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Purpose Leading causes of irreversible blindness such as age-related macular degeneration (AMD) and glaucoma can, respectively, lead to central or peripheral vision loss. The ability of sufferers to process visual motion information can be impacted even during early stages of eye disease. We used head-mounted display virtual reality as a tool to better understand how vision changes caused by eye diseases directly affect the processing of visual information critical for self-motion perception. Methods Participants with intermediate AMD or early manifest glaucoma with near-normal visual acuities and visual fields were recruited for this study. We examined their experiences of self-motion in depth (linear vection), spatial presence, and cybersickness when viewing radially expanding patterns of optic flow simulating different speeds of self-motion in depth. Viewing was performed with the head stationary (passive condition) or while making lateral-sway head movements (active conditions). Results Participants with AMD (i.e., central visual field loss) were found to have greater vection strength and spatial presence, compared to participants with normal visual fields. However, participants with glaucoma (i.e., peripheral visual field loss) were found to have lower vection strength and spatial presence, compared to participants with normal visual fields. Both AMD and glaucoma groups reported reduced severity in cybersickness compared to healthy normals. Conclusions These findings strongly support the view that perceived self-motion is differentially influenced by peripheral versus central vision loss, and that patients with different visual field defects are oppositely biased when processing visual cues to self-motion perception.

Purpose The purpose of this study was to examine changes in the ganglion cell layer (GCL) of individuals with intermediate age-related macular degeneration (AMD) using grid-wise analysis for macular optical coherence tomography (OCT) volume scans. We also aim to validate the use of age-correction functions for GCL thickness in diseased eyes. Methods OCT macular cube scans covering 30° × 25° were acquired using Spectralis spectral-domain OCT for 87 eyes with intermediate AMD, 77 age-matched normal eyes, and 254 non-age-matched normal eyes. The thickness of the ganglion cell layer (GCL) was defined after segmentation at 60 locations across an 8 × 8 grid centered on the fovea, where each grid location covered 0.74 mm2 (approximately 3° × 3°) within the macula. Each GCL location of normal eyes (n = 77) were assigned to a specific iso-ganglion cell density cluster in the macula, based on patterns of age-related GCL thickness loss. Analyses were then performed comparing AMD GCL grid-wise data against corresponding spatial clusters, and significant AMD GCL thickness changes were denoted as values outside the 95% distribution limits. Results Analysis of GCL thickness changes revealed significant differences between spatial clusters, with thinning toward the fovea, and thickening toward the peripheral macula. The direction of GCL thickness changes in AMD were associated more so with thickening than thinning in all analyses. Results were corroborated by the application of GCL thickness age-correction functions. Conclusions GCL thickness changed significantly and nonuniformly within the macula of intermediate AMD eyes. Further characterization of these changes is critical to improve diagnoses and monitoring of GCL-altering pathologies.

Purpose To investigate the effect of stimulus size and disease status on the structure-function relationship within the central retina, we correlated the differential light sensitivity (DLS) with Goldmann stimulus size I to V (GI-V) and optical coherence tomography (OCT) derived in vivo ganglion cell count per stimulus area (GCc) within the macular area in normal subjects and patients with early glaucoma. Methods Humphrey Field Analyzer 10-2 visual field data with GI through V and Spectralis OCT macular ganglion cell layer (GCL) thickness measurements were collected from normal and early glaucoma cohorts including 25 subjects each. GCc was calculated from GCL thickness data and correlated with DLSs for different stimulus sizes. Results Correlation coefficients attained with smaller stimulus size were higher compared to larger stimulus sizes in both normal (GI-GII: R2 = 0.41-0.43, GIII-GV: R2 = 0.16-0.41) and diseased cohorts (GI-GII: R2 = 0.33-0.41, GIII-GV: R2 = 0.19-0.36). Quadratic regression curves for combined GI to V data demonstrated high correlation (R2= 0.82-0.90) and differed less than 1 dB of visual sensitivity within the GCc range between cohorts. The established structure-function relationship was compatible with a histologically derived model correlation spanning the range predicted by stimulus sizes GI to GIII. Conclusions Stimulus sizes within critical spatial summation area (GI-II) improved structure-function correlations in the central visual field. The structure-function relationship was identical in both normal and diseased cohort when GI to GV data were combined. Congruency of GI and GII structure-function correlation with those previously derived with GIII from more peripheral locations further suggests that the structure-function relationship is governed by the number of ganglion cell per stimulus area.

