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1.  Endophenotypes for Age-Related Macular Degeneration: Extending Our Reach into the Preclinical Stages of Disease 
Journal of clinical medicine  2014;3(4):1335-1356.
The key to reducing the individual and societal burden of age-related macular degeneration (AMD)-related vision loss, is to be able to initiate therapies that slow or halt the progression at a point that will yield the maximum benefit while minimizing personal risk and cost. There is a critical need to find clinical markers that, when combined with the specificity of genetic testing, will identify individuals at the earliest stages of AMD who would benefit from preventive therapies. These clinical markers are endophenotypes for AMD, present in those who are likely to develop AMD, as well as in those who have clinical evidence of AMD. Clinical characteristics associated with AMD may also be possible endophenotypes if they can be detected before or at the earliest stages of the condition, but we and others have shown that this may not always be valid. Several studies have suggested that dynamic changes in rhodopsin regeneration (dark adaptation kinetics and/or critical flicker fusion frequencies) may be more subtle indicators of AMD-associated early retinal dysfunction. One can test for the relevance of these measures using genetic risk profiles based on known genetic risk variants. These functional measures may improve the sensitivity and specificity of predictive models for AMD and may also serve to delineate clinical subtypes of AMD that may differ with respect to prognosis and treatment.
PMCID: PMC4284143  PMID: 25568804
age-related macular degeneration; endophenotype; genetic risk; preclinical diagnostics; retinal function; predictive modeling
2.  Functional Burden of Strabismus 
JAMA ophthalmology  2013;131(11):1413-1419.
Binocular summation (BiS) is defined as the superiority of visual function for binocular over monocular viewing. Binocular summation decreases with age and large interocular differences in visual acuity. To our knowledge, BiS has not heretofore been well studied as a functional measure of binocularity in strabismus.
To evaluate the effect of strabismus on BiS using a battery of psychophysical tasks that are clinically relevant and easy to use and to determine whether strabismus is associated with binocular inhibition in extreme cases.
Case-control study.
University-based eye institute.
Strabismic patients recruited during 2010 to 2012 from a preoperative clinic and control participants with no history of eye disease other than refractive error.
A battery of psychophysical and electrophysiological tests including Early Treatment Diabetic Retinopathy Study visual acuity, Sloan low-contrast acuity (LCA) (2.5% and 1.25%), Pelli-Robson contrast sensitivity, and sweep visual evoked potential contrast sensitivity.
Binocular summation was calculated as the ratio between binocular and better-eye individual scores.
Sixty strabismic and 80 control participants were prospectively examined (age range, 8–60 years). Mean BiS was significantly lower in the strabismic patients than controls for LCA (2.5% and 1.25%, P = .005 and <.001, respectively). For 1.25% LCA, strabismic patients had a mean BiS score less than 1, indicating binocular inhibition (ie, the binocular score was less than that of the better eye’s monocular score). There was no significant difference in BiS for contrast thresholds on Early Treatment Diabetic Retinopathy Study visual acuity, Pelli-Robson contrast sensitivity, or sweep visual evoked potential contrast sensitivity. Regression analysis revealed a significant worsening of BiS with strabismus for 2.5% (P = .009) and 1.25% (P = .002) LCA, after accounting for age.
Strabismic patients demonstrate subnormal BiS and even binocular inhibition for LCA, suggesting that strabismus impairs visual function more than previously appreciated. This may explain why strabismic patients who are not diplopic close 1 eye in visually demanding situations. This finding clarifies the visual deficits impacting quality of life in strabismic patients and may represent a novel measure by which to evaluate and monitor function in strabismus.
PMCID: PMC4136417  PMID: 24052160
3.  Whole exome sequencing detects homozygosity for ABCA4 p.Arg602Trp missense mutation in a pediatric patient with rapidly progressive retinal dystrophy 
BMC Medical Genetics  2014;15:11.
A pediatric patient presented with rapidly progressive vision loss, nyctalopia and retinal dystrophy. This is the first report of homozygosity for the p.Arg602Trp mutation in the ABCA4 gene. The child became legally blind within a period of 2 years.
Case presentation
An eight year-old Hispanic female presented with bilateral decreased vision following a febrile gastrointestinal illness with nausea and vomiting. Extensive workup involved pediatric infectious disease and rheumatology consultations.
