An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease.

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

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.

Detailed functional and structural characterization of a macular lesion in a rhesus macaque.

PURPOSE: Animal models are powerful tools to broaden our understanding of disease mechanisms and to develop future treatment strategies. Here we present detailed structural and functional findings of a rhesus macaque suffering from a naturally occurring bilateral macular dystrophy (BMD), partial optic atrophy and corresponding reduction of central V1 signals in visual fMRI experiments when compared to data in a healthy macaque (CTRL) of similar age. METHODS: Retinal imaging included infrared and autofluorescence recordings, fluorescein and indocyanine green angiography and spectral domain optical coherence tomography (OCT) on the Spectralis HRA + OCT platform. Electroretinography included multifocal and Ganzfeld-ERG recordings. Animals were killed and eyes analyzed by immunohistochemistry. RESULTS: Angiography showed reduced macular vascularization with significantly larger foveal avascular zones (FAZ) in the affected animal (FAZBMD = 8.85 mm(2) vs. FAZCTRL = 0.32 mm(2)). OCT showed bilateral thinning of the macula within the FAZ (total retinal thickness, TRTBMD = 174 ± 9 µm) and partial optic nerve atrophy when compared to control (TRTCTRL = 303 ± 45 µm). Segmentation analysis revealed that inner retinal layers were primarily affected (inner retinal thickness, IRTBMD = 33 ± 9 µm vs. IRTCTRL = 143 ± 45 µm), while the outer retina essentially maintained its thickness (ORTBMD = 141 ± 7 µm vs. ORTCTRL = 160 ± 11 µm). Altered macular morphology corresponded to a preferential reduction of central signals in the multifocal electroretinography and to a specific attenuation of cone-derived responses in the Ganzfeld electroretinography, while rod function remained normal. CONCLUSION: We provided detailed characterization of a primate macular disorder. This study aims to stimulate awareness and further investigation in primates with macular disorders eventually leading to the identification of a primate animal model and facilitating the preclinical development of therapeutic strategies.

Suspected macular degeneration in a captive Western lowland gorilla (Gorilla gorilla gorilla).

The case of a 31-year-old captive female Western lowland gorilla (Gorilla gorilla gorilla) with decreased near vision but good distance vision is presented. Examination of the fundus revealed drusen-like bodies in the macula presumably because of an age-related macular degeneration (AMD).

Bestrophin detected in the basal membrane of the retinal epithelium and drusen of monkeys with drusenoid maculopathy.

PURPOSE: To determine if bestrophin is present in the basal membrane of macular retinal pigment epithelium (RPE) and in drusen of rhesus monkeys with age-related drusenoid maculopathy. METHODS: The macular region of three rhesus monkeys (Macaca mulatta), 23-24 years of age, with drusenoid maculopathy was dissected from eyes fixed with 4% paraformaldehyde. The macula was sectioned into rectangular pieces. The sclera was removed from each segment and the remainder separated into segments of neural retina with retinal epithelium or choroid with retinal epithelium. These segments were incubated with a goat polyclonal antibody to human bestrophin 1, reacted with gold-labeled rabbit antibody to goat IgG, silver-enhanced, and processed for transmission electron microscopy. RESULTS: Bestrophin-labeled gold particles were found in quasi-linear arrays on the basal surface of the macular RPE and also within drusen where bestrophin was found in segments of membranous-like material. The array density of the bestrophin-linked gold particles on the basal membrane of the epithelium had a maximal value of about 5-100 bestrophin molecules/micron(2). Immuno-detection of bestrophin was most effective when examined in an RPE layer that remained attached to the neural retina, where the basal surface of the epithelium is more directly exposed to the antibodies. CONCLUSION: Bestrophin is present on the basal membrane of macular RPE of rhesus monkeys with age-related drusenoid maculopathy, and also found in the membranous-like structures of drusen. The latter finding provides insight into the pathogenesis of drusen by indicating that segments of the basal membrane of RPE contribute to the material that accumulates within drusen.

Drusenoid maculopathy in rhesus monkeys: autofluorescence, lipofuscin and drusen pathogenesis.

