The Positive Role of the Carboxyl Terminus of the Gamma Subunit of Retinal CGMP-phosphodiesterase in Maintaining Phosphodiesterase Activity in Vivo

Vision Research. Feb, 2002  |  Pubmed ID: 11853759

The inhibitory rod cyclic GMP-phosphodiesterase gamma subunit, PDEgamma, is a key component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice that lack PDEgamma due to a targeted disruption of the gene encoding PDEgamma, (Pdeg) suffer from a very rapid and severe photoreceptor degeneration. Previously, deletions in the carboxyl-terminal domain of PDEgamma blocked its ability to inhibit trypsin-activated PDE activity, in vitro. In other words, these mutations eliminated PDEgamma's control on the catalytic activity of PDEalpha and PDEbeta. To study the in vivo effects resulting from the deletion of the last seven amino acids of the PDEgamma carboxyl terminal, this PDEgamma allele (Del7C) was introduced as a transgene Pdeg(tm1)/Pdeg(tm1) mice. These animals could only synthesize transgenic mutant PDEgamma. The mutant retinas were expected to display a higher basal level of PDE activity and lower cGMP levels in light and darkness than the PDEgamma knockout mice, which would allow the rescue of their photoreceptors. Instead, our results showed that the Del7C transgene could not complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. In fact, animals carrying the Del7C transgene have low PDE activity as well as reduced PDEalpha and PDEbeta content.

Retinal Degeneration and RPE Transplantation in Rpe65(-/-) Mice

Investigative Ophthalmology & Visual Science. Oct, 2002  |  Pubmed ID: 12356839

To determine whether transplanting normal retinal pigment epithelium (RPE) into the subretinal space influences photoreceptor function and degeneration in Rpe65(-/-) mice.

Stationary Night Blindness or Progressive Retinal Degeneration in Mice Carrying Different Alleles of PDE Gamma

Frontiers in Bioscience : a Journal and Virtual Library. May, 2003  |  Pubmed ID: 12700134

A challenge in genetics is to understand the molecular basis of genetic and allelic heterogeneity. Divergent phenotypes caused by different variants of the same gene determine allelic heterogeneity. In the past few years, we have been studying an allelic series of mutations in the gamma-subunit of the cGMP phosphodiesterase gene (Pdeg) that resulted in visual defects ranging from stationary night blindness to progressive retinal degeneration. Here we describe the morphology and physiology of the retina in mice carrying four different Pdeg alleles: Pdeg(tm), Del 7C, Y84G, and W70A and the effect that these mutations of PDE gamma have on components of the activation and deactivation phases of phototransduction.

Transgenic Mice Carrying the H258N Mutation in the Gene Encoding the Beta-subunit of Phosphodiesterase-6 (PDE6B) Provide a Model for Human Congenital Stationary Night Blindness

Human Mutation. Mar, 2007  |  Pubmed ID: 17044014

Mutations in the beta-subunit of cGMP-phosphodiesterase (PDE6beta) 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 Pde6b(rd1)/Pde6b(rd1) 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 x min(-1) x nmol(-1) rhodopsin in wild-type controls to 319.2 nmol x min(-1) x nmol(-1) rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6beta activity by the regulatory PDE6gamma subunit is blocked by this mutation. In the albino (B6CBA x 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.

Transducin Translocation in Rods is Triggered by Saturation of the GTPase-activating Complex

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2007  |  Pubmed ID: 17267570

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin alpha subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

Novel Phenotypic and Genotypic Findings in X-linked Retinoschisis

Archives of Ophthalmology. Feb, 2007  |  Pubmed ID: 17296904

To describe atypical phenotypes associated with the retinoschisis (X-linked, juvenile) 1 mutation (RS1).

Autofluorescence Imaging in a Case of Benign Familial Fleck Retina

Archives of Ophthalmology. May, 2007  |  Pubmed ID: 17502520

Electronegative Electroretinogram Associated with Topiramate Toxicity and Vitelliform Maculopathy

Documenta Ophthalmologica. Advances in Ophthalmology. Jan, 2008  |  Pubmed ID: 17912565

Topiramate is known to cause ocular side effects such as refractive changes and angle closure. We describe a patient with an electronegative electroretinogram (ERG) which may have been related to topiramate use. Electronegative ERG's have been associated with other drugs in humans as well as topiramate use in rabbits. However, this would be the first suggestion of causality in humans.