Initial visual acuity was 20/60 at distance and 20/30 at near in both eyes. Rapidly progressive vision loss occurred during a 2-year period resulting in visual acuities of 20/200 at distance in both eyes. Fundus exam disclosed attenuated vessels and multiple subretinal blister-like elevations. Optical coherence tomography showed far more lesions than were clinically evident with different levels of elevation. Autofluorescence imagery showed dramatic and widespread geographic areas of atrophy. The deposits that appeared drusen-like on clinical exam were hyperfluorescent, consistent with lipofuscin deposits containing A2e (N-retinylidene-N-retinylethanolamine) indicative of RPE cell dysfunction. Electroretinography was consistent with cone dystrophy, with relative preservation of rod function. Blood analysis and rheumatology evaluation found no evidence of a diffuse post-infectious/inflammatory process. The unique and rapid progression of her subretinal blister-like lesions was documented by fluorescein angiography, optical coherence tomography, autofluorescence imagery, and fundus photography. Family pedigree history disclosed consanguinity, her parents being first cousins. DNA analysis by whole exomic sequencing revealed homozygosity of p.Arg602Trp in the ABCA4 gene.
The pediatric patient presented with a striking clinical appearance and dramatic rate of progression that was clinically more characteristic of an infectious or inflammatory process. This case expands the diverse range of phenotypes attributed to ABCA4 mutations and further supports the role of whole exome sequencing as a powerful new tool available to aid clinicians in establishing diagnosis for challenging cases.
PMCID: PMC3905103  PMID: 24444108
ABCA4 retinopathy; Pediatric; Homozygosity; Consanguinity
4.  Neuronal Programmed Cell Death-1 Ligand Expression Regulates Retinal Ganglion Cell Number in Neonatal and Adult Mice 
During mouse retina maturation, the final number of retinal ganglion cells (RGCs) is determined by highly regulated programmed cell death. Previous studies demonstrated that the immunoregulatory receptor programmed cell death-1 (PD-1) promotes developmental RGC death. To identify the functional signaling partner(s) for PD-1, we identified retinal expression of PD-1 ligands and examined the effect of PD-1 ligand expression on RGC number. We also explored the hypothesis that PD-1 signaling promotes the development of functional visual circuitry.
Characterization of retinal and brain programmed cell death-1 ligand 1 (PD-L1) expression were examined by immunofluorescence on tissue sections. The contribution of PD-ligands, PD-L1, and programmed cell death-1 ligand 2 (PD-L2) to RGC number was examined in PD-ligand knockout mice lacking 1 or both ligands. Retinal architecture was assessed by spectral-domain optical coherence tomography, and retinal function was analyzed by electroretinography in wild-type and PD-L1/L2 double-deficient mice.
PD-L1 expression is found throughout the neonatal retina and persists in adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PD-ligands, there is a significant numerical increase in RGCs (34% at postnatal day 2 [P2] and 18% in adult), as compared to wild type, and PD-ligands have redundant function in this process. Despite the increased RGC number, adult PD-L1/L2 double-knockout mice have normal retinal architecture and outer retina function.
This study demonstrates that PD-L1 and PD-L2 together impact the final number of RGCs in adult mice and supports a novel role for active promotion of neuronal cell death through PD-1 receptor-ligand engagement.
PMCID: PMC3839320  PMID: 22635166
5.  Identification of DES1 as a Vitamin A Isomerase in Müller Glial Cells of the Retina 
Nature chemical biology  2012;9(1):30-36.
Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternate visual cycle for regenerating opsins in daylight. Here, we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternate pathway. DES1 is expressed in retinal Müller cells where it co-immunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in rpe65 −/− mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA-interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 possessed very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.
PMCID: PMC3522777  PMID: 23143414
6.  Comparative Regional Pupillography as a Noninvasive Biosensor Screening Method for Diabetic Retinopathy 
We describe infrared regional pupillometry as an objective comparative assessment of midperipheral to central retinal sensitivity and to correlate with midperipheral retinal ischemia in diabetic subjects.
