Publications by authors named "Stéphane Fouquet"

28 Publications

  • Page 1 of 1

Phototoxic damage to cone photoreceptors can be independent of the visual pigment: the porphyrin hypothesis.

Cell Death Dis 2020 08 29;11(8):711. Epub 2020 Aug 29.

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

Lighting is rapidly changing with the introduction of light-emitting diodes (LEDs) in our homes, workplaces, and cities. This evolution of our optical landscape raises major concerns regarding phototoxicity to the retina since light exposure is an identified risk factor for the development of age-related macular degeneration (AMD). In this disease, cone photoreceptors degenerate while the retinal pigment epithelium (RPE) is accumulating lipofuscin containing phototoxic compounds such as A2E. Therefore, it remains unclear if the light-elicited degenerative process is initiated in cones or in the RPE. Using purified cone photoreceptors from pig retina, we here investigated the effect of light on cone survival from 390 to 510 nm in 10 nm steps, plus the 630 nm band. If at a given intensity (0.2 mW/cm²), the most toxic wavelengths are comprised in the visible-to-near-UV range, they shift to blue-violet light (425-445 nm) when exposing cells to a solar source filtered by the eye optics. In contrast to previous rodent studies, this cone photoreceptor phototoxicity is not related to light absorption by the visual pigment. Despite bright flavin autofluorescence of cone inner segment, excitation-emission matrix of this inner segment suggested that cone phototoxicity was instead caused by porphyrin. Toxic light intensities were lower than those previously defined for A2E-loaded RPE cells indicating cones are the first cells at risk for a direct light insult. These results are essential to normative regulations of new lighting but also for the prevention of human retinal pathologies since toxic solar light intensities are encountered even at high latitudes.
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http://dx.doi.org/10.1038/s41419-020-02918-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456424PMC
August 2020

Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells.

Stem Cells Int 2019 15;2019:7858796. Epub 2019 Jul 15.

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

The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases.
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http://dx.doi.org/10.1155/2019/7858796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664555PMC
July 2019

Genetic Analysis of the Organization, Development, and Plasticity of Corneal Innervation in Mice.

J Neurosci 2019 02 26;39(7):1150-1168. Epub 2018 Dec 26.

Institut de la Vision, Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, F-75012 Paris, France,

The cornea has the densest sensory innervation of the body, originating primarily from neurons in the trigeminal ganglion. The basic principles of cornea nerve patterning have been established many years ago using classic neuroanatomical methods, such as immunocytochemistry and electrophysiology. Our understanding of the morphology and distribution of the sensory nerves in the skin has considerably progressed over the past few years through the generation and analysis of a variety of genetically modified mouse lines. Surprisingly, these lines were not used to study corneal axons. Here, we have screened a collection of transgenic and knockin mice (of both sexes) to select lines allowing the visualization and genetic manipulation of corneal nerves. We identified multiple lines, including some in which different types of corneal axons can be simultaneously observed with fluorescent proteins expressed in a combinatorial manner. We also provide the first description of the morphology and arborization of single corneal axons and identify three main types of branching pattern. We applied this genetic strategy to the analysis of corneal nerve development and plasticity. We provide direct evidence for a progressive reduction of the density of corneal innervation during aging. We also show that the semaphorin receptor neuropilin-1 acts cell-autonomously to control the development of corneal axons and that early axon guidance defects have long-term consequences on corneal innervation. We have screened a collection of transgenic and knockin mice and identify lines allowing the visualization and genetic manipulation of corneal nerves. We provide the first description of the arborization pattern of single corneal axons. We also present applications of this genetic strategy to the analysis of corneal nerve development and remodeling during aging.
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http://dx.doi.org/10.1523/JNEUROSCI.1401-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381234PMC
February 2019

Optogenetic Light Sensors in Human Retinal Organoids.

Front Neurosci 2018 2;12:789. Epub 2018 Nov 2.

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

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.
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http://dx.doi.org/10.3389/fnins.2018.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224345PMC
November 2018

Light action spectrum on oxidative stress and mitochondrial damage in A2E-loaded retinal pigment epithelium cells.

Cell Death Dis 2018 02 19;9(3):287. Epub 2018 Feb 19.

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

Aims: Blue light is an identified risk factor for age-related macular degeneration (AMD). We investigated oxidative stress markers and mitochondrial changes in A2E-loaded retinal pigment epithelium cells under the blue-green part of the solar spectrum that reaches the retina to better understand the mechanisms underlying light-elicited toxicity.