Non-vascular Vision Loss in Pseudoxanthoma Elasticum

Documenta Ophthalmologica. Advances in Ophthalmology. Jul, 2008  |  Pubmed ID: 18034271

Pseudoxanthoma elasticum patients with angioid streaks are well-known to have acute vision loss due to choroidal bleeding. However, chronic vision loss due to macular atrophy is less well characterized. We describe a patient with sub-acute vision loss in one eye due to loss of macular retinal pigment epithelium function. Autofluorescence and pattern electroretinogram were useful adjuncts to help diagnose the source of her vision loss.

Modulation of Phosphodiesterase6 Turnoff During Background Illumination in Mouse Rod Photoreceptors

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2008  |  Pubmed ID: 18305241

In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase gamma (PDEgamma) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEgamma (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEgamma is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin alpha GTP. In PDEgamma T35A rods, a change in the conformation of the PDEgamma subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.

Effects of Extracellular Matrix and Neighboring Cells on Induction of Human Embryonic Stem Cells into Retinal or Retinal Pigment Epithelial Progenitors

Experimental Eye Research. Jun, 2008  |  Pubmed ID: 18472095

To determine the effects of extracellular matrix and neighboring cells on the differentiation of human embryonic stem cells (hESC) into progenitors of retinal cells and/or retinal pigment epithelium (RPE). HESC were cultured on mouse PA6 stromal cells for approximately 2weeks to obtain neural progenitors. To induce photoreceptor marker expression, the neural progenitors were cultured on a confluent monolayer of ARPE19 or on laminin-coated dishes. To induce RPE markers, the neural progenitors were seeded onto human Bruch's membrane or Matrigel. Cells were examined morphologically and stained with different RPE or neural progenitor markers. Microarray techniques were used to compare the gene expression profiles of hESC cultured on mouse fibroblasts or neural progenitors on PA6 cells to the transcriptome of the adult neural retina and RPE. HESC cultured on PA6 cells expressed neural progenitor markers beta-tubulin III, PAX6, neural filament, GFAP and vimentin. Culturing these neural progenitors on confluent ARPE19 monolayer induced expression of the photoreceptor progenitor cell marker CRX; culturing neural progenitors on laminin substrates induced a neuronal phenotype with neurite formation. Neural progenitors expressed the RPE marker ZO-1 after culturing on Matrigel-coated dishes and the RPE marker Bestrophin after culturing on human Bruch's membrane explants. Hierarchical clustering analysis of samples suggested that when cultured on PA6 stromal cells hESC exhibited genetic characteristics towards differentiating into neural retina. Microarray analysis showed that after culturing on PA6 cells, stem cells expressed 117 new genes; among these there were 22 genes present in neural retina or RPE cells. The functions of these genes were highly related to cell proliferation, nervous system development and cell adhesion. HESC can be induced to differentiate into neural progenitors after culturing on PA6 cells. These neural progenitors can express RPE markers when cultured on Bruch's membrane or Matrigel, or photoreceptor markers when cultured on confluent ARPE19 or laminin. Additional studies are required to assess the function of hESC induced to express retinal or RPE markers prior to successful intraocular transplantation into animal models of retinal degeneration.

Preferred Retinal Locus in Macular Disease: Characteristics and Clinical Implications

Retina (Philadelphia, Pa.). Oct, 2008  |  Pubmed ID: 18628727

To investigate the location and fixation stability of preferred retinal locations (PRLs) in patients with macular disease, and the relationship among areas of abnormal fundus autofluorescence, the PRL and visual sensitivity.

Functional Rescue of Degenerating Photoreceptors in Mice Homozygous for a Hypomorphic CGMP Phosphodiesterase 6 B Allele (Pde6bH620Q)

Investigative Ophthalmology & Visual Science. Nov, 2008  |  Pubmed ID: 18658088

Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6alpha and PDE6beta) and two regulatory (PDE6gamma) subunits. In mice homozygous for a nonsense Pde6b(rd1) allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6b(rd1) phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6b(H620Q) was characterized further.