Bestrophin-1 gene (BEST1) mutations are responsible for a broad spectrum of human retinal phenotypes, jointly called bestrophinopathies. Canine multifocal retinopathy (cmr), caused by mutations in the dog gene ortholog, shares numerous phenotypic features with human BEST1-associated disorders. The purpose of this study was the assessment of molecular consequences and pathogenic outcomes of the cmr1 (C(73)T/R(25)X) premature termination and the cmr2 (G(482)A/G(161)D) missense mutation of the canine model compared with the C(87)G/Y(29)X mutation observed in human patients.Dogs carrying the BEST1 mutation were introduced into a breeding colony and used to produce either carrier or affected offspring. Eyes were collected immediately after euthanatization at the disease-relevant ages and were harvested for expression studies. In parallel, an in vitro cell culture model system was developed and compared with in vivoThe results demonstrate that cmr1 and human C(87)G-mutated transcripts bypass the nonsense-mediated mRNA decay machinery, suggesting the AUG proximity effect as an underlying transcriptional mechanism. The truncated protein, however, is not detectable in either species. The in vitro model accurately recapitulates transcriptional and translational expression events observed in vivo and, thus, implies loss of bestrophin-1 function in cmr1-dogs and Y(29)X-affected patients. Immunofluorescence microscopy of cmr2 mutant showed mislocalization of the protein.Molecular evaluation of cmr mutations in vivo and in vitro constitutes the next step toward elucidating genotype-phenotype interactions concerning human bestrophinopathies and emphasizes the importance of the canine models for studying the complexity of the BEST1 disease mechanism.

The RPGRIP1 gene encodes retinitis pigmentosa GTPase interacting protein 1, and mutations in the human gene are associated with Leber congenital amaurosis (LCA),1–3 juvenile retinitis pigmentosa,4 a late-onset cone–rod dystrophy,5 and cone–rod dystrophy type 1 in dogs.6,7 Human RPGRIP1 consists of 25 exons, of which 24 code for a 1259-amino-acid protein.1,3 Exons 6 to 13, 14 to 16, and 18 to 24 encode, respectively, the α-helical coil–coiled protein interaction motif of members of the structural maintenance of chromosomes (SMC) superfamily, two protein kinase C conserved region 2 motifs (C2), and conserved RPGR-interacting domain (RID).8–10 The biological functions of RPGRIP1 are complex. In the eye, it is expressed in amacrine neurons8,11 and photoreceptors,9,12 and in numerous other tissues, albeit at greatly reduced levels.9,12 Moreover, the existence of multiple isoforms, with species-specific subcellular localization patterns (e.g., connecting cilium,11,13 photoreceptor inner14 and outer8,11 segments, and basal bodies of cells with primary cilia15), suggests that different isoforms perform cell-specific functions. RPGRIP1 is required not only for disc morphogenesis of the outer segments (OS),16 but also for the formation of the OS itself, particularly in rods.17 A general role of RPGRIP1 as a scaffold protein has been suggested,13 and it interacts directly or indirectly with RPGR,9,16,18 NPHP4,10 and RanBP2.8 It has been shown that RPGRIP1 can be proteolytically processed, rendering its N-terminal domain competent for nuclear localization,19 suggesting that it may be involved in regulating gene expression. Although a mutation in RPGRIP1 is causally associated with canine cone–rod dystrophy,6,7 a potential large-animal model for gene-based therapies,20 little is known about the canine gene structure, organization, and expression, and the molecular basis of the disease. To assess the structure/function relationship of the RPGRIP1 isoforms, we characterized the full-length transcript of canine RPGRIP1 (cRPGRIP1) and its several alternatively spliced isoforms and evaluated the 5′- and 3′-UTRs of RPGRIP1 transcripts. Our results identified a novel complex 5′ and 3′ splicing pattern and further described the complete structure of six cRPGRIP1 alternatively spliced variants driven by two different promoters.