Results: Primary retinal pigment epithelium cells were loaded with a retinal photosensitizer, AE2, to mimic aging. Using a custom-made illumination device that delivers 10 nm-wide light bands, we demonstrated that A2E-loaded RPE cells generated high levels of both hydrogen peroxide (HO) and superoxide anion (O) when exposed to blue-violet light. In addition, they exhibited perinuclear clustering of mitochondria with a decrease of both their mitochondrial membrane potential and their respiratory activities. The increase of oxidative stress resulted in increased levels of the oxidized form of glutathione and decreased superoxide dismutase (SOD) and catalase activities. Furthermore, mRNA expression levels of the main antioxidant enzymes (SOD2, catalase, and GPX1) also decreased.

Conclusions: Using an innovative illumination device, we measured the precise action spectrum of the oxidative stress mechanisms on A2E-loaded retinal pigment epithelium cells. We defined 415-455 nm blue-violet light, within the solar spectrum reaching the retina, to be the spectral band that generates the highest amount of reactive oxygen species and produces the highest level of mitochondrial dysfunction, explaining its toxic effect. This study further highlights the need to filter these wavelengths from the eyes of AMD patients.
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http://dx.doi.org/10.1038/s41419-018-0331-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833722PMC
February 2018

Non-cell autonomous control of precerebellar neuron migration by Slit and Robo proteins.

Development 2018 01 17;145(2). Epub 2018 Jan 17.

Sorbonne Universités, UPMC Paris 06, INSERM, CNRS, Institut de la Vision 75012, Paris, France

During development, precerebellar neurons migrate tangentially from the dorsal hindbrain to the floor plate. Their axons cross it but their cell bodies stop their ventral migration upon reaching the midline. It has previously been shown that Slit chemorepellents and their receptors, Robo1 and Robo2, might control the migration of precerebellar neurons in a repulsive manner. Here, we have used a conditional knockout strategy in mice to test this hypothesis. We show that the targeted inactivation of the expression of and receptors in precerebellar neurons does not perturb their migration and that they still stop at the midline. The selective ablation of the expression of all three Slit proteins in floor-plate cells has no effect on pontine neurons and only induces the migration of a small subset of inferior olivary neurons across the floor plate. Likewise, we show that the expression of Slit proteins in the facial nucleus is dispensable for pontine neuron migration. Together, these results show that Robo1 and Robo2 receptors act non-cell autonomously in migrating precerebellar neurons and that floor-plate signals, other than Slit proteins, must exist to prevent midline crossing.
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http://dx.doi.org/10.1242/dev.150375DOI Listing
January 2018

The 3D Retinal Capillary Circulation in Pigs Reveals a Predominant Serial Organization.

Invest Ophthalmol Vis Sci 2017 11;58(13):5754-5763

Institut de la Vision, Sorbonne Universiteés, UPMC University Paris 06, INSERM, CNRS, Paris, France.

Purpose: To establish a model of the retinal capillary circulation in pigs, which in many aspects is close to the human retina.

Methods: Using high density confocal microscopy image stacks of immunolabeled porcine retinal whole mounts, microvessels close to the optic nerve head were traced in three dimensions. The direction of flow of individual capillaries was deduced from their arteriolar and/or venous connections.

Results: From major arteries, second-order arteries traversed the nerve fiber layer and resolved exclusively into the superficial vascular plexus (SVP), which dichotomized the blood flow between radial peripapillary capillaries (RPCs) on one side and the intermediate (IVP) and deep vascular plexus (DVP) on the other. Each RPC was supplied by one or several capillaries from the SVP and drained to the IVP or DVP. The DVP was a mosaic of approximately 300 to 600 μm wide anastomotic watersheds, each drained by one or two venules connected to major veins. A presumptive direction of flow could be determined for >90% of capillaries. These results suggest a model of the capillary circulation in which the three microvessel layers are serially organized with RPCs are in parallel between the SVP and IVP or DVP.

Conclusions: In the peripapillary retina of pigs, microvascular layers have a serial arrangement, with RPCs emerging from the SVP and draining to the IVP or DVP; hence, connected in parallel of this scheme. The bulk of flow, therefore, traverses the SVP and DVP successively. This organization contributes to the higher oxygen saturation in the SVP and RPCs than in the DVP. Physiopathologic implications of this model regarding retinal diseases are discussed.
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http://dx.doi.org/10.1167/iovs.17-22097DOI Listing
November 2017

Mutations in GREB1L Cause Bilateral Kidney Agenesis in Humans and Mice.

Am J Hum Genet 2017 Nov;101(5):803-814

Laboratory of Hereditary Kidney Diseases, INSERM UMR 1163, Imagine Institute, 75015 Paris, France; Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, 75015 Paris, France.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l embryos and a slight decrease in ureteric bud branching in Greb1l embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673669PMC
November 2017

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

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8.