A Novel Mutation and Phenotypes in Phosphodiesterase 6 Deficiency

American Journal of Ophthalmology. Nov, 2008  |  Pubmed ID: 18723146

To develop a systematic approach for the molecular diagnosis of retinitis pigmentosa (RP) and to report new genotype-phenotype correlations for phosphodiesterase 6 (PDE6)-based RP mutations.

Phenotype-genotype Correlations in Autosomal Dominant Retinitis Pigmentosa Caused by RHO, D190N

Current Eye Research. Nov, 2008  |  Pubmed ID: 19085385

To phenotype a family with RHO (Asp190Asn or D190N) dominantly inherited retinitis pigmentosa (RP) and to describe an approach to surveying affected families.

Benign Panretinal Uniform Radial Linear-shaped Flecks

Clinical & Experimental Ophthalmology. Dec, 2008  |  Pubmed ID: 19278486

A Comparison of Fundus Autofluorescence and Retinal Structure in Patients with Stargardt Disease

Investigative Ophthalmology & Visual Science. Aug, 2009  |  Pubmed ID: 19324865

To improve the understanding of Stargardt disease by comparing structural changes seen on spectral domain optical coherence tomography (SD-OCT) to those visible on fundus autofluorescence (FAF).

Fundus Autofluorescence in Cone Dystrophy

Documenta Ophthalmologica. Advances in Ophthalmology. Oct, 2009  |  Pubmed ID: 19340470

To describe fundus autofluorescence (FAF) finding in a case of cone dystrophy.

Case Report: Autofluorescence Imaging and Phenotypic Variance in a Sibling Pair with Early-onset Retinal Dystrophy Due to Defective CRB1 Function

Current Eye Research. May, 2009  |  Pubmed ID: 19401883

To phenotype two siblings with autosomal recessive early-onset retinal dystrophy due to CRB1 mutations.

Cellular and Molecular Origin of Circumpapillary Dysgenesis of the Pigment Epithelium

Ophthalmology. May, 2009  |  Pubmed ID: 19410955

We studied clinical phenotyping and TEAD1 expression in mice and humans to gain a better understanding of the primary origin in the pathogenesis of circumpapillary dysgenesis of the pigment epithelium.

Structural Assessment of Hyperautofluorescent Ring in Patients with Retinitis Pigmentosa

Retina (Philadelphia, Pa.). Jul-Aug, 2009  |  Pubmed ID: 19584660

To analyze the retinal structure underlying the hyperautofluorescent ring visible on fundus autofluorescence in patients with retinitis pigmentosa.

Light-dependent Phosphorylation of the Gamma Subunit of CGMP-phophodiesterase (PDE6gamma) at Residue Threonine 22 in Intact Photoreceptor Neurons

Biochemical and Biophysical Research Communications. Dec, 2009  |  Pubmed ID: 19878658

The gamma subunit of rod-specific cGMP phosphodiesterase 6 (PDE6gamma), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6gamma on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6gamma may increase or decrease the ability of PDE6gamma to deactivate phototransduction. To resolve role of phosphorylation of PDE6gamma in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6gamma (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6gamma, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6gamma is essential for the regulation of G-protein signaling.

Erythropoetin Receptor Expression in the Human Diabetic Retina

BMC Research Notes. 2009  |  Pubmed ID: 19930719

Recent evidence suggests erythropoietin (EPO) and the erythropoietin receptor (EPOR) may play a direct role in the pathogenesis of diabetic retinopathy. Better characterization of the EPO-EPOR signaling system in the ischemic retina may offer a new therapeutic modality for ischemic ophthalmic diseases. This study was performed to identify EPOR mRNA expression in the human diabetic eye.

