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

Recombinant AAV-Mediated <i>BEST1</i> Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

<div><p>Mutations in the <i>BEST1</i> gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for <i>BEST1</i>-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (<i>cmr</i>), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when <i>BEST1</i> and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or <i>BEST1</i> transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies.</p></div

Evaluation of rAAV2/1- and rAAV2/2-mediated <i>BEST1</i> transgene expression in the canine retina.

<p>(<b>A</b>) Comparison of two normal canine eyes that received subretinal injection of rAAV2/1 (1.94×10¹¹ vg) and a spike-in of corresponding vector expressing GFP (3.81×10⁹ vg) or rAAV2/2 (3.92×10¹¹ vg) expressing canine <i>BEST1</i> under control of human VMD2 promoter. The outlines of the injected areas detectable in NIR mode and more evident in AF mode for rAAV2/1 (arrowheads) corresponded to the bleb formed immediately after injection (insets). The arrows indicate retinotomy sites. (<b>B</b>) Retinal thickness profiling done by manual segmentation across the bleb boundaries revealed no significant changes 4 weeks p.i. with either vector construct. High-resolution OCT images were obtained using a 30° lens; NIR and AF images were captured using a 55° lens; vg: vector genomes injected; p.i.: post injection.</p

Bestrophin1 overexpression induced by rAAV2/2 in the wild-type canine retina.

<p>Confocal photomicrographs illustrating Best1 expression (red) in the wild-type canine RPE six months p.i. The endogenous expression of Best1 (boxed area left and corresponding magnification) was limited to the basolateral plasma membrane while the transgene protein was also observed in the cell cytoplasm as a result of overexpression mediated by rAAV2/2-hVMD2-c<i>BEST1</i> (3.92×10¹¹ vg) (boxed area right and corresponding magnification). Cell nuclei were stained with DAPI; vg: vector genomes injected; p.i.: post injection; scale bar: 40 µm.</p

Consequences of rAAV2/1- and rAAV2/2-induced <i>BEST1</i> transgene expression <i>in vivo</i>.

<p>Histological and immunohistochemical evaluation of wild-type canine retinae injected with rAAV2/1-hVMD2-c<i>BEST1</i> (2.63×10¹¹ vg) and a spike-in of corresponding vector expressing GFP (2.5×10⁹ vg) or rAAV2/2-hVMD2-c<i>BEST1</i> (4.44×10¹¹ vg) in comparison to the non-injected control. H&E staining did not reveal any histological changes with either vector serotype. Both vectors induced bestrophin1 overexpression in the RPE cells 4 weeks post injection (Best1, red). While no abnormalities were observed in rAAV2/2-transduced retina, the rAAV2/1 serotype caused fluorescence in individual photoreceptor cells (green), occasional mislocalization of cone and rod opsins (arrowheads) and patchy loss of cone photoreceptors (arrows) in the rAAV2/1-hVMD2-c<i>BEST1</i>-injected area. RPE: retinal pigment epithelium, OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer. Cell nuclei were stained with DAPI; vg: vector genomes injected; scale bar: 40 µm and applies to all panels.</p

CNGA3-R424W mutant channel is non-functional, but CNGA3-E306R/R424E double charge-reversal mutant rescues the phenotype.

<p><b>(A)</b> Cyclic nucleotide-activated currents of the wild-type CNGA3 and a set of R424- and E306-mutant channels recorded from excised membrane patches at -60mV and +60mV in a presence of saturating concentrations of cGMP (200μM) and cAMP (5000μM). No nucleotide-activated currents were recorded for R424W-, R424E- or E306R-mutant channels. E306R-R424E double mutant channel restored cGMP activation, producing large currents similar to the wild-type CNGA3. Detailed results from multiple patches are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138943#pone.0138943.s007" target="_blank">S2 Table</a>. <b>(B)</b> cGMP dose-response relationship of the wild-type and CNGA3-E306R-R424E double charge-reversal mutant channels. The plot shows the ligand concentration-dependent activation of the wild-type (black) and CNGA3 double mutant (blue) homomeric channels at -60 mV. Each data set was taken from a representative single patch. The cGMP K_0.5 is 14.2 μM for the wild-type and 118.4 μM for the mutant channel; Hill coefficient [N_h] value is 2.1 and 2.0, respectively. <b>(C)</b> Cellular localization of YFP-tagged wild-type canine CNGA3 and CNGA3-R424W mutant in HEK tsA201 cells. Cells transfected with the wild-type construct showed specific fluorescent signals in the plasma membrane and Golgi-like organelles (arrow); cells expressing the mutant protein exhibited augmented intracellular signals consistent with aggregate formation in addition to membrane and Golgi-like fluorescence. Scale bar: 10μm. <b>(D)</b> Histograms of averaged cellular localization patterns for R424- and E306-mutant constructs <i>versus</i> wild-type CNGA3. A significant increase in intracellular aggregates was found in R424W-transfected cells (red bars) <i>vs</i> CNGA3-WT (black bars), and an apparent reduction in aggregate formation in the E306R-R424E double mutant channels (blue bars). An unpaired t-test was used to compare individual mutants <i>vs</i> WT (mean% ± SD for n>300 cells). * p-value of <0.05; ** p-values <0.0001.</p

Histolopathology at the fovea-like area in two canine models of inherited macular degeneration.

<p>(a) Fovea-like area in a 112 week-old <i>BEST1</i>-mutant dog. (a₁) Epifluorescence microscopy image (with DIC/Nomarski optics) showing at the fovea-like area increased autofluorescence (yellow) in the retinal pigment epithelium (RPE). (a₂) Immunohistochemistry on the same section as (b₁) shows focal separation (diamond) of cone (red) outer segments (OS) from the underlying RPE. Note: Red fluorescent signal originating from the RPE is endogenous autofluorescence (see a₁). (a₃) Immunohistochemistry shows focal separation (diamond) of rod OS (green) from hypertrophied RPE cells (red; arrowheads), and (a₄) extension of RPE apical processes (arrowheads). (b) Fovea-like areas in <i>RPGR</i> mutant dogs. (b₁) Horizontal retinal cross-section (H&E stained) shows the abrupt ONL thinning and shortened inner segments (IS) at the fovea-like area of a 4 week-old mutant dog while ONL thickness and structure of photoreceptors is preserved in the immediate peri-foveal regions. (b₂) Horizontal retinal cross-section (H&E stained) shows more prominent ONL thinning at the fovea-like area which has now extended peri-foveally in a 22 week-old dog. (b3) Early reduction in the number of cones is seen at the fovea-like area on retinal cross sections (left; cone nuclei are highlighted in yellow for visibility) and quantitative comparison to wildtype results (right). CA: cone arrestin; Rho: rhodopsin.</p