We tested 12 normal and 17 diabetic subjects using bilateral infrared pupillometry. The diabetic cohort included seven subjects without, five with mild, three with moderate, and two with severe non-proliferative diabetic retinopathy (NPDR). Central and annular stimuli of varying intensity were presented to one eye, and pupillary amplitude and constriction velocity were measured from both eyes. Light stimulus of increasing intensity was presented as 20 consecutive trials (stimulus duration of 300 ms with 3000 ms intervals). The ratio of central to peripheral responses (Q values) was calculated for each stimulus configuration. Average responses with respect to the stimulus strength were regressed with Gompertz sigmoid function.
Control and moderate/severe NPDR cases comparison showed statistically significant differences in amplitude (QA) and constriction velocity (QCV) (Wilcoxon rank sum test P = 0.002, respectively). Age difference for these groups was not statistically significant (Wilcoxon rank sum test P = 0.15). The comparison of control and diabetic subjects without NPDR/mild NPDR was statistically significant for QA and QCV (Wilcoxon rank sum test P = 0.0002 and P = 0.001, respectively). QA and QCV differences were statistically significant between moderate/severe NPDR cases and subjects without or mild NPDR cases (Wilcoxon rank sum test P = 0.013).
QA and QCV values correlated highly with the severity of diabetic retinopathy, but not with the duration of diabetes. ( number, NCT01546766.)
We describe a novel methodology based on the pupillary response to evaluate midperipheral and central retinal sensitivity. In diabetic subjects, the ratios of peripheral to central amplitude and constriction velocity of the pupillary response are reduced, correlating with severity of retinopathy.
PMCID: PMC3541948  PMID: 23154459
7.  Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells 
Visual neuroscience  2012;29(2):83-93.
Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch’s membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host’s cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.
PMCID: PMC3694443  PMID: 22391151
Retina; Spectral domain optical coherence tomography; Cell transplantation; Lipofuscin; Electroretinography
8.  PTEN regulates retinal interneuron morphogenesis and synaptic layer formation 
The lipid phosphatase PTEN is a critical negative regulator of extracellular signal-induced PI3K activities, yet the roles of PTEN in the neural retina remain poorly understood. Here, we investigate the function of PTEN during retinal development. Deletion of Pten at the onset of neurogenesis in retinal progenitors results in the reduction of retinal ganglion cells and rod photoreceptors, but increased Müller glial genesis. In addition, PTEN deficiency leads to elevated phosphorylation of Akt, especially in the developing inner plexiform layer, where high levels of PTEN are normally expressed. In Pten mutant retinas, various subtypes of amacrine cells show severe dendritic overgrowth, causing specific expansion of the inner plexiform layer. However, the outer plexiform layer remains relatively undisturbed in the Pten deficient retina. Physiological analysis detects reduced rod function and augmented oscillatory potentials originating from amacrine cells in Pten mutants. Furthermore, deleting Pten or elevating Akt activity in individual amacrine cells is sufficient to disrupt dendritic arborization, indicating that Pten activity is required cell autonomously to control neuronal morphology. Moreover, inhibiting endogenous Akt activity attenuates inner plexiform layer formation in vitro. Together, these findings demonstrate that suppression of PI3K/Akt signaling by PTEN is crucial for proper neuronal differentiation and normal retinal network formation.
PMCID: PMC3278565  PMID: 22155156
Mouse retinal development; PTEN function; retinal specific knockout; PI3K/Akt signaling; amacrine cell morphogenesis
9.  Retinoid Content, Visual Responses, and Ocular Morphology Are Compromised in the Retinas of Mice Lacking the Retinol-Binding Protein Receptor, STRA6 
We report generation of a mouse model in which the STRA6 gene has been disrupted functionally to facilitate the study of visual responses, changes in ocular morphology, and retinoid processing under STRA6 protein deficiency.
A null mouse line, stra6 −/−, was generated. Western Blot and immunocytochemistry were used to determine expression of STRA6 protein. Visual responses and morphological studies were performed on 6-week, 5-month and 10-month-old mice. The retinoid content of eye tissues was evaluated in dark-adapted mice by high performance liquid chromatography.