PURPOSE: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys. METHODS: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy. RESULTS: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen. Electron microscopy revealed that all retinal epithelial cells had large concentrations of lipofuscin bodies. The epithelial cells overlying drusen, however, tended to have less lipofuscin than epithelial cells not associated with drusen. Electron microscopy revealed that the epithelial cells overlying drusen were losing segments of cytoplasm containing lipofuscin bodies. Macrophage-like cells were consistently present in Bruch's membrane microns away from this lipofuscin-containing cytoplasmic material. CONCLUSIONS: Retinal epithelial cells overlying drusen have less lipofuscin than neighboring epithelial cells. The loss of lipofuscin seems due to a loss of cytoplasm containing lipofuscin that contributes to drusen formation. Macrophages in Bruch's membrane may be responsible for removing this lipofuscin debris. The results support in vivo studies showing reduced autofluorescence over drusen and support the "budding" of epithelial cytoplasm as a source of drusen material.

Drusenoid maculopathy in rhesus monkeys (Macaca mulatta): effects of age and gender.

PURPOSE: To compare drusenoid maculopathy in monkeys with human age-related macular degeneration, and evaluate the influence of age, gender and caloric restriction. METHODS: Examination by indirect ophthalmoscopy, slit-lamp biomicroscopy and fundus photography, including in some cases fluorescein angiography, was performed on 61 male and 60 female rhesus macaques of ages 10-39 years. Fifty-four of the monkeys were maintained on a calorically restricted diet (approximately 30% lower than control levels) and 67 on an approximately ad libitum diet for 2-19 years, with all other environmental factors held constant. Maculopathies were graded on a 5-point scale and the effects of age, sex, and diet on prevalence and severity were examined. The retinas of six monkeys with macular drusen, 19-28 years old, were examined histologically. RESULTS: Rhesus monkeys showed a high prevalence (61%) of drusenoid maculopathy. The prevalence and severity of the maculopathy increased with age (p = 0.012). Fully half of all monkeys aged 10-12 years had some detectable degree of drusen. This high prevalence in young adulthood indicates that drusen develop much earlier in rhesus monkeys than in humans, who develop early maculopathy most rapidly at 50-60 years of age, even when correcting for the 3-fold difference in lifespan. No neovascularization or geographic atrophy was found. Females had a higher prevalence and severity than males (p = 0.019). Calorically restricted monkeys had a slightly lower prevalence and severity at 10-12 years than controls, but the difference was not statistically significant. This is an on-going project, and differences between the caloric restricted and ad-lib groups may emerge as the animals age. Some monkeys developed severe maculopathy in their 20s, with others unaffected in their 30s. The histology of drusen resembled those in human retina. CONCLUSION: Drusenoid maculopathy is common in rhesus monkeys, even in young adult life. Half of the rhesus monkeys examined have drusen at a much younger age than in humans. Severity of maculopathy was greater in female monkeys, a gender difference not consistently found in humans. No differences were detected due to caloric restriction, but a definitive test of this intervention will require a larger sample, longer period of observation, and/or an earlier institution of caloric restriction. Genetic factors are implied because with similar environments, some monkeys are affected at an early age, while older ones are not.

IMPG1 gene variation in rhesus macular drusen.

Drusen is a hallmark of human age-related maculopathy. Rhesus macaques (Macaca mulatta) represent a natural model of age-related maculopathy with drusen. We have already mapped the macular drusen susceptibility locus in rhesus macaques to the homolog of human chromosome 6q14-15 and shown that a particular IMPG1 gene SNP haplotype was apparently associated with drusen formation in the rhesus macaques maintained by the Caribbean Primate Research Center (CPRC), Puerto Rico, USA. The aim of the present study was to verify this finding in the macaques kept at the German Primate Research Center (DPZ), Germany. The study group comprised 64 animals (34 affected, 30 unaffected). These monkeys were genotyped for all known variations in the IMPG1 gene and haplotype analysis was performed. A total absence of the previously identified risk haplotype of the IMPG1 gene, and a much lower drusen prevalence in comparison to the CPRC group, was observed in the DPZ samples. This prompted a re-analysis of the original disease association in the CPRC, which revealed that the implied risk haplotype was in fact a sequencing artifact. Taken together, the data highlight that additional factors, other than IMPG1 variation, must play a role in drusen pathogenesis in rhesus macaques.

Familial early onset macular degeneration in cynomolgus monkeys (Macaca fascicularis).

