J. Tosi

Cellular Origin of Fundus Autofluorescence in Patients and Mice with Defective NR2E3 Gene

Aim: To characterise new clinical features in a family with enhanced S-cone syndrome (ESCS) and investigate the pathogenesis of these clinical features in the homozygous Nr2e3rd7 (rd7) mutant mice. Methods: Four patients from an affected family were included for genotypic and phenotypic study. Eye tissues from rd7 mice were used to detect a possible relationship between macrophages and autofluorescent material by immunohistochemistry (IHC) staining. Results: Homozygous mutation in R311Q in NR2E3 was detected in this family. Colour photographs revealed that white dots do not correlate to hyperautofluorescent spots seen in autofluorescence imaging of the macula. OCT showed rosette-like lesions similar to those found in rd7 mice histology sections. From IHC analysis, we observed that F4/80 (a pan macrophage marker) and autofluorescence were colocalised to the same cells within the retina rosettes. Conclusions: The retinal structure of a young ESCS patient with homozygous R311Q mutation in the NR2E3 gene is similar to that seen in the rd7 mice. The macrophages were found to contain autofluorescent materials in the retinal rosettes of rd7 mice. These data are consistent with macrophage infiltration contributing to the hyperautofluorescent spots found in our patients.

A Novel Mutation and Phenotypes in Phosphodiesterase 6 Deficiency

PURPOSE 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. DESIGN Clinical and molecular studies on a retrospective case series. METHODS We screened 40 unrelated RP patients with an autosomal recessive RP microarray. Individuals with RP caused by PDE6 deficiency underwent genetic segregation and phenotype analysis. RESULTS A disease-associated allele was identified in 32% of patients. Two probands (5%) had PDE6 mutations. The first proband was a compound heterozygote for known R102C and N216S alleles in PDE6A (MIM#180071). Pedigree analysis determined that the N216S variant was benign and direct sequencing discovered a novel, S303C allele. The second proband had a homozygous D600N mutation in the PDE6B gene (MIM#180072). Visual acuities of PDE6-deficient patients ranged from 20/40 to 20/200. Clinical studies showed unusual vitreomacular traction, cystoid macular edema, macular atrophy, and ring hyperfluorescence in PDE6-deficient patients. Such extensive vitreoretinal degeneration is not characteristic of photoreceptor-specific enzyme deficiencies. CONCLUSION High-throughput deoxyribonucleic acid microarray chips can be used in combination with clinical imaging to precisely characterize patients with RP. Identifying the precise mutation in RP may become the standard of care as gene therapy emerges.

Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.

Mutations in Pde6b lead to high levels of signaling molecules cyclic guanosine monophosphate (cGMP) and Ca2+, 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 Ca2+ 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.

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.

In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca2+) 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 Ca2+ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na+ and Ca2+ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca2+ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca2+ 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 Pde6bH620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6bH620Q 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.

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

Purpose: To phenotype two siblings with autosomal recessive early-onset retinal dystrophy due to CRB1 mutations. Methods: Autofluorescence (AF) imaging, high resolution optical coherence tomography (OCT), and full-field electroretinography (ERG) were performed. The results of DNA sequencing from polymerase chain reaction (PCR) products of the CRB1 gene were obtained from hospital records. Results: Two siblings, 14 years old and 17 years old, were compound heterozygotes for 749 del Ser and C948Y mutations in the gene encoding CRB1. AF imaging documented the preservation of retinal pigment epithelium (RPE) along the arterioles. High-resolution OCT showed abnormally thick retinae with increased lamination. Conclusion: Leber congenital amaurosis caused by CRB1 is a unique form of early-onset retinal dystrophy because it spares the para-arteriolar RPE and causes abnormal retinal lamination with thickening. These findings, detectable with AF imaging and high-resolution OCT, can be combined with electrophysiology and genetic testing to molecularly classify retinal degenerations efficiently.

