Publications by authors named "Thierry Leveillard"

76 Publications

Mutated Coding for a Mitochondrial Protein, MITOK Is a Candidate Gene Defect for Autosomal Recessive Rod-Cone Dystrophy.

Int J Mol Sci 2021 Jul 23;22(15). Epub 2021 Jul 23.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 75012 Paris, France.

The purpose of this work was to identify the gene defect underlying a relatively mild rod-cone dystrophy (RCD), lacking disease-causing variants in known genes implicated in inherited retinal disorders (IRD), and provide transcriptomic and immunolocalization data to highlight the best candidate. The DNA of the female patient originating from a consanguineous family revealed no large duplication or deletion, but several large homozygous regions. In one of these, a homozygous frameshift variant, c.244_246delins17 p.(Trp82Valfs*4); predicted to lead to a nonfunctional protein, was identified in . encodes the mitochondrial coiled-coil domain containing 51 protein, also called MITOK. MITOK ablation causes mitochondrial dysfunction. Here we show for the first time that CCDC51/MITOK localizes in the retina and more specifically in the inner segments of the photoreceptors, well known to contain mitochondria. Mitochondrial proteins have previously been implicated in IRD, although usually in association with syndromic disease, unlike our present case. Together, our findings add another ultra-rare mutation implicated in non-syndromic IRD, whose pathogenic mechanism in the retina needs to be further elucidated.
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http://dx.doi.org/10.3390/ijms22157875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346125PMC
July 2021

Assessing Photoreceptor Status in Retinal Dystrophies: From High-Resolution Imaging to Functional Vision.

Am J Ophthalmol 2021 Oct 14;230:12-47. Epub 2021 May 14.

From the Department of Ophthalmology, University of Pittsburgh School of Medicine and Medical Center, Pittsburgh, Pennsylvania, USA (J.-A.S.); Sorbonne Université, Paris, France (J.-A.S.); Quinze Vingts National Ophthalmology Hospital, Paris, France (J.A.S., K.G., S.M.-S., M.P., I.A., A.-E.C.-R., L.A.); StreetLab - Institut de la Vision, Paris, France (C.P., C.A., K.B., E.G.); and Institut de la Vision, Sorbonne Université, Inserm, CNRS Paris, France (M.P., I.A., T.L., C.Z., S.P., D.D., K.M.).

Purpose: To describe the value of integrating phenotype/genotype data, disease staging, and evaluation of functional vision in patient-centered management of retinal dystrophies.

Methods: (1) Cross-sectional structure-function and retrospective longitudinal studies to assess the correlations between standard fundus autofluorescence (FAF), optical coherence tomography, visual acuity (VA), and perimetry (visual field [VF]) examinations to evaluate photoreceptor functional loss in a cohort of patients with rod-cone dystrophy (RCD); (2) flood-illumination adaptive optics (FIAO) imaging focusing on photoreceptor misalignment and orientation of outer segments; and (3) evaluation of the impact of visual impairment in daily life activities, based on functional (visual and mobility) vision assessment in a naturalistic environment in visually impaired subjects with RCD and subjects treated with Luxturna for RPE65-related Leber congenital amaurosis before and after therapy.

Results: The results of the cross-sectional transversal study showed that (1) VA and macular sensitivity were weakly correlated with the structural variables; and (2) functional impairment (VF) was correlated with reduction of anatomical markers of photoreceptor structure and increased width of autofluorescent ring. The dimensions of the ring of increased FAF evolved faster. Other criteria that differed among groups were the lengths of the ellipsoid zone, the external limiting membrane, and the foveal thickness. FIAO revealed a variety of phenotypes: paradoxical visibility of foveal cones; heterogeneous brightness of cones; dim, inner segment-like, and RPE-like mosaic. Directional illumination by varying orientation of incident light (Stiles-Crawford effect) and the amount of side illumination (gaze-dependent imaging) affected photoreceptor visibility. Mobility assessment under different lighting conditions showed correlation with VF, VA, contrast sensitivity (CS), and dark adaptation, with different predictive values depending on mobility study paradigms and illumination level. At high illumination level (235 lux), VF was a predictor for all mobility performance models. Under low illumination (1 and 2 lux), VF was the most significant predictor of mobility performance variables, while CS best explained the number of collisions and segments. In subjects treated with Luxturna, a very favorable impact on travel speed and reduction in the number of collisions, especially at low luminance, was observable 6 months following injection, in both children and adults.

Conclusions: Our results suggest the benefit of development and implementation of quantitative and reproducible tools to evaluate the status of photoreceptors and the impact of both visual impairment and novel therapies in real-life conditions. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.
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http://dx.doi.org/10.1016/j.ajo.2021.04.013DOI Listing
October 2021

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions.

J Vis Exp 2021 03 20(169). Epub 2021 Mar 20.

Department of Genetics - Sorbonne Université, INSERM, CNRS, Institut de la Vision;

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration. This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa. Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization. This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.
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http://dx.doi.org/10.3791/61998DOI Listing
March 2021

The role of RdCVFL in a mathematical model of photoreceptor interactions.

J Theor Biol 2021 07 23;520:110642. Epub 2021 Feb 23.

INSERM, U968, Paris F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris F-75012, France; CNRS, UMR_7210, Paris F-75012, France.

Recent experimental and mathematical work has shown the interdependence of the rod and cone photoreceptors with the retinal pigment epithelium in maintaining sight. Accelerated intake of glucose into the cones via the theoredoxin-like rod-derived cone viability factor (RdCVF) is needed as aerobic glycolysis is the primary source of energy production. Reactive oxidative species (ROS) result from the rod and cone metabolism and recent experimental work has shown that the long form of RdCVF (RdCVFL) helps mitigate the negative effects of ROS. In this work we investigate the role of RdCVFL in maintaining the health of the photoreceptors. The results of our mathematical model show the necessity of RdCVFL and also demonstrate additional stable modes that are present in this system. The sensitivity analysis shows the importance of glucose uptake, nutrient levels, and ROS mitigation in maintaining rod and cone health in light-damaged mouse models. Together, these suggests areas on which to focus treatment in order to prolong the photoreceptors, especially in situations where ROS is a contributing factor to their death such as retinitis pigmentosa.
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http://dx.doi.org/10.1016/j.jtbi.2021.110642DOI Listing
July 2021

A Splice Variant in Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells.

Cells 2021 01 18;10(1). Epub 2021 Jan 18.

Institut de la Vision, CNRS, INSERM, Sorbonne Université, 17 rue Moreau, F-75012 Paris, France.

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.
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http://dx.doi.org/10.3390/cells10010179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831140PMC
January 2021

Insulin inhibits inflammation-induced cone death in retinal detachment.

J Neuroinflammation 2020 Nov 26;17(1):358. Epub 2020 Nov 26.

Institut de la Vision, INSERM, UMR_S 968, CNRS, Sorbonne Université, 17 rue Moreau, F-75012, Paris, France.

Background: Rhegmatogenous retinal detachment (RD) involving the macula is a major cause of visual impairment despite high surgical success rate, mainly because of cone death. RD causes the infiltration of activated immune cells, but it is not clear whether and how infiltrating inflammatory cells contribute to cone cell loss.

Methods: Vitreous samples from patients with RD and from control patients with macular hole were analyzed to characterize the inflammatory response to RD. A mouse model of RD and retinal explants culture were then used to explore the mechanisms leading to cone death.

Results: Analysis of vitreous samples confirms that RD induces a marked inflammatory response with increased cytokine and chemokine expression in humans, which is closely mimicked by experimental murine RD. In this model, we corroborate that myeloid cells and T-lymphocytes contribute to cone loss, as the inhibition of their accumulation by Thrombospondin 1 (TSP1) increased cone survival. Using monocyte/retinal co-cultures and TSP1 treatment in RD, we demonstrate that immune cell infiltration downregulates rod-derived cone viability factor (RdCVF), which physiologically regulates glucose uptake in cones. Insulin and the insulin sensitizers rosiglitazone and metformin prevent in part the RD-induced cone loss in vivo, despite the persistence of inflammation CONCLUSION: Our results describe a new mechanism by which inflammation induces cone death in RD, likely through cone starvation due to the downregulation of RdCVF that could be reversed by insulin. Therapeutic inhibition of inflammation and stimulation of glucose availability in cones by insulin signaling might prevent RD-associated cone death until the RD can be surgically repaired and improve visual outcome after RD.

Trial Registration: ClinicalTrials.gov NCT03318588.
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http://dx.doi.org/10.1186/s12974-020-02039-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694924PMC
November 2020

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

Clin Genet 2021 02 9;99(2):298-302. Epub 2020 Nov 9.

Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

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.
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http://dx.doi.org/10.1111/cge.13872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049445PMC
February 2021

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

Immunity 2020 08;53(2):429-441.e8

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France. Electronic address:

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
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http://dx.doi.org/10.1016/j.immuni.2020.07.021DOI Listing
August 2020

Metabolic and Redox Signaling of the Nucleoredoxin-Like-1 Gene for the Treatment of Genetic Retinal Diseases.

Int J Mol Sci 2020 Feb 27;21(5). Epub 2020 Feb 27.

Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France.

The loss of cone photoreceptor function in retinitis pigmentosa (RP) severely impacts the central and daily vision and quality of life of patients affected by this disease. The loss of cones follows the degeneration of rods, in a manner independent of the causing mutations in numerous genes associated with RP. We have explored this phenomenon and proposed that the loss of rods triggers a reduction in the expression of rod-derived cone viability factor (RdCVF) encoded by the nucleoredoxin-like 1 () gene which interrupts the metabolic and redox signaling between rods and cones. After providing scientific evidence supporting this mechanism, we propose a way to restore this lost signaling and prevent the cone vision loss in animal models of RP. We also explain how we could restore this signaling to prevent cone vision loss in animal models of the disease and how we plan to apply this therapeutic strategy by the administration of both products of encoding the trophic factor RdCVF and the thioredoxin enzyme RdCVFL using an adeno-associated viral vector. We describe in detail all the steps of this translational program, from the design of the drug, its production, biological validation, and analytical and preclinical qualification required for a future clinical trial that would, if successful, provide a treatment for this incurable disease.
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http://dx.doi.org/10.3390/ijms21051625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084304PMC
February 2020

Functional Genomics of the Retina to Elucidate its Construction and Deconstruction.

Int J Mol Sci 2019 Oct 4;20(19). Epub 2019 Oct 4.

Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France.

The retina is the light sensitive part of the eye and nervous tissue that have been used extensively to characterize the function of the central nervous system. The retina has a central position both in fundamental biology and in the physiopathology of neurodegenerative diseases. We address the contribution of functional genomics to the understanding of retinal biology by reviewing key events in their historical perspective as an introduction to major findings that were obtained through the study of the retina using genomics, transcriptomics and proteomics. We illustrate our purpose by showing that most of the genes of interest for retinal development and those involved in inherited retinal degenerations have a restricted expression to the retina and most particularly to photoreceptors cells. We show that the exponential growth of data generated by functional genomics is a future challenge not only in terms of storage but also in terms of accessibility to the scientific community of retinal biologists in the future. Finally, we emphasize on novel perspectives that emerge from the development of redox-proteomics, the new frontier in retinal biology.
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http://dx.doi.org/10.3390/ijms20194922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801968PMC
October 2019

A Mathematical Analysis of Aerobic Glycolysis Triggered by Glucose Uptake in Cones.

Sci Rep 2019 03 11;9(1):4162. Epub 2019 Mar 11.

Institut de la Vision: Department of genetics Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012, Paris, France.

Patients affected by retinitis pigmentosa, an inherited retinal disease, experience a decline in vision due to photoreceptor degeneration leading to irreversible blindness. Rod-derived cone viability factor (RdCVF) is the most promising mutation-independent treatment today. To identify pathologic processes leading to secondary cone photoreceptor dysfunction triggering central vision loss of these patients, we model the stimulation by RdCVF of glucose uptake in cones and glucose metabolism by aerobic glycolysis. We develop a nonlinear system of enzymatic functions and differential equations to mathematically model molecular and cellular interactions in a cone. We use uncertainty and sensitivity analysis to identify processes that have the largest effect on the system and their timeframes. We consider the case of a healthy cone, a cone with low levels of glucose, and a cone with low and no RdCVF. The three key processes identified are metabolism of fructose-1,6-bisphosphate, production of glycerol-3-phosphate and competition that rods exert on cone resources. The first two processes are proportional to the partition of the carbon flux between glycolysis and the pentose phosphate pathway or the Kennedy pathway, respectively. The last process is the rods' competition for glucose, which may explain why rods also provide the RdCVF signal to compensate.
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http://dx.doi.org/10.1038/s41598-019-39901-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411757PMC
March 2019

Is Retinal Metabolic Dysfunction at the Center of the Pathogenesis of Age-related Macular Degeneration?

Int J Mol Sci 2019 Feb 11;20(3). Epub 2019 Feb 11.

. Department of Therapeutics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France.

The retinal pigment epithelium (RPE) forms the outer blood⁻retina barrier and facilitates the transepithelial transport of glucose into the outer retina via GLUT1. Glucose is metabolized in photoreceptors via the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) but also by aerobic glycolysis to generate glycerol for the synthesis of phospholipids for the renewal of their outer segments. Aerobic glycolysis in the photoreceptors also leads to a high rate of production of lactate which is transported out of the subretinal space to the choroidal circulation by the RPE. Lactate taken up by the RPE is converted to pyruvate and metabolized via OXPHOS. Excess lactate in the RPE is transported across the basolateral membrane to the choroid. The uptake of glucose by cone photoreceptor cells is enhanced by rod-derived cone viability factor (RdCVF) secreted by rods and by insulin signaling. Together, the three cells act as symbiotes: the RPE supplies the glucose from the choroidal circulation to the photoreceptors, the rods help the cones, and both produce lactate to feed the RPE. In age-related macular degeneration this delicate ménage à trois is disturbed by the chronic infiltration of inflammatory macrophages. These immune cells also rely on aerobic glycolysis and compete for glucose and produce lactate. We here review the glucose metabolism in the homeostasis of the outer retina and in macrophages and hypothesize what happens when the metabolism of photoreceptors and the RPE is disturbed by chronic inflammation.
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http://dx.doi.org/10.3390/ijms20030762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387069PMC
February 2019

Mechanisms Underlying the Visual Benefit of Cell Transplantation for the Treatment of Retinal Degenerations.

Int J Mol Sci 2019 Jan 28;20(3). Epub 2019 Jan 28.

Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France.

The transplantation of retinal cells has been studied in animals to establish proof of its potential benefit for the treatment of blinding diseases. Photoreceptor precursors have been grafted in animal models of Mendelian-inherited retinal degenerations, and retinal pigmented epithelial cells have been used to restore visual function in animal models of age-related macular degeneration (AMD) and recently in patients. Cell therapy over corrective gene therapy in inherited retinal degeneration can overcome the genetic heterogeneity by providing one treatment for all genetic forms of the diseases. In AMD, the existence of multiple risk alleles precludes a priori the use of corrective gene therapy. Mechanistically, the experiments of photoreceptor precursor transplantation reveal the importance of cytoplasmic material exchange between the grafted cells and the host cells for functional rescue, an unsuspected mechanism and novel concept. For transplantation of retinal pigmented epithelial cells, the mechanisms behind the therapeutic benefit are only partially understood, and clinical trials are ongoing. The fascinating studies that describe the development of methodologies to produce cells to be grafted and demonstrate the functional benefit for vision are reviewed.
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http://dx.doi.org/10.3390/ijms20030557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387096PMC
January 2019

Maintaining Cone Function in Rod-Cone Dystrophies.

Adv Exp Med Biol 2018 ;1074:499-509

Sorbonne Universités, UPMC Univ Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, Paris, France.

Retinal degenerative diseases are a major cause of untreatable blindness due to a loss of photoreceptors. Recent advances in genetics and gene therapy for inherited retinal dystrophies (IRDs) showed that therapeutic gene transfer holds a great promise for vision restoration in people with currently incurable blinding diseases. Due to the huge genetic heterogeneity of IRDs that represents a major obstacle for gene therapy development, alternative therapeutic approaches are needed. This review focuses on the rescue of cone function as a therapeutic option for maintaining central vision in rod-cone dystrophies. It highlights recent developments in better understanding the mechanisms of action of the trophic factor RdCVF and its potential as a sight-saving therapeutic strategy.
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http://dx.doi.org/10.1007/978-3-319-75402-4_62DOI Listing
May 2019

Cell Signaling with Extracellular Thioredoxin and Thioredoxin-Like Proteins: Insight into Their Mechanisms of Action.

Oxid Med Cell Longev 2017 12;2017:8475125. Epub 2017 Sep 12.

INSERM, U968, Sorbonne Universités, 75012 Paris, France.

Thioredoxins are small thiol-oxidoreductase enzymes that control cellular redox homeostasis. Paradoxically, human thioredoxin (TXN1) was first identified as the adult T cell leukemia-derived factor (ADF), a secreted protein. ADF has been implicated in a wide variety of cell-to-cell communication systems acting as a cytokine or a chemokine. TRX80 is a truncated TXN1 protein with cytokine activity. The unconventional secretion mechanism of these extracellular thioredoxins is unknown. The thioredoxin system is relying on glucose metabolism through the pentose phosphate pathway that provides reducing power in the form of NADPH, the cofactor of thioredoxin reductase (TXNRD). While a complete extracellular TXN system is present in the blood in the form of circulating TXN1 and TXNDR1, the source of extracellular NADPH remains a mystery. In the absence of redox regenerating capacity, extracellular thioredoxins may rather be prooxidant agents. Rod-derived cone viability factor (RdCVF) is the product of intron retention of the nucleoredoxin-like 1 () gene, a secreted truncated thioredoxin-like protein. The other product encoded by the gene, RdCVFL, is an enzymatically active thioredoxin. This is a very singular example of positive feedback of a superthioredoxin system encoded by a single gene likely emerging during evolution from metabolic constraints on redox signaling.
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http://dx.doi.org/10.1155/2017/8475125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613632PMC
July 2018

Further Insights into the Ciliary Gene and Protein KIZ and Its Murine Ortholog PLK1S1 Mutated in Rod-Cone Dystrophy.

Genes (Basel) 2017 Oct 18;8(10). Epub 2017 Oct 18.

Sorbonne Universités, UPMC University Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, 75012 Paris, France.

We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in , encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in . KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in , the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in -mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of mutations.
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http://dx.doi.org/10.3390/genes8100277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664127PMC
October 2017

Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation.

Mol Ther 2018 01 8;26(1):219-237. Epub 2017 Sep 8.

INSERM, U968, Paris 75012, France; Sorbonne Universités, UPMC Univ Paris 06 UMR_S 968, Institut de la Vision, Paris 75012, France; CNRS, UMR_7210, Paris 75012, France. Electronic address:

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.
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http://dx.doi.org/10.1016/j.ymthe.2017.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762984PMC
January 2018

Metabolic and redox signaling in the retina.

Cell Mol Life Sci 2017 10 20;74(20):3649-3665. Epub 2016 Aug 20.

Department of Genetics, Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06 UMR_S968, INSERM_U968, CNRS UMR_7210, 17 rue Moreau, 75012, Paris, France.

Visual perception by photoreceptors relies on the interaction of incident photons from light with a derivative of vitamin A that is covalently linked to an opsin molecule located in a special subcellular structure, the photoreceptor outer segment. The photochemical reaction produced by the photon is optimal when the opsin molecule, a seven-transmembrane protein, is embedded in a lipid bilayer of optimal fluidity. This is achieved in vertebrate photoreceptors by a high proportion of lipids made with polyunsaturated fatty acids, which have the detrimental property of being oxidized and damaged by light. Photoreceptors cannot divide, but regenerate their outer segments. This is an enormous energetic challenge that explains why photoreceptors metabolize glucose through aerobic glycolysis, as cancer cells do. Uptaken glucose produces metabolites to renew that outer segment as well as reducing power through the pentose phosphate pathway to protect photoreceptors against oxidative damage.
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http://dx.doi.org/10.1007/s00018-016-2318-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597695PMC
October 2017

Mathematical Model of the Role of RdCVF in the Coexistence of Rods and Cones in a Healthy Eye.

Bull Math Biol 2016 07 21;78(7):1394-409. Epub 2016 Jul 21.

School of Mathematical and Natural Sciences, Arizona State University, Glendale, AZ, 85306, USA.

Understanding the essential components and processes for coexistence of rods and cones is at the forefront of retinal research. The recent discovery on RdCVF's mechanism and mode of action for enhancing cone survival brings us a step closer to unraveling key questions of coexistence and codependence of these neurons. In this work, we build from ecological and enzyme kinetic work on functional response kinetics and present a mathematical model that allows us to investigate the role of RdCVF and its contribution to glucose intake. Our model results and analysis predict a dual role of RdCVF for enhancing and repressing the healthy coexistence of the rods and cones. Our results show that maintaining RdCVF above a threshold value allows for coexistence. However, a significant increase above this value threatens the existence of rods as the cones become extremely efficient at uptaking glucose and begin to take most of it for themselves. We investigate the role of natural glucose intake and that due to RdCVF in both high and low nutrient levels. Our analysis reveals that under low nutrient levels coexistence is not possible regardless of the amount of RdCVF present. With high nutrient levels coexistence can be achieved with a relative small increase in glucose uptake. By understanding the contributions of rods to cones survival via RdCVF in a non-diseased retina, we hope to shed light on degenerative diseases such as retinitis pigmentosa.
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http://dx.doi.org/10.1007/s11538-016-0185-xDOI Listing
July 2016

The Thioredoxin Encoded by the Rod-Derived Cone Viability Factor Gene Protects Cone Photoreceptors Against Oxidative Stress.

Antioxid Redox Signal 2016 06 12;24(16):909-23. Epub 2016 May 12.

1 INSERM , U968, Paris, France .

Aims: Rod-derived cone viability factor long (RdCVFL) is an enzymatically active thioredoxin encoded by the nucleoredoxin-like-1 (Nxnl1) gene. The second product of the gene, RdCVF, made by alternative splicing is a novel trophic factor secreted by rods that protects cones in rodent models of retinitis pigmentosa, the most prevalent inherited retinal disease. It acts on cones by stimulating aerobic glycolysis through its interaction with a complex containing basigin-1 and the glucose transporter GLUT1. We studied the role of Nxnl1 in cones after its homologous recombination using a transgenic line expressing Cre recombinase under the control of a cone opsin promoter.

Results: We show that the cones of these mice are dysfunctional and degenerate by 8 months of age. The age-related deficit in cones is exacerbated in young animals by exposure to high level of oxygen. In agreement with this phenotype, we found that the cones express only one of the two Nxnl1 gene products, the thioredoxin RdCVFL. Administration of RdCVFL to the mouse carrying a deletion of the Nxnl1 gene in cones reduces the damage produced by oxidative stress. Silencing the expression of RdCVFL in cone-enriched culture reduces cell viability, showing that RdCVFL is a cell-autonomous mechanism of protection.

Innovation: This novel mode of action is certainly relevant for the therapy of retinitis pigmentosa since the delivery into cones of the rd10 mouse, a recessive model of the disease, rescues cones.

Conclusion: Our work highlights the duality of the Nxnl1 gene, which protects the cones by two distinct mechanisms. Antioxid. Redox Signal. 24, 909-923.
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http://dx.doi.org/10.1089/ars.2015.6509DOI Listing
June 2016

Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

PLoS One 2016 17;11(3):e0150758. Epub 2016 Mar 17.

INSERM, U968, Paris, F-75012, France.

To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795653PMC
August 2016

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

Nat Genet 2016 Feb 21;48(2):134-43. Epub 2015 Dec 21.

Department of Ophthalmology, Erasmus Medical Center, Rotterdam, the Netherlands.

Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.
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http://dx.doi.org/10.1038/ng.3448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745342PMC
February 2016

[Altruism in the retina: sticks feed cones].

Med Sci (Paris) 2015 Oct 19;31(10):828-30. Epub 2015 Oct 19.

Inserm U968, Paris, F-75012, France - Sorbonne Universités, UPMC université Paris 06, UMR_S 968, Institut de la Vision, 17, rue Moreau, Paris, F-75012, France - CNRS, UMR_7210, Paris, F-75012, France.

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http://dx.doi.org/10.1051/medsci/20153110005DOI Listing
October 2015

Cancer metabolism of cone photoreceptors.

Oncotarget 2015 Oct;6(32):32285-6

INSERM, U968, Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, CNRS, UMR_7210, Paris, France.

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http://dx.doi.org/10.18632/oncotarget.5963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741680PMC
October 2015

Genotypic and phenotypic characterization of P23H line 1 rat model.

PLoS One 2015 26;10(5):e0127319. Epub 2015 May 26.

INSERM, U968, Paris, France; CNRS, UMR_7210, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, DHU ViewMaintain, INSERM-DHOS CIC 1423, Paris, France; Institute of Ophthalmology, University College of London, London, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127319PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444340PMC
April 2016

Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.

Cell 2015 May;161(4):817-32

INSERM, U968, 75012 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, 75012 Paris, France; CNRS, UMR_7210, 75012 Paris, France. Electronic address:

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.
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http://dx.doi.org/10.1016/j.cell.2015.03.023DOI Listing
May 2015

Vibratome sectioning mouse retina to prepare photoreceptor cultures.

J Vis Exp 2014 Dec 22(94). Epub 2014 Dec 22.

Department of Genetics, UMR_S 968, Institut de la Vision; Sorbonne Universités, Paris 06, UMR_S 968, Institut de la Vision; INSERM, U968, Institut de la Vision; CNRS, UMR_7210, Institut de la Vision;

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10(5) cells/cm(2). The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.
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http://dx.doi.org/10.3791/51954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354458PMC
December 2014

Viral-mediated RdCVF and RdCVFL expression protects cone and rod photoreceptors in retinal degeneration.

J Clin Invest 2015 Jan 21;125(1):105-16. Epub 2014 Nov 21.

Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease.
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http://dx.doi.org/10.1172/JCI65654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382269PMC
January 2015

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration.

Hum Mol Genet 2014 Nov 4;23(21):5827-37. Epub 2014 Jun 4.

The Fred Hollows Foundation, Auckland, New Zealand, School of Social Sciences, University of New South Wales, Sydney, Australia.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.
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http://dx.doi.org/10.1093/hmg/ddu276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189898PMC
November 2014

Therapeutic strategy for handling inherited retinal degenerations in a gene-independent manner using rod-derived cone viability factors.

C R Biol 2014 Mar 20;337(3):207-13. Epub 2014 Feb 20.

Inserm, U968, département de génétique, Institut de la vision, 17, rue Moreau, 75012 Paris, France; UMR_S 968, département de génétique, université Pierre-et-Marie-Curie (Paris-6), Institut de la vision, 17, rue Moreau, 75012 Paris, France; CNRS, UMR_7210, 75012 Paris, France.

The most common hereditary retinal degeneration, retinitis pigmentosa (RP), leads to blindness by degeneration of cone photoreceptors. Meanwhile, genetic studies have shown that a significant proportion of RP genes is expressed only by rods, which raises the question of the mechanism leading to the degeneration of cones. Following the concept of sustainability factor cones, rods secrete survival factors that are necessary to maintain the cones, named Rod-derived Cone Viability Factors (RdCVFs). In patients suffering from RP, loss of rods results in the loss of RdCVFs expression and followed by cone degeneration. We have identified the bifunctional genes nucleoredoxin-like 1 and 2 that encode for, by differential splicing, a thioredoxin enzyme and a cone survival factor, respectively RdCVF and RdCVF2. The administration of these survival factors would maintain cones and central vision in most patients suffering from RP.
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http://dx.doi.org/10.1016/j.crvi.2013.12.002DOI Listing
March 2014
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