The term retinitis pigmentosa (RP) refers to a group of many different inherited retinal diseases characterized by progressive rod or rod–cone photoreceptor degeneration that causes subsequent visual impairment and blindness. Some of the causative genes have clear, well-identified roles (e.g., involvement in phototransduction, in maintaining photoreceptor structure, or in RPE retinoid metabolism; RetNet: http://www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). However, there remain a large number of diseases caused by genes with poorly understood functions and for which the mechanism linking the genes and/or mutations with photoreceptor disease and degeneration is unknown. Among these is the RP3 form of X-linked RP (XLRP), a uniformly severe, early-onset retinal disease in humans that is caused by mutations in the RP GTPase regulator (RPGR) gene.1 Although estimates vary depending on the sample population and methods of testing, it is generally accepted that mutations in RPGR account for >70% of XLRP cases.2–4 Furthermore, the carboxyl-terminal exon open reading frame 15 (ORF15) of RPGR, a mutational hot spot, has been shown to be mutated in 22% to 60% of XLRP patients.2,5,6 RPGR is essential for the maintenance of photoreceptor viability.7 The protein, which has a series of six RCC1-like domains (RLDs) characteristic of the highly conserved guanine nucleotide exchange factors, is found in the rod and cone photoreceptor connecting cilia.8 RPGR has complex interactions with other proteins that have microtubular-based transport functions in the retina and that are presumed to function in the photoreceptor centrosome, inner and outer segments, and ciliary axoneme region.9,10 Among these, the genes coding for nephrocystin-4,11 -5,12 and -69; PDE6D13; RPGR interacting protein (RPGRIP1)11; and RPGRIP1L14 cause retinal disease when mutated, thus emphasizing the critical importance of this protein complex in maintaining photoreceptor structure, function, and viability. One approach to developing insights into the cell- or tissue-specific functions of genes or to examining the molecular mechanisms of disease is microarray-based global profiling of gene expression in combination with bioinformatic analysis. In several studies, the transcriptome of the mouse and human retinas has been analyzed by characterizing changes in expression profiles during development and aging.15–17 More recently, transcriptomic data of distinct retinal cells18–20 and a web-based platform containing numerous retinal gene expression studies have been made available (http://alnitak.u-strasbg.fr/RetinoBase/ provided in the public domain by University Louis Pasteur, Strasbourg, France). In addition, studies based on differential gene expression in mouse retinal disease models provide useful information to aid in discerning the role of disease-causing genes with respect to other genes and in evaluating their involvement in gene pathways and cascades.21–23 These approaches have specific limitations in terms of human retinal diseases, not the least being the lack of adequate sample sizes at the appropriate disease stages. However, the constraints can be overcome by using animal models of homologous diseases. These models provide a powerful tool for translational studies, provided that the human disease modeled and the corresponding animal disease are comparable. Natural mutations in RPGRORF15 occur in humans and dogs,2,24 and X-linked progressive retinal atrophy (XLPRA) is the dog homolog of human XLRP. In dogs, two different ORF15 microdeletions have been identified: XLPRA1 is a postdevelopmental, slowly progressive photoreceptor degeneration resulting from a 5-bp deletion in ORF15 that truncates the translated protein, whereas XLPRA2 is an early-onset, progressive rod and cone photoreceptor disease caused by a 2-bp deletion that creates a frameshift and premature stop in the translated protein. The deduced peptide sequence is changed by the inclusion of 34 additional basic residues that increase the isoelectric point of the truncated protein.25 Beltran et al.26,27 described in detail the course of retinal disease in canine XLPRA2, the phenotype of which replicates the salient features of RPGR-XLRP.28,29 The purpose of the present study was to identify the genes and molecular mechanisms associated with disease onset and progression in normal and XLPRA2 mutant canine retinas. We examined the global retinal gene expression profiles at 7 and 16 weeks, the most relevant disease-related ages. Kinetics of photoreceptor cell death show a burst of dying cells between 6 and 7 weeks, whereas at 16 weeks, when the retina has lost approximately 40% of its photoreceptors, there is a constant but decreased rate of cell death.26 For this, we used a validated custom retinal cDNA microarray30,31 and performed real-time quantitative reverse transcription-PCR (qRT-PCR), Western blot analysis, and immunohistochemistry, to confirm and expand the microarray results. We detected several genes that were differentially expressed (DE) at critical time points in the degenerating XLPRA2 retina and that are specific for the disease stages examined. The downregulation of rod-specific genes also suggests the differential and preferential damage of rods in the early stages of the disease.

Purpose Identification of causative mutations for retinal blinding disorders is often limited by restricted understanding of gene expression and underlying molecular mechanisms that trigger degenerative processes. This study was conducted to develop a catalog of canine retina-expressed genes that would provide a unique tool to investigate normal and altered function in the adult retina. Because of the conserved syntenies between the dog and human, this approach would identify new potential disease candidate genes for both species. Methods A canine normalized retinal cDNA library was produced and analyzed by using a modified PhredPhrap algorithm. Computerized annotation provided gene homology and chromosomal location for individual clones and contigs in a Web-accessible database. Results From 6316 cDNA clones, 3980 retinal expressed sequence tags (ESTs) were derived. Homology to the canine genome draft sequence was found for more than 99% of all ESTs, but only for 32% when compared with annotated canine cDNAs. Functional analysis suggests an enrichment of this library for genes involved with eye function and development, chaperone, or ribosomal functions when compared with mouse and human National Center for Biotechnology Information (NCBI) RefSeq entries. Conclusions A combination of annotation approaches with ongoing mapping and expression studies provide functional data covering at least 27% to 30% of the currently proposed canine catalog of genes expressed in the retina. This is an essential first step toward establishing an integrated network for gene identification and expression patterns suitable for functional genetics, comparative genomics and evolutionary analysis of genes and gene families with respect to the developmental and degenerative processes of the retina.