Cellular and Molecular Origin of Circumpapillary Dysgenesis of the Pigment Epithelium

PURPOSE 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. DESIGN Observational case series and experimental study. PARTICIPANTS Three female patients from an affected family were included for phenotypic study. Mice and human tissues were used for biochemistry and immunohistochemistry studies. METHODS We performed genetic analyses and longitudinal clinical, imaging, and electrophysiologic studies in a 3-generation family. Western blotting and immunohistochemistry were used to detect TEAD1 expression in mice and human retinal tissues. MAIN OUTCOME MEASURES Autofluorescence and optical coherence tomography (OCT) imaging were compared and reviewed from 3 patients. TEAD1 expression was compared in different tissues from mice and human samples. RESULTS A point mutation at T1261 in TEAD1 was detected in the mother. Autofluorescence and OCT imaging studies revealed choroid is involved earlier than retinal pigment epithelium (RPE). From immunoblot analysis, we discovered that TEAD1 and its cofactors YAP65 and FOXA2 are expressed in the choroid. Immunohistochemical analysis on frozen sections of mouse retina supports immunoblot results. CONCLUSIONS The primary cellular origin of circumpapillary dysgenesis of the pigment epithelium is within the choroid instead of the pigment epithelium. The loss of the RPE and photoreceptors in later stages of the disease is a secondary consequence of choroidal degeneration. Studies of the downstream targets of TEAD1 in choroidal cells will provide promising new research opportunities for the development of treatments for choroidal diseases. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Vigabatrin-Induced Retinal Toxicity Is Partially Mediated by Signaling in Rod and Cone Photoreceptors

Vigabatrin (VGB) is a commonly prescribed antiepileptic drug designed to inhibit GABA-transaminase, effectively halting seizures. Unfortunately, VGB treatment is also associated with the highest frequencies of peripheral visual field constriction of any of the antiepileptic drugs and the mechanisms that lead to these visual field defects are uncertain. Recent studies have demonstrated light exposure exacerbates vigabatrin-induced retinal toxicity. We further assessed this relationship by examining the effects of vigabatrin treatment on the retinal structures of mice with genetically altered photoreception. In keeping with previous studies, we detected increased toxicity in mice exposed to continuous light. To study whether cone or rod photoreceptor function was involved in the pathway to toxicity, we tested mice with mutations in the cone-specific Gnat2 or rod-specific Pde6g genes, and found the mutations significantly reduced VGB toxicity. Our results confirm light is a significant enhancer of vigabatrin toxicity and that a portion of this is mediated, directly or indirectly, by phototransduction signaling in rod and cone photoreceptors.

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

PurposeWe report a noninvasive method for the monitoring of retinal ganglion cell (RGC) survival in live mice utilizing standard fluorescence microscopy.ProceduresTransgenic mice expressing cyan fluorescent protein (CFP) under the regulation of an RGC-specific promoter Thy1 were used in this study.ResultsWe established that Thy1-CFP expression is a quantitative reflection of the number of surviving RGCs, the fluorescence emission is stable for at least a year and that the loss of fluorescence correlates directly to glaucomatous damage. In high pressure glaucoma model, the peripheral retina is preferentially affected.ConclusionsOur live-imaging technique allows for the longitudinal assessment of RGC survival from the same animal. Noninvasive monitoring of neuronal cell death and survival is a powerful technique that would allow investigators to validate new potential glaucoma therapy based on neuroprotection.

Function of the asparagine 74 residue of the inhibitory γ-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo.

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.

