Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179

The rd1 mouse with a mutation in the Pde6b gene was the first strain of mice identified with a retinal degeneration. However, AAV-mediated gene supplementation of rd1 mice only results in structural preservation of photoreceptors, and restoration of the photoreceptor-mediated a-wave, but not in restoration of the bipolar cell-mediated b-wave. Here we show that a mutation in Gpr179 prevents the full restoration of vision in rd1 mice. Backcrossing rd1 with C57BL6 mice reveals the complete lack of b-wave in a subset of mice, consistent with an autosomal recessive Mendelian inheritance pattern. We identify a mutation in the Gpr179 gene, which encodes for a G-protein coupled receptor localized to the dendrites of ON-bipolar cells. Gene replacement in rd1 mice that are devoid of the mutation in Gpr179 successfully restores the function of both photoreceptors and bipolar cells, which is maintained for up to 13 months. Our discovery may explain the failure of previous gene therapy attempts in rd1 mice, and we propose that Grp179 mutation status should be taken into account in future studies involving rd1 mice

Dark-adapted red flash ERGs in healthy adults

Purpose: The x-wave of the dark-adapted (DA) ERG to a red flash reflects DA cone function. This exploratory study of healthy adults aimed to investigate changes in the DA red ERG with flash strength and during dark adaptation to optimise visualisation and therefore quantification of the x-wave. Methods: The effect of altering red flash strength was investigated in four subjects by recording ERGs after 20 minutes dark adaptation to red flashes (0.2–2.0 cd s m-2) using skin electrodes and natural pupils. The effect of dark adaptation duration was investigated in 16 subjects during 20 minutes in the dark, by recording DA 1.5 red ERGs at 1, 2, 3, 4, 5, 10, 15 and 20 minutes. Results: For a dark adaption period of 20 minutes, the x-wave was more clearly visualised to weaker (< 0.6 cd s m-2) red flash strengths: to stronger flashes it became obscured by the b-wave. For red flashes of 1.5 cd s m-2, the x-wave was most prominent in ERGs recorded after 1–5 minutes of dark adaptation: with longer dark-adaptation, it was subsumed into the b-wave’s rising edge. Conclusions: This small study suggests that x-wave visibility in healthy subjects after 20 minutes dark adaptation is improved by using flashes weaker than around 0.6 cd s m-2; for flash strengths of 1.5 cd s m-2, x-wave visibility is enhanced by recording after only around 5 minutes of dark adaptation. No evidence was found that interim red flash ERGs affecting the dark-adapted state of the normal retina

In situ regeneration of retinal pigment epithelium by gene transfer of E2F2: a potential strategy for treatment of macular degenerations

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration

Identification of the Photoreceptor Transcriptional Co-Repressor SAMD11 as Novel Cause of Autosomal Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP), the most frequent form of inherited retinal dystrophy is characterized by progressive photoreceptor degeneration. Many genes have been implicated in RP development, but several others remain to be identified. Using a combination of homozygosity mapping, whole-exome and targeted next-generation sequencing, we found a novel homozygous nonsense mutation in SAMD11 in five individuals diagnosed with adult-onset RP from two unrelated consanguineous Spanish families. SAMD11 is ortholog to the mouse major retinal SAM domain (mr-s) protein that is implicated in CRX-mediated transcriptional regulation in the retina. Accordingly, protein-protein network analysis revealed a significant interaction of SAMD11 with CRX. Immunoblotting analysis confirmed strong expression of SAMD11 in human retina. Immunolocalization studies revealed SAMD11 was detected in the three nuclear layers of the human retina and interestingly differential expression between cone and rod photoreceptors was observed. Our study strongly implicates SAMD11 as novel cause of RP playing an important role in the pathogenesis of human degeneration of photoreceptors.This work was supported by several grants from the Spanish Centre for Biomedical Network Research on Rare Diseases (CIBERER)(06/07/0036), Instituto de Salud Carlos III (ISCIII, Spanish Ministry of Health)/FEDER, including FIS (PI013/00226) and RETICS (RD09/0076/00101 and RD12/0034/0010), Ministry of Economy and Competitiveness (MINECO), including FEDER (BFU2012-36845), and BIO2011-27069, Conselleria de Educació of the Valencia Community (PROMETEOII/2014/025), Spanish National Organization of the Blind (ONCE) and the Spanish Fighting Blindness Foundation (FUNDALUCE). M.C. was sponsored by the Miguel Servet Program for Researchers in the Spanish National Health Service (CP12/03256) and RSA by Sara Borrel Postdoctoral Program (CD12/00676), both from the ISCIII/FEDER. A.A-F. was sponsored by CIBERER, RPC is supported by Fundación Conchita Rábago (FCR), L.C is sponsored by RETICS (RD12/0034/0010) from ISCIII and L.d.S. was supported by CAPES Foundation, Ministry of Education of Brazil