G1961E Mutant Allele in the Stargardt Disease Gene ABCA4 Causes Bull's Eye Maculopathy

Experimental Eye Research. Jun, 2009  |  Pubmed ID: 19217903

The aim of this study was to characterize the pathological and functional consequences of the G1961E mutant allele in the Stargardt disease gene ABCA4. Data from 15 patients were retrospectively reviewed and all the patients had at least one G1961E mutation. Comprehensive ophthalmic examination, full-field and pattern electroretinograms, and fundus autofluorescence (FAF) imaging were performed on all patients. Microperimetry, spectral-domain optical coherence tomography (OCT), and fluorescein angiography were performed in selected cases. Genetic screening was performed using the ABCR400 micro-array that currently detects 496 distinct ABCA4 variants. All patients had normal full-field scotopic and photopic electroretinograms (ERGs) and abnormal pattern electroretinograms (PERGs) performed on both eyes, and all the fundi had bull's eye maculopathy without retinal flecks on FAF. On OCT, 1 patient had disorganization of photoreceptor outer segment, 2 had outer nuclear layer (ONL) thinning likely due to photoreceptor atrophy proximal to the foveal center, and 3 had additional retinal pigment epithelium (RPE) atrophy. On microperimetry, 6 patients had eccentric superior fixation and amongst this group, 5 had an absolute scotoma in the foveal area. DNA analysis revealed that 3 patients were homozygous G1961E/G1961E and the rest were compound heterozygotes for G1961E and other ABCA4 mutations. The G1961E allele in either homozygosity or heterozygosity is associated with anatomical and functional pathologies limited to the parafoveal region and a trend to delayed onset of symptoms, relative to other manifestations of ABCA4 mutations. Our observations support the hypothesis that the G1961E allele contributes to localized macular changes rather than generalized retinal dysfunction, and is a cause of bull's eye maculopathy in either the homozygosity or heterozygosity state. In addition, genetic testing provides precise diagnosis of the underlying maculopathy, and current non-invasive imaging techniques could be used to detect photoreceptor damage at the earliest clinical onset of the disease.

Rapid and Noninvasive Imaging of Retinal Ganglion Cells in Live Mouse Models of Glaucoma

Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. Aug, 2010  |  Pubmed ID: 19937134

We report a noninvasive method for the monitoring of retinal ganglion cell (RGC) survival in live mice utilizing standard fluorescence microscopy.

Transplantation of Reprogrammed Embryonic Stem Cells Improves Visual Function in a Mouse Model for Retinitis Pigmentosa

Transplantation. Apr, 2010  |  Pubmed ID: 20164818

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice.

Fundus Autofluorescence, Optical Coherence Tomography, and Electroretinogram Findings in Choroidal Sclerosis

Retina (Philadelphia, Pa.). Jul-Aug, 2010  |  Pubmed ID: 20224472

The purpose of this study was to describe fundus autofluorescence (FAF), optical coherence tomography, and electroretinogram findings in choroidal sclerosis.

Autofluorescence and High-Resolution OCT Findings Revealed Ciliopathy in Senior-Loken Syndrome

Ophthalmic Surgery, Lasers & Imaging : the Official Journal of the International Society for Imaging in the Eye. Mar, 2010  |  Pubmed ID: 20337316

To describe novel findings on fundus autofluorescence (FAF) and high-resolution optical coherence tomography (OCT) in a 27-year-old woman with the Senior-Loken syndrome (SLSN) emphasizing the photoreceptors' cilia appearance in the macula. The patient had renal transplantation early in life and poor visual acuity due to advanced autosomal recessive retinitis pigmentosa. FAF showed diffuse spots of decreased autofluorescence in the mid-periphery and a perifoveal ring of increased autofluorescence suggesting a bull's eye maculopathy. High-resolution OCT revealed a barely detectable inner-outer photoreceptor segment junction in the central macula corresponding to the area inside of the ring of increased autofluorescence, suggesting initial ciliary junction disorganization before photoreceptors death. Non-invasive technologies can monitor central photoreceptors cilliary anatomy enabling early detection of cell disorganization in diseases involving ciliopathy such as the Senior-Loken syndrome are concluded.

Fundus Autofluorescence and Optical Coherence Tomography of Congenital Grouped Albinotic Spots

Retina (Philadelphia, Pa.). Sep, 2010  |  Pubmed ID: 20539258

The purpose of this study was to describe the findings of fundus autofluores-cence (FAF) and optical coherence tomography in a series of patients with congenital grouped albinotic spots.