STRA6 protein was not detectable in stra6 −/− null mice, which had a consistent reduction, but not total ablation of their visual responses. The mice also showed significant depletion of their retinoid content in retinal pigment epithelium (RPE) and neurosensory retina, including a 95% reduction in retinyl esters. At the morphological level, a reduction in thickness of the neurosensory retina due to shortening of the rod outer and inner segments was observed when compared to control litter mates with a commensurate reduction in rod a- and b-wave amplitudes. In addition, there was a reduction in cone photoreceptor cell number and cone b-wave amplitude. A typical hallmark in stra6 −/− null eyes was the presence of a persistent primary hypertrophic vitreous, an optically dense vascularized structure located in the vitreous humor between the posterior surface of the lens and neurosensory retina.
Our studies of stra6 −/− null mice established the importance of the STRA6 protein for the uptake, intracellular transport, and processing of retinol by the RPE. In its absence, rod photoreceptor outer and inner segment length was reduced, and cone cell numbers were reduced, as were scotopic and photopic responses. STRA6 also was required for dissolution of the primary vitreous. However, it was clear from these studies that STRA6 is not the only pathway for retinol uptake by the RPE.
stra6 −/− mice display an eye phenotype remarkably different from control litter mates, but less severe than the pathological abnormalities observed in humans with mutations in the same gene.
PMCID: PMC3378086  PMID: 22467576
10.  Molecular diagnosis of putative Stargardt disease probands by exome sequencing 
BMC Medical Genetics  2012;13:67.
The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases.
We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis.
Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified.
Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.
PMCID: PMC3459799  PMID: 22863181
Stargardt Disease; Macular Degeneration; Exome; Mutation Screening; Molecular Diagnostics; ABCA4; PRPH2
11.  Dysfunction of Heterotrimeric Kinesin-2 in Rod Photoreceptor Cells and the Role of Opsin Mislocalization in Rapid Cell Death 
Molecular Biology of the Cell  2010;21(23):4076-4088.
Loss of kinesin-2 function causes rapid death of rod photoreceptors. The cell death is dependent on the expression of opsin, which first accumulates along the route to the outer segment, but not on signaling by opsin-arrestin complexes or by light activation; the key element appears to be the accumulation of excessive protein in the wrong place.
Due to extensive elaboration of the photoreceptor cilium to form the outer segment, axonemal transport (IFT) in photoreceptors is extraordinarily busy, and retinal degeneration is a component of many ciliopathies. Functional loss of heterotrimeric kinesin-2, a major anterograde IFT motor, causes mislocalized opsin, followed by rapid cell death. Here, we have analyzed the nature of protein mislocalization and the requirements for the death of kinesin-2-mutant rod photoreceptors. Quantitative immuno EM showed that opsin accumulates initially within the inner segment, and then in the plasma membrane. The light-activated movement of arrestin to the outer segment is also impaired, but this defect likely results secondarily from binding to mislocalized opsin. Unlike some other retinal degenerations, neither opsin–arrestin complexes nor photoactivation were necessary for cell loss. In contrast, reduced rod opsin expression provided enhanced rod and cone photoreceptor survival and function, as measured by photoreceptor cell counts, apoptosis assays, and ERG analysis. The cell death incurred by loss of kinesin-2 function was almost completely negated by Rho−/−. Our results indicate that mislocalization of opsin is a major cause of photoreceptor cell death from kinesin-2 dysfunction and demonstrate the importance of accumulating mislocalized protein per se, rather than specific signaling properties of opsin, stemming from photoactivation or arrestin binding.
PMCID: PMC2993738  PMID: 20926680
12.  The Mouse Model of Down Syndrome Ts65Dn Presents Visual Deficits as Assessed by Pattern Visual Evoked Potentials 
The authors characterized the visual system at the retina and the visual cortex using the ERG and VEP in a Ts65Dn mouse model of Down syndrome (DS), and these tests provided support to the hypothesis that the visual cortex is the primary site responsible for the visual defect shown by these animals. These results should validate further the use of Ts65Dn mice as a mouse model of DS and raise awareness of potential confounders in the interpretation of behavioral assessments in these mice.
The Ts65Dn mouse is the most complete widely available animal model of Down syndrome (DS). Quantitative information was generated about visual function in the Ts65Dn mouse by investigating their visual capabilities by means of electroretinography (ERG) and patterned visual evoked potentials (pVEPs).
pVEPs were recorded directly from specific regions of the binocular visual cortex of anesthetized mice in response to horizontal sinusoidal gratings of different spatial frequency, contrast, and luminance generated by a specialized video card and presented on a 21-in. computer display suitably linearized by gamma correction.
