WDR34, a candidate gene for non-syndromic rod-cone dystrophy

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.Doctoral funding from the Ministère de l'Enseignement Supérieur et de la Recherche; Europe exchange 2018 Erasmus; European Reintegration Grant, Grant/Award Number: PERG04-GA-2008-231125; Fondation de France-Berthe Fouassier; Foundation Fighting Blindness, Grant/Award Number: Grant # CD-CL-0808-0466-CHNO CIC503 recogn; Foundation Voir et Entendre; French Agence Nationale de la Recherche, Grant/Award Numbers: IHU FOReSIGHT: ANR-18-IAHU-0001, LIFESENSES: ANR-10-LABX-65; National Eye Institute [R01EY012910 (EAP), R01EY026904 (KMB/EAP) and P30EY014104 (MEEI core support)], the Foundation Fightin

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme (ANR-11-IDEX-0004-0), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency

International audienceMutations in GPR179 lead to autosomal recessive complete congenital stationary night blindness (cCSNB). This condition represents a signal transmission defect from the photoreceptors to the ON-bipolar cells. To confirm the phenotype, better understand the pathogenic mechanism in vivo, and provide a model for therapeutic approaches, a Gpr179 knock-out mouse model was genetically and functionally characterized. We confirmed that the insertion of a neo/lac Z cassette in intron 1 of Gpr179 disrupts the same gene. Spectral domain optical coherence tomography reveals no obvious retinal structure abnormalities. Gpr179 knock-out mice exhibit a so-called no-b-wave (nob) phenotype with severely reduced b-wave amplitudes in the electroretinogram. Optomotor tests reveal decreased optomotor responses under scotopic conditions. Consistent with the genetic disruption of Gpr179, GPR179 is absent at the dendritic tips of ON-bipolar cells. While proteins of the same signal transmission cascade (GRM6, LRIT3, and TRPM1) are correctly localized, other proteins (RGS7, RGS11, and GNB5) known to regulate GRM6 are absent at the dendritic tips of ON-bipolar cells. These results add a new model of cCSNB, which is important to better understand the role of GPR179, its implication in patients with cCSNB, and its use for the development of therapies

LRIT3 Differentially Affects Connectivity and Synaptic Transmission of Cones to ON- and OFF-Bipolar Cells

International audiencePurpose: Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). Using a cCSNB mouse model lacking Lrit3 (nob6), we recently have shown that LRIT3 has a role in the correct localization of TRPM1 (transient receptor potential melastatin 1) to the dendritic tips of ON-bipolar cells (BCs), contacting both rod and cone photoreceptors. Furthermore, postsynaptic clustering of other mGluR6 cascade components is selectively eliminated at the dendritic tips of cone ON-BCs. The purpose of this study was to further define the role of LRIT3 in structural and functional organization of cone synapses.Methods: Exhaustive electroretinogram analysis was performed in a patient with LRIT3 mutations. Multielectrode array recordings were performed at the level of retinal ganglion cells in nob6 mice. Targeting of GluR1 and GluR5 at the dendritic tips of OFF-BCs in nob6 retinas was assessed by immunostaining and confocal microscopy. The ultrastructure of photoreceptor synapses was evaluated by electron microscopy in nob6 mice.Results: The patient with LRIT3 mutations had a selective ON-BC dysfunction with relatively preserved OFF-BC responses. In nob6 mice, complete lack of ON-pathway function with robust, yet altered signaling processing in OFF-pathways was detected. Consistent with these observations, molecules essential for the OFF-BC signaling were normally targeted to the synapse. Finally, synaptic contacts made by ON-BC but not OFF-BC neurons with the cone pedicles were disorganized without ultrastructural alterations in cone terminals, horizontal cell processes, or synaptic ribbons.Conclusions: These results suggest that LRIT3 is likely involved in coordination of the transsynaptic communication between cones and ON-BCs during synapse formation and function

Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

<div><h3>Background</h3><p>In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids.</p> <h3>Methodology</h3><p>To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP₃) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP₃ production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells.</p> <h3>Results</h3><p>Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP₃ production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP₃ production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin.</p> <h3>Conclusion</h3><p>Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.</p> </div

EC50s of insulin, insulin analogues and IGF1 for PIP₃ production in MCF-7 and MDA-MB231 cells.

<p>Results are the means ± S.E.M. of 5 to 6 independent experiments.</p>*, **<p>, p<0.05 or p<0.01 respectively, when compared to insulin.</p

EC50s of insulin, IGF1, glargine and glargine metabolites towards IR homodimers and IR/IGF1R hybrids.

<p>Results are the means ± S.E.M. of 4 to 6 independent experiments.</p>*, **<p>, p<0.05 or p<0.01 respectively, when compared to insulin.</p

Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB).

Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade

Effect of insulin, glargine and IGF1 on PIP₃ production in intact living cells.

<p>Activation of tyrosine kinase receptors by their ligands stimulates the activity of PI-3 kinase, leading to increased phosphorylation of phosphatidyl-inositol 2 phosphate (PIP₂) into phosphatidyl-inositol 3 phosphate (PIP₃) and subsequent recruitment of Akt to the plasma membrane through its pleckstrin homology (PH) domain. To monitor the production of PIP₃ induced by receptor activation, cells were co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to the membrane localization sequence of neuromodulin. Cells were pre-incubated for 10 min with coelenterazine and then stimulated with increasing ligand concentrations. (A) Typical experiment showing real-time insulin or IGF1 effects on PIP₃ production in MCF-7 cells. (B) Dose-dependent effect of insulin, glargine and IGF1 on PIP₃ production in MCF-7 and MDA-MB231 cells. Ligand-induced BRET (BRET above basal at the plateau) was determined for each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 5 to 6 independent experiments. EC50 for insulin, IGF1, glargine and other insulin analogues are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t002" target="_blank">Table 2</a>. (C) Left panel: Receptors were partially purified from MDA-MB231 and MCF-7 cells by WGL chromatography. WGL eluates (12 µg of protein) were submitted to electrophoresis and western-blotting. IR and IGF1R expression levels were evaluated by immunoblotting (IB) using anti-IR (C-19) and anti-IGF1R (C-20) antibodies. Right panel: After normalization of the eluates for IR content, immunoprecipitation (IP) was performed using anti-IR antibody (CT1) and hybrid receptors were detected using anti-IGF1R antibody. Blots were then stripped and reprobed with anti-IR antibody. Results are representative of 6 immunoprecipitation experiments performed on three independent batches of receptor preparations (**, p<0.01).</p

Effect of insulin, glargine and IGF1 on IR homodimers and IR/IGF1R hybrid receptors.

<p>HEK-293 cells were co-transfected with IRA-Luc/IRA-YFP, IRB-Luc/IRB-YFP, IRA-Luc/IGF1R-YFP or IRB-Luc/IGF1R-YFP. Receptors were partially purified by WGL chromatography. Ligand binding induces a conformational change that brings the two ß-subunits in close proximity, resulting in an energy transfer between the luciferase and YFP. BRET assays were performed in the presence of increasing ligand concentrations. (A) Typical experiments showing basal and insulin or IGF1 stimulated BRET signals. (B) Dose-response curves showing the effect of insulin, IGF1 and glargine on IR homodimers and IR/IGF1R hybrids. Ligand-induced BRET (BRET above basal) was determined at each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 4 to 6 independent experiments. EC50 for insulin, IGF1, glargine and other insulin analogues are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t001" target="_blank">Table 1</a>.</p