Vitamin A Deficiency in New York City

Lancet. Jul, 2010  |  Pubmed ID: 20663549

Loss of Peripapillary Sparing in Non-group I Stargardt Disease

Experimental Eye Research. Nov, 2010  |  Pubmed ID: 20696155

The aim of this study was to assess peripapillary sparing in patients with non-group I Stargardt disease. We suggest this as a useful clinical sign for formulating disease severity. Patients with a diagnosis of Stargardt disease were grouped by electroretinogram (ERG). Fundus autofluorescence was used to assess the peripapillary area for involvement in the Stargardt disease process. From a cohort of 32 patients (64 eyes), 17 patients (33 eyes) demonstrated loss of peripapillary sparing. One of 15 patients in Group I, six of 7 patients in group II and 9 of 10 patients in group III demonstrated peripapillary atrophy. One patient in group II had peripapillary flecks. All patients had at least one mutation detected in the ABCA4 gene. Both mutations were detected in 21 patients. Patients in groups II and III had the earliest ages of onset and the poorest visual acuities. Two novel disease causing mutation in the ABCA4 gene were detected. Our data supports the observation that peripapillary sparing is not universal finding for Stargardt disease and peripapillary atrophy is a useful clinical sign for identifying patients with Stargardt disease who fall into the more severe ERG groups, i.e. groups II and III. The presence of atrophy suggests a continuum of disease between groups II and III. Loss of peripapillary sparing is likely associated with the more deleterious mutations of the ABCA4 gene.

Unilateral Electronegative ERG in a Presumed Central Retinal Artery Occlusion

Clinical Ophthalmology (Auckland, N.Z.). 2010  |  Pubmed ID: 21139671

A unilateral electronegative electroretinogram (ERG) was seen in a 94-year-old man with presumed central retinal artery occlusion. Goldmann perimetry revealed central scotoma in the right eye and no abnormalities in the left eye. Full-field ERG in the right eye described a reduction of the b-wave with a relative preservation of the a-wave which is characteristic of electronegative ERG. Hence, our case illustrates that ERG testing is essential for the work-up of individuals with suspected retinal vascular disorders.

Macular Dystrophy in Heimler Syndrome

Ophthalmic Genetics. Jun, 2011  |  Pubmed ID: 21366429

To describe the retinal imaging findings in the index patient with Heimler syndrome (OMIM #234580).

Allelic and Phenotypic Heterogeneity in ABCA4 Mutations

Ophthalmic Genetics. Sep, 2011  |  Pubmed ID: 21510770

Since the discovery of the ABCA4 gene as the cause of autosomal recessive Stargardt disease/fundus flavimaculatus much has been written of the phenotypic variability in ABCA4 retinopathy. In this review the authors discuss the findings seen on examination and the disease features detected using various clinical tests. Important differential diagnoses are presented and unusual presentations of ABCA4 disease highlighted.

Function of the Asparagine 74 Residue of the Inhibitory γ-subunit of Retinal Rod CGMP-phophodiesterase (PDE) in Vivo

Cellular Signalling. Oct, 2011  |  Pubmed ID: 21616145

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g(tm1) mice. This transgenic line was crossed with Pde6g(tm1)/Pde6g(tm1) mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.

Quantification of Peripapillary Sparing and Macular Involvement in Stargardt Disease (STGD1)

Investigative Ophthalmology & Visual Science. 2011  |  Pubmed ID: 21873672

To quantify and compare structure and function across the macula and peripapillary area in Stargardt disease (STGD1).