ERG assessments indicated no significant deficit in retinal physiology in Ts65Dn mice compared with euploid control mice. The Ts65Dn mice were found to exhibit deficits in luminance threshold, spatial resolution, and contrast threshold, compared with the euploid control mice. The behavioral counterparts of these parameters are luminance sensitivity, visual acuity, and the inverse of contrast sensitivity, respectively.
DS includes various phenotypes associated with the visual system, including deficits in visual acuity, accommodation, and contrast sensitivity. The present study provides electrophysiological evidence of visual deficits in Ts65Dn mice that are similar to those reported in persons with DS. These findings strengthen the role of the Ts65Dn mouse as a model for DS. Also, given the historical assumption of integrity of the visual system in most behavioral assessments of Ts65Dn mice, such as the hidden-platform component of the Morris water maze, the visual deficits described herein may represent a significant confounding factor in the interpretation of results from such experiments.
PMCID: PMC2874110  PMID: 20130276
13.  Involvement of OA1, an Intracellular GPCR, and Gαi3, Its Binding Protein, in Melanosomal Biogenesis and Optic Pathway Formation 
Ocular albinism type 1 (OA1) is characterized by abnormalities in retinal pigment epithelium (RPE) melanosomes and misrouting of optic axons. The OA1 gene encodes a G-protein–coupled receptor (GPCR) that coimmunoprecipitates with the Gαi-subunit of heterotrimeric G-proteins from human melanocyte extracts. This study was undertaken to test whether one of the Gαi proteins, Gαi3, signals in the same pathway as OA1 to regulate melanosome biogenesis and axonal growth through the optic chiasm.
Adult Gαi3−/− and Oa1−/− mice were compared with their respective control mice (129Sv and B6/NCrl) to study the effects of the loss of Gαi3 or Oa1 function. Light and electron microscopy were used to analyze the morphology of the retina and the size and density of RPE melanosomes, electroretinograms to study retinal function, and retrograde labeling to investigate the size of the uncrossed optic pathway.
Although Gαi3−/− and Oa1−/− photoreceptors were comparable to those of the corresponding control retinas, the density of their RPE melanosomes was significantly lower than in control RPEs. In addition, the RPE cells of Gαi3−/− and Oa1−/− mice showed abnormal melanosomes that were far larger than the largest 129Sv and B6/NCrl melanosomes, respectively. Although Gαi3−/− and Oa1−/− mice had normal results on electroretinography, retrograde labeling showed a significant reduction from control in the size of their ipsilateral retinofugal projections.
These results indicate that Gαi3, like Oa1, plays an important role in melanosome biogenesis. Furthermore, they suggest a common Oa1-Gαi3 signaling pathway that ultimately affects axonal growth through the optic chiasm.
PMCID: PMC2881626  PMID: 18378571
14.  The Mouse Model of Down Syndrome Ts65Dn Presents Visual Deficits as Assessed by Pattern Visual Evoked Potentials 
The Ts65Dn mouse is the most complete widely available animal model for Down syndrome (DS). To generate quantitative information about visual function in the Ts65Dn mouse, we investigated their visual capabilities by means of electroretinography (ERG) and patterned visual evoked potentials (pVEPs).
pVEPs were recorded directly from specific regions of the binocular visual cortex of anesthetized mice in response to horizontal sinusoidal gratings of different spatial frequency, contrast, and luminance generated by a specialized video card and presented on a 21” computer display suitably linearized by gamma correction.
ERG assessments indicated no significant deficit in retinal physiology in Ts65Dn mice compared to euploid controls mice. We found, however, that Ts65Dn mice exhibit deficits in luminance threshold, spatial resolution, and contrast threshold compared to euploid control mice. The behavioral counterparts of these parameters are luminance sensitivity, visual acuity, and the inverse of contrast sensitivity, respectively.