The morphology of human rod ERGs obtained by silent substitution stimulation

YesPurpose To record transient ERGs from the lightadapted human retina using silent substitution stimuli which selectively reflect the activity of rod photoreceptors. We aim to describe the morphology of these waveforms and examine how they are affected by the use of less selective stimuli and by retinal pathology. Methods Rod-isolating stimuli with square-wave temporal profiles (250/250 ms onset/offset) were presented using a 4 primary LED ganzfeld stimulator. Experiment 1: ERGs were recorded using a rodisolating stimulus (63 ph Td, rod contrast, Crod = 0.25) from a group (n = 20) of normal trichromatic observers. Experiment 2: Rod ERGs were recorded from a group (n = 5) using a rodisolating stimulus (Crod = 0.25) which varied in retinal illuminance from 40 to 10,000 ph Td. Experiment 3: ERGs were elicited using 2 kinds of nonisolating stimuli; (1) broadband and (2) rod-isolating stimuli which contained varying degrees of L- and M-cone excitation. Experiment 4: Rod ERGs were recorded from two patient groups with rod monochromacy (n = 3) and CSNB (type 1; n = 2). Results The rod-isolated ERGs elicited from normal subjects had a waveform with a positive onset component followed by a negative offset. Response amplitude was maximal at retinal illuminances\100 ph Td and was virtually abolished at 400 ph Td. The use of non-selective stimuli altered the ERG waveform eliciting more photopic-like ERG responses. Rod ERGs recorded from rod monochromats had similar features to those recorded from normal trichromats, in contrast to those recorded from participants with CSNB which had an electronegative appearance. Conclusions Our results demonstrate that ERGs elicited by silent substitution stimuli can selectively reflect the operation of rod photoreceptors in the normal, light-adapted human retina.Deutsche Forschungsgemeinschaft (DFG) (KR1317/13-1) and Bundesministerium für Bildung und Forschung (BMBF) (01DN14009) provided financial support for JK

Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases

Zebrafish: a vertebrate tool for studying basal body biogenesis, structure, and function.

Understanding the role of basal bodies (BBs) during development and disease has been largely overshadowed by research into the function of the cilium. Although these two organelles are closely associated, they have specific roles to complete for successful cellular development. Appropriate development and function of the BB are fundamental for cilia function. Indeed, there are a growing number of human genetic diseases affecting ciliary development, known collectively as the ciliopathies. Accumulating evidence suggests that BBs establish cell polarity, direct ciliogenesis, and provide docking sites for proteins required within the ciliary axoneme. Major contributions to our knowledge of BB structure and function have been provided by studies in flagellated or ciliated unicellular eukaryotic organisms, specifically Tetrahymena and Chlamydomonas. Reproducing these and other findings in vertebrates has required animal in vivo models. Zebrafish have fast become one of the primary organisms of choice for modeling vertebrate functional genetics. Rapid ex-utero development, proficient egg laying, ease of genetic manipulation, and affordability make zebrafish an attractive vertebrate research tool. Furthermore, zebrafish share over 80 % of disease causing genes with humans. In this article, we discuss the merits of using zebrafish to study BB functional genetics, review current knowledge of zebrafish BB ultrastructure and mechanisms of function, and consider the outlook for future zebrafish-based BB studies