Lentivirus-mediated Expression of CDNA and ShRNA Slows Degeneration in Retinitis Pigmentosa

Experimental Biology and Medicine (Maywood, N.J.). Oct, 2011  |  Pubmed ID: 21885480

Mutations in Pde6b lead to high levels of signaling molecules cyclic guanosine monophosphate (cGMP) and Ca(2+), which ultimately result in photoreceptor cell death in certain forms of retinitis pigmentosa (RP). The level of cGMP, which is controlled by opposing activities of guanylate cyclase (GUCY) and photoreceptor phosphodiesterase-6 (PDE6), regulates the opening of cyclic nucleotide-gated ion channels [CNG] and thereby controls Ca(2+) influx into the outer segments. Using a lentiviral gene therapy approach, we have previously shown that degeneration can be temporarily slowed either by introducing wild-type PDE6β or knocking down expression of GUCY2E and CNGA1 in photoreceptors of Pde6b(H620Q), a mouse model for RP. Rescue was transient with either approach. Therefore, we tested a novel combination therapy using bipartite lentiviral vectors designed to both introduce wild-type PDE6β expression and knockdown GUCY2E or CNGA1. Immunoblot analysis shows simultaneous increases in PDE6β and decreases in GUCY2E or CNGA1 in retinas transduced by the vectors, indicating successful transduction. In Pde6b(H620Q) mutants, we observe rescue of photoreceptor function and an increase in photoreceptor rows as compared with untreated controls. However, no evidence of prolonged rescue beyond the limit of the previously tested single therapy was observed.

Analysis of the ABCA4 Gene by Next-generation Sequencing

Investigative Ophthalmology & Visual Science. Oct, 2011  |  Pubmed ID: 21911583

To find all possible disease-associated variants in coding sequences of the ABCA4 gene in a large cohort of patients diagnosed with ABCA4-associated diseases.

The Inner Segment/outer Segment Border Seen on Optical Coherence Tomography is Less Intense in Patients with Diminished Cone Function

Investigative Ophthalmology & Visual Science. Dec, 2011  |  Pubmed ID: 22110066

PURPOSE; The integrity of the inner segment ellipsoid (ISe) band, previously called the inner segment/outer segment (IS/OS) border, seen on optical coherence tomography (OCT) scans is of clinical significance. To better understand the influence of cones on the appearance of this band, the intensity of its signal in patients with diminished cone function was examined.

Progressive Constriction of the Hyperautofluorescent Ring in Retinitis Pigmentosa

American Journal of Ophthalmology. Dec, 2011  |  Pubmed ID: 22137208

PURPOSE: To evaluate the constriction of the hyperautofluorescent ring over time in patients with retinitis pigmentosa (RP). DESIGN: Prospective study. METHODS: Fourteen eyes of 14 RP patients with a hyperautofluorescent ring were studied. Ring constriction was evaluated by measurements of its external and internal boundaries along the vertical and horizontal axes at baseline and at 12-, 24-, 36-, and 48-month follow-ups. Repeat fundus autofluorescence was obtained at 12, 24, 36, and 48 months in 13, 7, 5, and 1 eyes respectively. Spectral-domain optical coherence tomography (SD-OCT) images were obtained on 8 eyes and the horizontal extent of the inner segment/outer segment (IS/OS) junction was measured. SD-OCT was repeated at 12 and 24 months in 6 and 4 eyes respectively. RESULTS: The external boundaries of the ring were identified along the horizontal axis in 12 eyes and along the vertical axis in 13. Internal boundaries were identified in 7 eyes. Constriction was demonstrated in all patients except 1 who demonstrated minimal expansion of the internal boundary along the horizontal axis. SD-OCT measurements showed a decrease in the IS/OS junction length. CONCLUSION: Progressive constriction of the hyperautofluorescent ring and a concordant decrease in IS/OS junction length were observed over time.

Unilateral Retinitis Pigmentosa: a Proposal of Genetic Pathogenic Mechanisms

European Journal of Ophthalmology. Nov, 2011  |  Pubmed ID: 22139616

Purpose. To investigate and integrate anatomic and physiologic findings from a group of patients who present retinitis pigmentosa affecting just one eye and use this information to propose mechanisms of disease pathogenesis. Methods. This prospective cross-sectional study examined 5 patients, all female, from 8 to 60 years old. The study was conducted in 4 university hospitals. The patients were selected according to the characteristics of ocular involvement, notably unilateral presentation of similar anatomic and functional abnormalities. Full-field electroretinogram, fundus photography, fundus autofluorescence, infrared imaging, optical coherence tomography, and genetic testing were performed. Results. Full-field electroretinogram showed unilateral decrease in amplitude and increase in implicit time; autofluorescence showed unilateral areas of decreased intensity. The USH2AW4149R mutation was confirmed in one patient. Conclusions. Imaging and functional testing are important in elucidating the unilateral pattern of the disease and in monitoring these individuals. Mosaicism or somatic mutation may cause unilateral genetic disease presentation.