DS includes various phenotypes associated with the visual system, including deficits in visual acuity, accommodation, and contrast sensitivity. The present study provides electrophysiological evidence of visual deficits in Ts65Dn mice that are similar to those reported in persons with DS. These findings strengthen the role of the Ts65Dn mouse as a model for DS. Also, given the historical assumption of integrity of the visual system most behavioral assessments of Ts65Dn mice, such as the hidden platform component of the Morris water maze, the visual deficits described here might represent a significant confounding factor in the interpretation of results from such experiments.
PMCID: PMC2874110  PMID: 20130276
15.  Accelerated Accumulation of Lipofuscin Pigments in the RPE of a Mouse Model for ABCA4-Mediated Retinal Dystrophies following Vitamin A Supplementation 
Dietary supplementation with vitamin A is sometimes prescribed as a treatment for retinitis pigmentosa, a group of inherited retinal degenerations that cause progressive blindness. Loss-of-function mutations in the ABCA4 gene are responsible for a subset of recessive retinitis pigmentosa. Other mutant alleles of ABCA4 cause the related diseases, recessive cone-rod dystrophy, and recessive Stargardt macular degeneration. Mice with a knockout mutation in the abca4 gene massively accumulate toxic lipofuscin pigments in the retinal pigment epithelium. Treatment of these mice with fenretinide, an inhibitor of vitamin A delivery to the eye, blocks formation of these toxic pigments. Here the authors tested the hypothesis that dietary supplementation with vitamin A may accelerate lipofuscin pigment formation in abca4−/− mice.
Wild-type and abca4−/− mice were fed normal or vitamin A–supplemented diets. Tissues from these mice were analyzed biochemically for retinoids and lipofuscin pigments. Eyes from these mice were analyzed morphologically for lipofuscin in the retinal pigment epithelium and for degeneration of photoreceptors. Visual function in these mice was analyzed by electroretinography.
Mice that received vitamin A supplementation had dramatically higher levels of retinyl esters in the liver and retinal pigment epithelium. Lipofuscin pigments were significantly increased by biochemical and morphologic analysis in wild-type and abca4−/− mice fed the vitamin A–supplemented diet. Photoreceptor degeneration was observed in 11-month-old albino, but not pigmented, abca4−/− mice on both diets.
Vitamin A supplementation should be avoided in patients with ABCA4 mutations or other retinal or macular dystrophies associated with lipofuscin accumulation in the retinal pigment epithelium.
PMCID: PMC2851626  PMID: 18515570
16.  Transgenic Mice Carrying the H258N Mutation in the Gene Encoding the β-Subunit of Phosphodiesterase-6 (PDE6B) Provide a Model for Human Congenital Stationary Night Blindness 
Human mutation  2007;28(3):243-254.
Mutations in the β-subunit of cGMP-phosphodiesterase (PDE6β) can lead to either progressive retinal disease, such as human retinitis pigmentosa (RP), or stationary disease, such as congenital stationary night blindness (CSNB). Individuals with CSNB in the Rambusch pedigree were found to carry the H258N allele of PDE6B (MIM# 180072); a similar mutation was not found in RP patients. This report describes an individual carrying the H258N allele, who presented with generalized retinal dysfunction affecting the rod system and a locus of dysfunction at the rod-bipolar interface. Also described are preclinical studies in which transgenic mice with the H258N allele were generated to study the pathophysiological mechanisms of CSNB. While Pde6brd1/Pde6brd1 mice have severe photoreceptor degeneration, as in human RP, the H258N transgene rescued these cells. The cGMP-PDE6 activity of dark-adapted H258N mice showed an approximate three-fold increase in the rate of retinal cGMP hydrolysis: from 130.1 nmol × min−1 × nmol−1 rhodopsin in wild-type controls to 319.2 nmol × min−1 × nmol−1 rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6β activity by the regulatory PDE6γ subunit is blocked by this mutation. In the albino (B6CBA × FVB) F2 hybrid background, electroretinograms (ERG) from H258N mice were similar to those obtained from affected Rambusch family members, as well as humans with the most common form of CSNB (X-linked), demonstrating a selective loss of the b-wave with relatively normal a-waves. When the H258N allele was introduced into the DBA background, there was no evidence of selective reduction in b-wave amplitudes; rather a- and b-wave amplitudes were both reduced. Thus, factors other than the PDE6B mutation itself could contribute to the variance of an electrophysiological response. Therefore, caution is advisable when interpreting physiological phenotypes associated with the same allele on different genetic backgrounds. Nevertheless, such animals should be of considerable value in further studies of the molecular pathology of CSNB.
