Involvement of Cyclic GMP Phosphodiesterase Activator in an Hereditary Retinal Degeneration

CYCLIC NUCLEOTIDES mediate many aspects of normal cellular metabolism; thus, degradation as well as synthesis of these intracellular mediators must be strictly regulated. Phosphodiesterase (PDE), the enzyme of cyclic nucleotide catabolism, is present in mammalian tissues in multiple forms, which differ in substrate specificity, kinetic characteristics and sub-cellular localisation. Moreover, a calcium-dependent protein activator (now called calmodulin) has been characterised that specifically activates at least one of the PDE types although other types of PDE are known to be activator independent. Thus, several mechanisms are present in vivo which allow strict control of PDE. A unique cyclic GMP-PDE is compartmentalised in the outer segments of retinal photoreceptor cells; its activity is low in the dark-adapted state but increases dramatically on light adaptation. The resulting drop in cyclic GMP content could serve as a chemical ‘signal’ in the normal visual process. However, despite much investigation of various cyclic nucleotide systems, no definitive information has been obtained which clearly links a disorder of cyclic nucleotide metabolism with a disease process elsewhere than in retina. We have recently presented preliminary evidence that an abnormality in cyclic GMP metabolism could be present in the retinas of Irish setter dogs with inherited rod–cone dysplasia that could lead to greatly increased cyclic GMP content, as had been reported in mice with inherited retinal degeneration. We now report that the basic defect in the disease seems to be a failure to switch PDE type and a concomitant decrease in protein activator concentration early in postnatal development, at the time of photoreceptor differentiation

Genetic and Phenotypic Variations of Inherited Retinal Diseases in Dogs: The Power of Within- and Across-Breed Studies

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20 years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision

Operating in the Dark: A Night-Vision System for Surgery in Retinas Susceptible to Light Damage

A standard operating microscope was modified with a bandpass infrared filter in the light path and infrared image intensifiers for each of the 2 eyepieces. We evaluated this system for subretinal injections in normal control dogs and those with a mutation in the rhodopsin gene. Rhodopsin-mutant dogs are a model for human autosomal dominant retinitis pigmentosa, and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light but not the infrared light. The modified operating microscope provided an excellent view of the ocular fundus under infrared illumination and allowed us to perform subretinal injections in the retinas of the rhodopsin-mutant dogs without any subsequent light-induced retinal degeneration. The first description of light-induced retinal damage showed that exposure of albino rats to visible light intensities ordinarily encountered in the laboratory led to irreversible retinal damage.1 This finding soon was extended to other species, including rabbits2 and monkeys.3 Studies in animal models of retinal degeneration (eg, Royal College of Surgeons rats, ABCA4-mutant mice, or rhodopsin-mutant mice and dogs) have emphasized the interplay between the gene mutation and environmental light and demonstrated acceleration of the disease process by light.4- 10 The effect of environmental light on disease severity in humans with retinitis pigmentosa has been suggested by case reports,11 although definitive proof is lacking. However, patients with class B1 rhodopsin mutations may be at risk of accelerated vision loss with increased light exposures because they have defects in dark (bleaching) adaptation similar to those found in rhodopsin-mutant dogs that demonstrate a high susceptibility to retinal light damage.8,12,13 Many of the emerging therapies for retinal degeneration require the intraocular placement of a reagent or device with the use of an operating microscope.14,15 The damaging effect of the microscope light on the normal retina, even with the appropriate filtering of UV light, has been described previously,16,17 and there is increased emphasis on reducing the intensity and duration of the exposures. Herein, we describe a modification of an operating microscope with an infrared bandpass filter and a night-vision system to perform surgical interventions in the posterior segment of the eyes of rhodopsin-mutant dogs without the risk of photochemical retinal damage. This modification prevents acceleration of the photoreceptor degeneration that occurs with exposure to modest light levels as used in routine clinical practice.8 If humans with retinitis pigmentosa are shown to have similar light damage susceptibility, this microscope modification could be considered for surgical procedures such as the subretinal application of gene therapy vectors