ShRNA Knockdown of Guanylate Cyclase 2e or Cyclic Nucleotide Gated Channel Alpha 1 Increases Photoreceptor Survival in a CGMP Phosphodiesterase Mouse Model of Retinitis Pigmentosa

Journal of Cellular and Molecular Medicine. Aug, 2011  |  Pubmed ID: 20950332

In vertebrate rods, dark and light conditions produce changes in guanosine 3',5'-cyclic monophosphate (cGMP) and calcium (Ca(2+) ) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP-gated calcium sodium channels (CNG) to increase Na(+) and Ca(2+) influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na(+) and Ca(2+) levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca(2+) levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca(2+) to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b(H620Q) photoreceptors prior to degeneration. Gucy2e- or Cnga1-shRNA lentiviral-mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b(H620Q) mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.

HYPERAUTOFLUORESCENT RING IN AUTOIMMUNE RETINOPATHY

Retina (Philadelphia, Pa.). Jan, 2012  |  Pubmed ID: 22218149

PURPOSE: To report the presence of a hyperautofluorescent ring and corresponding spectral-domain optical coherence tomography (SD-OCT) features seen in patients with autoimmune retinopathy. METHODS: All eyes were evaluated by funduscopic examination, full-field electroretinography, fundus autofluorescence, and SD-OCT. Further confirmation of the diagnosis was obtained with immunoblot and immunohistochemistry testing of the patient's serum. Humphrey visual fields and microperimetry were also performed. RESULTS: Funduscopic examination showed atrophic retinal pigment epithelium (RPE) associated with retinal artery narrowing but without pigment deposits. The scotopic and photopic full-field electroretinograms were nondetectable in three patients and showed a cone-rod pattern of dysfunction in one patient. Fundus autofluorescence revealed a hyperautofluorescent ring in the parafoveal region, and the corresponding SD-OCT demonstrated loss of the photoreceptor inner segment-outer segment junction with thinning of the outer nuclear layer from the region of the hyperautofluorescent ring toward the retinal periphery. The retinal layers were generally intact within the hyperautofluorescent ring, although the inner segment-outer segment junction was disrupted, and the outer nuclear layer and photoreceptor outer segment layer were thinned. CONCLUSION: This case series revealed the structure of the hyperautofluorescent ring in autoimmune retinopathy using SD-OCT. Fundus autofluorescence and SD-OCT may aid in the diagnosis of autoimmune retinopathy and may serve as a tool to monitor its progression.

Mice with a D190N Mutation in the Gene Encoding Rhodopsin: a Model for Human Autosomal Dominant Retinitis Pigmentosa

Molecular Medicine (Cambridge, Mass.). Jan, 2012  |  Pubmed ID: 22252712

Rhodopsin is the G protein-coupled receptor in charge of initiating signal transduction in rod photoreceptor cells upon the arrival of the photon. D190N, a missense mutation in rhodopsin, causes autosomal dominant retinitis pigmentosa (adRP) in humans. Affected patients present hyperfluorescent retinal rings and progressive rod photoreceptor degeneration. Studies in humans cannot reveal the molecular processes causing the earliest stages of the condition, thus necessitating the creation of an appropriate animal model. A knock-in mouse model with the D190N mutation was engineered to study the pathogenesis of the disease. Electrophysiological and histological findings in the mouse were similar to those observed in human patients, and the hyperfluorescence pattern was analogous to that seen in humans, confirming that the D190N mouse is an accurate model for the study of adRP.