PMCID: PMC2753261  PMID: 17044014
congenital stationary night blindness; retinal dystrophy; animal model; PDE6
17.  Non-Invasive Gene Transfer by Iontophoresis for Therapy of an Inherited Retinal Degeneration 
Experimental eye research  2008;87(3):168-175.
Despite extensive research on many of the genes responsible for inherited retinal degenerations leading to blindness, no effective treatment is currently available for patients affected with these diseases. Among the therapeutic approaches tested on animal models of human retinal degeneration, gene therapy using different types of viral vectors as delivery agents has yielded promising results. We report here our results on a non-invasive, non-viral delivery approach using transscleral iontophoresis for transfer of plasmid DNA into mouse retina. Proof of principle experiments were carried out using plasmid containing GFP cDNA to demonstrate expression of the transferred gene in the retina after single applications of iontophoresis. Various parameters for multiple applications of iontophoresis were optimized to sustain GFP gene expression in mouse photoreceptors. Subsequently, repeated iontophoresis of plasmid containing normal β-phosphodiesterase (β-PDE) cDNA was performed in the rd1 mouse, an animal model of autosomal recessive retinitis pigmentosa caused by a mutant β-PDE gene.
In normal mice, transscleral iontophoresis of the GFP plasmid provided a significant increase in fluorescence of the retina in the treated versus non-treated eyes. In rd1 mice, repeated iontophoresis of β-PDE cDNA plasmid partially rescued photoreceptors morphologically, as observed by microscopy, and functionally, as recorded on ERG measurements, without adverse effects. Therefore, transscleral iontophoresis of plasmid DNA containing therapeutic genes may be an efficient, safe and non-invasive method for the treatment of retinal degenerations.
PMCID: PMC2713253  PMID: 18653181
gene transfer; gene therapy; iontophoresis; retina; retinitis pigmentosa; rd1 mouse
18.  The Role of Interphotoreceptor Retinoid-Binding Protein on the Translocation of Visual Retinoids and Function of Cone Photoreceptors 
The first event in light perception is absorption of a photon by the retinaldehyde chromophore of an opsin pigment in a rod or cone photoreceptor cell. This induces isomerization of the chromophore, rendering the bleached pigment insensitive to light. Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE). Interphotoreceptor retinoid-binding protein (IRBP) is present in the extracellular space between photoreceptors and the RPE. IRBP is known to bind visual retinoids. Previous studies on irbp−/− mice suggested that IRBP plays an insignificant role in opsin-pigment regeneration. However, the mice in these studies were uncontrolled for a severe mutation in the rpe65 gene. Rpe65 catalyzes the rate-limiting step in the visual cycle. Here, we examined the phenotype in irbp−/− mice homozygous for the wild-type (Leu450) rpe65 gene. We show that lack of IRBP causes delayed transfer of newly synthesized chromophore from RPE to photoreceptors. Removal of bleached chromophore from photoreceptors is also delayed in irbp−/− retinas after light exposure. It was previously shown that rods degenerate in irbp−/− mice. Here,weshow that cones and rods degenerate at similar rates. However, cones are more affected functionally and show greater reductions in outer segment length than rods in irbp−/− mice. The disproportionate reductions in cone function and outer-segment length appear to result from mistrafficking of cone opsins due to impaired delivery of retinaldehyde chromophore, which functions as a chaperone for cone opsins but not rhodopsin.
PMCID: PMC2680234  PMID: 19193895
opsin; pigment epithelium; retinal degeneration; trafficking; visual cycle; vitamin A
19.  Rescue of retinal degeneration by intravitreally injected adult bone marrow–derived lineage-negative hematopoietic stem cells 
Journal of Clinical Investigation  2004;114(6):765-774.
Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow–derived stem cells (lineage-negative hematopoietic stem cells [Lin– HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin– HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.
PMCID: PMC516263  PMID: 15372100
20.  Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure 
BMC Genetics  2001;2:18.
Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma.
Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve.
We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.
PMCID: PMC59999  PMID: 11722794

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