverse phenotypes that display different cell-surface and intracellular markers, secrete different factors, and exhibit different functions. Two extreme microglial phenotypes are shown: the classically activated (M1) phenotype that promotes a pro-inflammatory response, and the alternatively activated (M2) phenotype that facilitates an anti-inflammatory response. Microglia can control their own polarization through autocrine and paracrine mechanisms. M1-polarized microglial phenotype is promoted by several cytokines, including TNF, IL1B and IL18. On the other hand, microglia can be driven to M2 phenotype by stimuli like IL4, IL13, IL-10, TGFB, CX3CL1 and MANF. Microglia, using receptors and signals, are in constant communication with neurons and other retinal cells. In pathological conditions this tight communication between cells mediate adaptive responses within the retina. Comments: Three basic types of glial cell in the retina are shown: Müller cells, astroglia, and microglia phenotypes are resting or activated, M1 or M2. Pro-inflammatory cytokines/receptors are in red and anti-inflammatory/neuroprotective factors are marked in blue. In addition, since MANF receptor is not identified yet, it’s marked by “?”.</p

Retinal localization of pro-inflammatory proteins in late-onset xlpra1-affected retinas with different disease severity phenotypes.

<p>Immunolabeling of normal and late-onset disease (xlpra1) retinas with <i>Mild</i>, <i>Moderate</i> and <i>Severe</i> phenotypes was done using IBA1, CD18, PYCARD, TNF and TLR4 antibodies. Double immunolabeling with microglial markers CD18 (green) and IBA1 (red) antibodies was done (A1–A4) to describe the phagocytes response characterized by their morphological changes and intraretinal migration. IBA1 and CD18 show significant co-localization in xlpra1 retinas displaying a well-developed disease phenotype that includes reduction of ONL (A2-A4). Both IBA1 and CD18 immunolabeling are robustly upregulated in disease retinas although highest expression is seen in <i>Moderate</i> severity. Double staining (A1-A4) of IBA1 and CD18 shows three subpopulation of phagocytes are seen in xlpra1 retina: CD18⁺/IBA1⁺ (yellow), CD18⁺/IBA1^- (green) and CD18^-/IBA1⁺ (red). Additionally, aggressive invasion of retinal layers by activated microglia/macrophages (arrowhead) is noticeable (A2-A4), particularly at <i>Mild</i> stage. Double immunolabeling of CD18 (green) with PYCARD (red) antibodies (B1-B4) shows PYCARD expression in CD18⁺ positive cells (yellow), with upregulation of both proteins in affected retinas. Robust infiltration of ONL by activated microglia/macrophages (B2) is very prominent in <i>Mild</i> stage (arrow). TNF labeling in normal and diseased retinas (C1-C4) shows TNF present in all retinal layers following the same pattern at all disease stages analyzed, however in <i>Mild</i> disease labeling signal was highest while intensity decreases as disease progresses. TLR4 labeling is present in all layers of normal and diseased retinas with labeling higher in <i>Severe</i> phenotype. PR = photoreceptors; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cells layer. Scale bar 40 μm.</p

Comparative analysis of differentially expressed genes in study models: Neuroprotective and anti-inflammatory group.

<p>DE genes between rcd1, xlpra2, erd and xlpra1 mutants compared to normal at different ages.</p

Retinal localization of selected pro-inflammatory proteins in early-onset disease models.

<p>Immunolabeling of normal and early-onset disease (rcd1, erd, xlpra2) retinas at various ages using IBA1, PYCARD and CD18 antibodies. IBA1 (red) labeling, a well-known marker of reactive microglia, increases in rcd1 and xlpra2 at 7 and 16 wks (A2, A3, C2, C3) when compared to normal control (A1, C1), suggesting microglial activation. IBA⁺ cells demonstrate migration of microglia from inner retina towards the outer retinal layers of rcd1 (A2, C2) and xlpra2 (A3, C3) at all ages tested. Additionally, double immunolabeling of the same retinal samples were also done with microglia/macrophage marker CD18 (green) and the inflammasome component PYCARD (red) antibodies. PYCARD is expressed primarily in CD18⁺ positive cells, as shown by labeling overlapping (B2-B4, D2-D4) thus demonstrating inflammasome component expression specifically in retinal microglia, albeit in two distinct subpopulations; CD18⁺/PYCARD⁺ (yellow; arrow) and CD18^-/PYCARD⁺ (red; arrowhead) cells. PYCARD expression is observed at all studied ages in diseased (B2-B4, D2-D4) and normal (B1, D1) retinas, and is significantly upregulated in rcd1 and xlpra2, especially at 16 wks of age. Conversely, IBA1 staining in erd retinas at 8 wks (A4) and 14.1 wks (C4) do not significantly change when compared to normal (A1, C1) indicating no additional microglial activation is occurring. Similarly, PYCARD/CD18 staining in erd at 8 and 14.1 wks remains the same as normal retinas. Although two subpopulations of CD18/PYCARD cells are also observed in erd retinas, increase in microglia migration to the outer retina layers as seen in rcd1 and xlpra2 was noticeably absent. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; GCL = ganglion cells layer. Scale bar 40 μm.</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

Protein quantification in retinas of rcd1 (A), xlpra2 (B), erd (C) and xlpra1 (D) models.

<p>Quantification of pro-inflammatory proteins on western blots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177224#pone.0177224.g002" target="_blank">Fig 2A</a>) normalized against the corresponding housekeeping protein (ACTB) was done using Li-COR Odyssey Fc and represented as fold-changes compared to the normal tissue values. Differences in relative fluorescence (Y-axis) for the proteins analyzed (X-axis) show CASP1, NLRP3, IL1B-precursor, IL1R1, TLR4 and CSF1R in disease (rcd1, xlpra2, erd, xlpra1) were similar to normal levels at all ages. In contrast, two proteins were increased over normal levels: mature ILIB-17 KDa (7 wks xlpra2, 16 wks rcd1) and PYCARD (7 wks xlpra2, 16 wks xlpra2, 16 wks rcd1). Moreover, three proteins were below normal levels: MYD88 (7 wks rcd1, 16 wks rcd1), IRAK4 (7 wks rcd1, 16 wks rcd1) and IL18-precursor (7 wks rcd1, 16 wks rcd1, 8 wks erd, 7 wks xlpra2). * indicates significance level 5% (p<0.05).</p

Western blot analysis of pro-inflammatory (A) and histone acetylation proteins (B) in study models of retinal degeneration.

<p>(A) Representative western immunoblot was performed for the expression of 11 proteins involved in pro-inflammatory signaling in total retinal protein extracts for normal and mutant retinas at 7 wks (rcd1, xlpra2), 8 wks (erd), 12 wks (erd), and 16 wks (rcd1, xlpra2, xlpra1). The following proteins were analyzed: inflammasome components (CASP1, NLRP3 and PYCARD), inflammasome substrates (IL1B and IL18) and their receptors (IL1R1 and IL18R1), inflammasome receptor (TLR4), common components of IL1B-, IL18- and TLR4-pathways (MYD88 and IRAK4) and macrophages expressing protein (CSF1R). (B) Level of histone acetylation in retinal protein extracts from the same four disease models was evaluated with acetylated-Lysine and acetyl-Histone H3 antibodies at the indicated time points.</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