Biotechnol Bioeng 2016 12 30;113(12):2712-2724. Epub 2016 Jun 30.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, Paris 75012, France.

Recently, we described a modified AAV2 vector-AAV2-7m8-having a capsid-displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high-level gene delivery into deep layers of the retina when virus is delivered into the eye's vitreous. Here, we further characterize AAV2-7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2-7m8 and AAV9-7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV. Biotechnol. Bioeng. 2016;113: 2712-2724. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26031DOI Listing
December 2016

PrP(c) deficiency and dasatinib protect mouse intestines against radiation injury by inhibiting of c-Src.

Radiother Oncol 2016 07 9;120(1):175-83. Epub 2016 Jul 9.

Institut de Radioprotection et de Sûreté Nucléaire, PRP-HOM, Fontenay-aux-Roses, France.

Background & Aim: Despite extensive study of the contribution of cell death and apoptosis to radiation-induced acute intestinal injury, our knowledge of the signaling mechanisms involved in epithelial barrier dysfunction remains inadequate. Because PrP(c) plays a key role in intestinal homeostasis by renewing epithelia, we sought to study its role in epithelial barrier function after irradiation.

Design: Histology, morphometry and plasma FD-4 levels were used to examine ileal architecture, wound healing, and intestinal leakage in PrP(c)-deficient (KO) and wild-type (WT) mice after total-body irradiation. Impairment of the PrP(c) Src pathway after irradiation was explored by immunofluorescence and confocal microscopy, with Caco-2/Tc7 cells. Lastly, dasatinib treatment was used to switch off the Src pathway in vitro and in vivo.

Results: The decrease in radiation-induced lethality, improved intestinal wound healing, and reduced intestinal leakage promoted by PrP(c) deficiency demonstrate its involvement in acute intestinal damage. Irradiation of Cacao2/Tc7 cells induced PrP(c) to target the nuclei associated with Src activation. Finally, the protective effect triggered by dasatinib confirmed Src involvement in radiation-induced acute intestinal toxicity.

Conclusion: Our data are the first to show a role for the PrP(c)-Src pathway in acute intestinal response to radiation injury and offer a novel therapeutic opportunity.
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http://dx.doi.org/10.1016/j.radonc.2016.06.009DOI Listing
July 2016

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Hum Mol Genet 2016 07 10;25(14):3070-3079. Epub 2016 Jun 10.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 75012 Paris, France.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).
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http://dx.doi.org/10.1093/hmg/ddw159DOI Listing
July 2016

Synthetic 3D diamond-based electrodes for flexible retinal neuroprostheses: Model, production and in vivo biocompatibility.

Biomaterials 2015 Oct 14;67:73-83. Epub 2015 Jul 14.

INSERM, U968, Institut de la Vision, Paris F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S968, Institut de la Vision, Paris F-75012, France; CNRS UMR 7210, Institut de la Vision, Paris 75012, France. Electronic address:

Two retinal implants have recently received the CE mark and one has obtained FDA approval for the restoration of useful vision in blind patients. Since the spatial resolution of current vision prostheses is not sufficient for most patients to detect faces or perform activities of daily living, more electrodes with less crosstalk are needed to transfer complex images to the retina. In this study, we modelled planar and three-dimensional (3D) implants with a distant ground or a ground grid, to demonstrate greater spatial resolution with 3D structures. Using such flexible 3D implant prototypes, we showed that the degenerated retina could mould itself to the inside of the wells, thereby isolating bipolar neurons for specific, independent stimulation. To investigate the in vivo biocompatibility of diamond as an electrode or an isolating material, we developed a procedure for depositing diamond onto flexible 3D retinal implants. Taking polyimide 3D implants as a reference, we compared the number of neurones integrating the 3D diamond structures and their ratio to the numbers of all cells, including glial cells. Bipolar neurones were increased whereas there was no increase even a decrease in the total cell number. SEM examinations of implants confirmed the stability of the diamond after its implantation in vivo. This study further demonstrates the potential of 3D designs for increasing the resolution of retinal implants and validates the safety of diamond materials for retinal implants and neuroprostheses in general.
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http://dx.doi.org/10.1016/j.biomaterials.2015.07.018DOI Listing
October 2015

Multicolor analysis of oligodendrocyte morphology, interactions, and development with Brainbow.

Glia 2015 Apr 21;63(4):699-717. Epub 2014 Dec 21.

INSERM, UMRS_U968, Institut de la Vision, Paris, F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, Institut de la Vision, Paris, F-75012, France; CNRS, UMR_7210, Paris, F-75012, France.

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single-out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre-lox recombination, previously used for visualizing axons and neurons. We used tamoxifen-inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single-cell resolution.
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http://dx.doi.org/10.1002/glia.22779DOI Listing
April 2015

Multiplex cell and lineage tracking with combinatorial labels.