Benign panretinal uniform radial linear-shaped flecks

Our case illustrates a new presentation of benign retina flecks with line-shaped and panretinal, uniform and radial distribution. Compared with previous reports of fleck retina disorders, our case does not show increased autofluorescence deposits, no delay in dark adaptation, neither significant macular nor nasal disc involvement.1–5 A 51-year-old woman with consanguinous parents was referred for evaluation of bilateral retinal lesions. On examination, best-corrected visual acuity was 6/7.5 with moderate hyperopic correction in both eyes. Visual fields were normal. The anterior segments were unremarkable. Fundus examination revealed multiple yellow-creamy discrete round and mostly linear-shaped flecks scattered throughout the retina in both eyes with relative sparing of the fovea. The flecks distributed in a radial pattern centred around the posterior pole (Fig. 1) and were located well posterior to the retinal vasculature. The discs, maculae and retinal vessels appeared unremarkable. Fundus autofluorescence imaging (cSLO, Heidelberg Retina Angiograph 2; Heidelberg Engineering, Dossenheim, Germany) did not show hyper- or hypofluorescent flecks (Fig. 2a), but multiple discrete round lesions above the retinal pigment epithelium were apparent in the infrared imaging (Fig. 2b). Corresponding spectral optical coherence tomography (Optivue SD-OCT, Fremont, CA, and Cirrus, Carl Zeiss Meditec Inc., Dublin, CA, USA) revealed that these lesions span across the retinal pigment epithelium complex and photoreceptor inner–outer segment junction layers, without involving the choroid (Figs 3,​,4).4). To assess retinal function, ISCEV (International Society for Clinical Electrophysiology of Vision) standardized full-field electroretinography were performed following 20 min and overnight dark adaptation. The electroretinography traces showed that both scotopic and photopic responses were symmetric and within normal limits (Fig. 5). Figure 1 A wide-angle montage of the right (a) and left (b) fundi demonstrates multiple yellow-cream discrete flecks widely distributed in the equator and far periphery of the retina. A few flecks are centred in the posterior pole. Figure 2 (a) Fundus autofluorescence of the right eye showing absence of hyper- or hypofluorescent flecks; (b) infrared imaging of the right eye showing multiple discrete round lesions in the para-foveal area. Figure 3 Spectral-domain OCT of the right eye (Optivue). (a) Multiple flecks in the posterior pole (yellow and green arrows represent OCT scans on (b) and (c), respectively, and white square represents longitudinal SLO scan on (d); (b) normal appearing macula; ... Figure 4 Spectral-domain OCT of the right eye (Cirrus Zeiss). (a) Multiple flecks in the posterior pole (yellow and green arrows represent OCT scans on (b) and (c), respectively, and white square represents longitudinal scan on (d); (b) normal appearing macula; ... Figure 5 Representative photopic and scotopic electroretinogram recordings compared with normal control after 20 min in the right eye and overnight dark adaptation in the left eye. Full-field electroretinographies were performed with Ganzfeld stimulation on this ... In contrast to previous reports,1–3 the retinal lesions in our patient appeared to be significantly smaller, more of a linear shape(s) and uniform in size(s). These lesions were distributed ubiquitously in the equator and mid and far periphery of the fundus. The centrally located flecks in the posterior pole appeared sparse, small, round and dot-like. Unfortunately, the patient’s family members were not available for eye examination. In contrast to the classic findings reported by Audo et al.,4 where the retinal flecks demonstrated increased autofluorescence, the fundus imaging of our patient did not show any specks of abnormal autofluorescence. Infrared imaging (Fig. 2b) and optical coherence tomography (Figs 3,​,4)4) revealed numerous subretinal dot-like lesions, which correspond to those observed in funduscopy. Different types of flecked retinal disorders such as dominant radial drusen, fleck retina of Kandori,1 fundus albipunctatus, retinitis punctata albescens, fundus flavimaculatus and vitamin A deficiency5 may be considered as differential diagnosis. However, the lack of drusen on the nasal side of the optic disc, extension of flecks to peripheral retina, absence of high-density autofluorescent deposits on scanning laser ophthalmoscopy imaging and presence of intact EFEMP1 gene sequence made the diagnosis of dominant radial drusen unlikely. Moreover, normal scotopic electroretinography response excludes fundus albipunctatus. In addition to clinical assessment, electrophysiology testing was essential to exclude fundus albipunctatus and autofluorescent imaging can be a helpful tool to characterize hyper-autofluorescent deposits observed in different variants of benign fleck retina. The normal electrophysiology and visual field testing, in addition to good visual acuity and lack of symptoms justify the condition as benign. Hence, our case represents a different presentation of benign fleck retina,4 with linear-shaped flecks and a panretinal, uniform and radial distribution.