Age-Dependent Disease Expression Determines Remodeling of the Retinal Mosaic in Carriers of \u3cem\u3eRPGR\u3c/em\u3e Exon ORFn\u3csub\u3e15\u3c/sub\u3e Mutations

PURPOSE. To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS. Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS. A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared so that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (≥8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina. In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age; however, only patchy degeneration was seen by 1.4 years and was still apparent at 7.8 years. CONCLUSIONS. The time of onset and the progression of the disease differed between the two models. In the early-onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late-onset form (XLPRA1), patches of disease persisted until later ages

Intravitreal Injection of Ciliary Neurotrophic Factor (CNTF) Causes Peripheral Remodeling and Does Not Prevent Photoreceptor Loss in Canine \u3cem\u3eRPGR\u3c/em\u3e Mutant Retina

Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 μg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the central and mid-peripheral retina. CNTF also caused ocular side-effects and morphologic alterations in the periphery that were consistent with cell dedifferentiation and proliferation. Our findings suggest that some inherited forms of retinal degeneration may not respond to CNTF\u27s neuroprotective effects

A Non-Stop S-Antigen Gene Mutation Is Associated With Late Onset Hereditary Retinal Degeneration in Dogs

Purpose: To identify the causative mutation of canine progressive retinal atrophy (PRA) segregating as an adult onset autosomal recessive disorder in the Basenji breed of dog. Methods: Basenji dogs were ascertained for the PRA phenotype by clinical ophthalmoscopic examination. Blood samples from six affected cases and three nonaffected controls were collected, and DNA extraction was used for a genome-wide association study using the canine HD Illumina single nucleotide polymorphism (SNP) array and PLINK. Positional candidate genes identified within the peak association signal region were evaluated. Results: The highest -Log10(P) value of 4.65 was obtained for 12 single nucleotide polymorphisms on three chromosomes. Homozygosity and linkage disequilibrium analyses favored one chromosome, CFA25, and screening of the S-antigen (SAG) gene identified a non-stop mutation (c.1216T\u3eC), which would result in the addition of 25 amino acids (p.*405Rext*25). Conclusions: Identification of this non-stop SAG mutation in dogs affected with retinal degeneration establishes this canine disease as orthologous to Oguchi disease and SAG-associated retinitis pigmentosa in humans, and offers opportunities for genetic therapeutic intervention

Exonic SINE Insertion in \u3cem\u3eSTK38L\u3c/em\u3e Causes Canine Early Retinal Degeneration (\u3cem\u3eerd\u3c/em\u3e)

Fine mapping followed by candidate gene analysis of erd — a canine hereditary retinal degeneration characterized by aberrant photoreceptor development — established that the disease cosegregates with a SINE insertion in exon 4 of the canine STK38L/NDR2 gene. The mutation removes exon 4 from STK38L transcripts and is predicted to remove much of the N terminus from the translated protein, including binding sites for S100B and Mob proteins, part of the protein kinase domain, and a Thr-75 residue critical for autophosphorylation. Although known to have roles in neuronal cell function, the STK38L pathway has not previously been implicated in normal or abnormal photoreceptor development. Loss of STK38L function in erd provides novel potential insights into the role of the STK38L pathway in neuronal and photoreceptor cell function, and suggests that genes in this pathway need to be considered as candidate genes for hereditary retinal degenerations

Identification of Genetic Variation and Haplotype Structure of the Canine \u3cem\u3eABCA4\u3c/em\u3e Gene for Retinal Disease Association Studies

Over 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype–phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed sequence diversity of ABCA4 in the dog, identifying 80% of novel variations. While none of the observed polymorphisms have been associated with blinding disorders to date, breed and potentially disease specific haplotypes have been identified. Moreover, a tag SNP map of 17 (15) markers has been established that accurately predicts common ABCA4 haplotypes (frequency \u3e 5%) explaining \u3e85% (\u3e80%) of the observed genetic diversity and will considerably advance future studies. Our sequence analysis of the complete canine ABCA4 coding region will clearly provide a baseline and tools for future association studies and comparative genomics to further delineate the role of ABCA4 in canine blinding disorders

Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina?

PURPOSE. The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. METHODS. Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. RESULTS. Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. CONCLUSIONS. The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa