Publications by authors named "Géraldine Millet-Puel"

11 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

Nxnl2 splicing results in dual functions in neuronal cell survival and maintenance of cell integrity.

Hum Mol Genet 2012 May 15;21(10):2298-311. Epub 2012 Feb 15.

Institut de la vision, INSERM, U968, Paris F-75012, France.

The rod-derived cone viability factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration, arises due to the loss of trophic support from RdCVF2. In contrary, the progressive loss of rod visual function of the Nxnl2-/- mouse results from a decrease in outer segment length, mediated by a cell autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2-/- mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.
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http://dx.doi.org/10.1093/hmg/dds050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664437PMC
May 2012

The thioredoxin-like protein rod-derived cone viability factor (RdCVFL) interacts with TAU and inhibits its phosphorylation in the retina.

Mol Cell Proteomics 2009 Jun 11;8(6):1206-18. Epub 2009 Mar 11.

double daggerInstitut de la Vision, INSERM UMR592, 17 rue Moreau, 75012 Paris, France.

Rod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1(-/-) mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress.
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http://dx.doi.org/10.1074/mcp.M800406-MCP200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2690495PMC
June 2009