Infrared Imaging and Optical Coherence Tomography Reveal Early-Stage Astrocytic Hamartomas Not Detectable by Fundoscopy

American Journal of Ophthalmology. Feb, 2012  |  Pubmed ID: 22310082

PURPOSE: To describe and correlate the features of astrocytic hamartomas using multimodal imaging. DESIGN: Prospective, noncomparative, observational case series. METHODS: This was a single-center study of 4 patients (8 eyes) with tuberous sclerosis complex. A complete ophthalmologic examination, fundus photography, fundus autofluorescence (FAF), infrared imaging, and spectral-domain optical coherence tomography (SD-OCT) were performed for each patient. Images from each modality were analyzed and compared. RESULTS: In 2 patients, infrared imaging and SD-OCT detected occult retinal astrocytic hamartomas that were not observed on clinical examination or color fundus photography. FAF demonstrated the greatest contrast between lesions and surrounding retina but failed to identify 1 occult lesion that was detected with infrared imaging and SD-OCT. SD-OCT revealed lesions arising from the retinal nerve fiber layer with overlying vitreous adhesions, hyperreflective dots, and optically empty spaces at all depths of the tumor. Hamartomas were hyporeflective on infrared imaging and hypoautofluorescent on FAF. FAF of some lesions demonstrated hyperautofluorescent spots. CONCLUSIONS: Infrared imaging and SD-OCT aid in the detection of astrocytic hamartomas that are not visible on clinical examination or color fundus photography. SD-OCT enhances visualization of structural details. FAF is a useful adjunctive test to obtain greater contrast between lesions and surrounding retina. The ability to monitor structural changes over time in astrocytic hamartomas using SD-OCT may be beneficial for monitoring the success of systemic chemotherapy in the treatment of various tuberous sclerosis tumors.

Familial Discordance in Stargardt Disease

Molecular Vision. 2012  |  Pubmed ID: 22312191

To report genetic and phenotypic discordance across two generations of a family with autosomal recessive Stargardt disease (STGD1) and to compare pathogenicities of the G1961E and A1038V alleles of the ATP-binding cassette transporter, subfamily A, member 4 (ABCA4) gene.

Effect of the ILE86TER Mutation in the γ Subunit of CGMP Phosphodiesterase (PDE6) on Rod Photoreceptor Signaling

Cellular Signalling. Jan, 2012  |  Pubmed ID: 21920434

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100μM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.

Autofluorescence Imaging and Spectral-domain Optical Coherence Tomography in Incomplete Congenital Stationary Night Blindness and Comparison with Retinitis Pigmentosa

American Journal of Ophthalmology. Jan, 2012  |  Pubmed ID: 21920492

To test the hypothesis that the evaluation of retinal structure can have diagnostic value in differentiating between incomplete congenital stationary night blindness (CSNB2) and retinitis pigmentosa (RP). To compare retinal thickness differences between patients with CSNB2 and myopic controls.

Disruption in Bruch Membrane in Patients with Stargardt Disease

Ophthalmic Genetics. Mar, 2012  |  Pubmed ID: 22060670

Purpose: To describe the spectral domain-optical coherence tomography (SD-OCT) findings of two patients with complete defects in the retinal pigment epithelium (RPE) with disruptions in Bruch membrane in Stargardt disease (STGD1). Methods: Two patients with STGD1 were referred to our clinic for further evaluation. Fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT), electroretinography (ERG) and Microperimetry (MP-1) were performed to assess the retinal anatomy and function. Screening for mutations in the ABCA4 gene was carried out and detected mutations were confirmed by direct sequencing. Results: Both patients had bilateral macular geographic atrophy (GA) and yellowish subretinal pisciform flecks and mutations were detected in the ABCA4 gene by chip screening. SD-OCT revealed marked atrophy of the retina in the central macula, with focal defects in the RPE with disruptions in Bruch membrane and herniation of the retina through the defect in three of four eyes. Conclusion: This case report highlights the necessity for a detailed ophthalmic examination including SD-OCT of patients with STGD1.

Structural and Functional Changes Associated with Normal and Abnormal Fundus Autofluorescence in Patients with Retinitis Pigmentosa

Retina (Philadelphia, Pa.). Feb, 2012  |  Pubmed ID: 21909055

To analyze the structure and visual function of regions bordering the hyperautofluorescent ring/arcs in retinitis pigmentosa.