The mode of inheritance of macular degeneration was determined with 45 cynomolgus monkeys (18 females and 27 males) who were the offspring of one breeding male with typical macular degeneration. In the first generation, 27 offspring (10 females and 17 males) were born from mating between the macular degeneration-affected founder male and 5 normal female breeders. Among them, 18 monkeys (9 females and 9 males) were judged as having macular degeneration (affected). Next, the distribution of affected offspring was examined with 18 offspring who were born from 3 different mating pairs, normal vs normal, affected vs normal and affected vs affected, when they became 2 years old. All of the 9 monkeys (4 females and 5 males) obtained from the 2 pairs of normal vs normal were normal. On the other hand, 6 affected monkeys (3 females and 3 males) were detected in 8 offspring from the mating pair of affected vs normal, and the single offspring produced by the mating pair of affected vs affected was affected. These results showed that this degeneration must be early onset familial macular degeneration controlled by autosomal dominant gene(s).

Trace element status and free radical defense in elderly rhesus macaques (Macaca mulatta) with macular drusen.

Research into the mechanisms underlying the development of age-related macular degeneration (AMD), the leading cause of visual loss in the United States and Europe in people over 60 years old, has been limited in part by the lack of animal models for this disease. In the current study, we examined 62 elderly (> or = 20 years old) rhesus macaques (Macaca mulatta) for the presence and severity of macular drusen. Drusen were observed in 47% of the macaques; they were similar histologically and in clinical appearance to the drusen observed in humans with AMD. It has been proposed that excessive tissue free radical damage may contribute to the development of AMD. Thus, circulating levels of select components of the free radical defense system and plasma thiobarbituric acid reactive substances (TBARS), an estimate of lipid peroxides, were measured in the above animals. Macaques diagnosed with drusen were characterized by alterations in concentrations and activities of several components of the free radical defense system. Alterations were most evident with respect to those enzymes associated with copper. The concept that excessive oxidative lipid damage might be a factor contributing to the occurrence of this disease is suggested by the findings of higher plasma TBARS concentrations in animals with > 10 drusen compared with animals without drusen.

Adult-onset macular degeneration in the Cayo Santiago macaques.

Since 1985 a group from the University of Florida has examined 136 rhesus monkeys from the Cayo Santiago colony. From the sample, 97 are older than nine years (approximately 30 human years) and 39 are younger. Drusen were found in 17% of the younger eyes and in 46% of the older eyes. All animals over 25 years of age had drusen in the central fundus. The incidence of drusen varied from 19-77% between five social groups. Incidence reported in random-source colonies in the continental U.S.A. is about six percent. Compared to near-age matched controls without drusen, selected rhesus exhibited visual resolution losses amounting to two Snellen-lines or more. The end-stage disciform changes and ultrastructural similarities are comparable with human macular disease. Future prospective studies may include therapies, surgical intervention, environmental manipulation and genetic research.

Degenerative changes in maculas of rhesus monkeys.

In the hope of identifying an animal model for age-related macular degeneration (AMD) we undertook a pilot investigation of aged rhesus monkeys. Twenty-nine monkeys from a seminatural colony were examined at the Caribbean Primate Research Center. Macular drusen were found in 74% of the monkey eyes. Alterations of the retinal pigment epithelium within the macula were noted in 45% of the eyes. Fluorescein angiography in selected animals revealed window defects consistent with drusen. None of this sample showed the exudative form of AMD or disciform scarring. One typical monkey underwent special studies including measurement of visual resolution by electrophysiological study of the retinal and visual cortex. Application of human criteria to this animal supported the diagnosis of early AMD. Histopathologic study of one eye by transmission electron microscopy confirmed the presence of drusen with nearly identical ultrastructural features to those found in the human pigment epithelium in AMD.

[Abnormal findings in the ocular fundi of colony-born cynomolgus monkeys].

The ocular fundi of 1,151 apparently healthy colony-born cynomolgus monkeys (Macaca fascicularis) ranging in age from newborn to 19 years were examined using an ophthalmoscope. Two hundred and thirty-eight abnormal findings were recorded in 219 of the 1,151 monkeys. Of these, 23 were related to the optic disc and 115 to the retinal vessels. Of the remaining 100 abnormal findings, 91 were retinal degenerations and 9 were retinal hemorrhages. The 23 optic disc abnormalities consisted of 3 cases of micropapilla, 4 of ectasia and 16 of myelination of the retinal nerve fibers. Of the 115 retinal vascular abnormalities, 87 were arterial tortuosity, one was venous tortuosity, 2 were tortuosity of both artery and vein, 2 were artery-vein crossing, 20 were copper-wire artery, one was inosculation of the artery, one was vascularization of the vein and one was persistent hyaloid artery. Of the 91 retinal degenerations, one was degeneration of the periphery of the macular and the optic disc, 8 were macular degeneration and 82 were peripheral degeneration. Nine cases of retinal hemorrhages appeared under 6 years of age.