Neuron 2014 Feb;81(3):505-20

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:

We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.
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http://dx.doi.org/10.1016/j.neuron.2013.12.016DOI Listing
February 2014

Src family kinases involved in CXCL12-induced loss of acute morphine analgesia.

Brain Behav Immun 2014 May 18;38:38-52. Epub 2013 Nov 18.

Centre de Recherche Institut de la Vision, Université Pierre et Marie Curie, INSERM, UMR_S968, CNRS, UMR_7210, Paris, France. Electronic address:

Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occurred in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu-(MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1μg) significantly attenuated the subcutaneous morphine (4mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5μg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10μg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12-induced loss of acute morphine analgesia is linked to Src family kinases activation.
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http://dx.doi.org/10.1016/j.bbi.2013.11.010DOI Listing
May 2014

Taurine provides neuroprotection against retinal ganglion cell degeneration.

PLoS One 2012 24;7(10):e42017. Epub 2012 Oct 24.

Institut National de la Santé et de la Recherche Médicale UMR 968, Institut de la Vision, Paris, France.

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042017PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480351PMC
April 2013

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination.

Glia 2012 Oct 9;60(10):1590-604. Epub 2012 Jul 9.

CNRS, UMR 7102, Paris, France.

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone-treated mice. The analysis of the optic nerve and anterior commissure of Sema6A-deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild-type oligodendrocytes. In myelinating co-cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation.
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http://dx.doi.org/10.1002/glia.22378DOI Listing
October 2012

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells.

Amino Acids 2012 Nov 4;43(5):1979-93. Epub 2012 Apr 4.

INSERM, U-968, Insitut de la Vision Retinal Information Processing: Pharmacology and Pathologies, 17, rue Moreau, 75012 Paris, France.

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20 %) as the number of retinal ganglion cells (19 %). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.
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http://dx.doi.org/10.1007/s00726-012-1273-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472058PMC
November 2012

Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors.

PLoS One 2011 Mar 7;6(3):e17621. Epub 2011 Mar 7.

Inserm, U1016, Institut Cochin, Paris, France.

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049782PMC
March 2011

Progressive upregulation of PD-1 in primary and metastatic melanomas associated with blunted TCR signaling in infiltrating T lymphocytes.

J Invest Dermatol 2011 Jun 24;131(6):1300-7. Epub 2011 Feb 24.

Institut Cochin, Inserm, U1016, Paris, France.

Programmed death-1 (PD-1) is involved in T-cell tolerance to self-antigens. For some cancers, it has been suggested that the expression of a ligand of PD-1, namely PD-L1, could contribute to tumor escape from immune destruction. Nevertheless, the relationship between PD-1 expression on tumor-infiltrating T lymphocytes (TILs), disease stage, and TIL responsiveness is still poorly documented. In this study, we show that freshly isolated CD4(+) and CD8(+) TILs express substantial levels of PD-1 in primary melanomas. The expression of PD-1 was further increased at later stages in distant cutaneous metastases, especially on CD8(+) TILs. The expression of PD-1 ligands was frequent only in metastases, on both tumor cells and tumor-derived myeloid cells. TILs isolated from these cutaneous tumors are poorly reactive ex vivo, with blunted calcium response and IFN-γ production after TCR stimulation. Surprisingly, in distinct parts of a primary melanoma, either invasive or regressing, we show that TILs similarly express PD-1 and remain dysfunctional. The expressions of PD-1 and PD-L1 in metastatic melanoma lesions could be considered as witnesses of an unsuccessful anti-tumoral immune response, but the direct involvement of PD-1 in the severity of the disease, and the importance of TILs in tumor regression, remain to be established.
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http://dx.doi.org/10.1038/jid.2011.30DOI Listing
June 2011

Taurine deficiency damages photoreceptors and retinal ganglion cells in vigabatrin-treated neonatal rats.

Mol Cell Neurosci 2010 Apr 1;43(4):414-21. Epub 2010 Feb 1.

INSERM, UMR_S968, Institut de la Vision, Paris, France; UPMC Univ Paris 06, UMR_S968, Institut de la Vision, Paris, France.

The anti-epileptic drug vigabatrin induces an irreversible constriction of the visual field, but is still widely used to treat infantile spasms and some forms of epilepsy. We recently reported that vigabatrin-induced cone damage is due to a taurine deficiency. However, optic atrophy and thus retinal ganglion cell degeneration was also reported in children treated for infantile spasms. We here show in neonatal rats treated from postnatal days 4 to 29 that the vigabatrin treatment triggers not only cone photoreceptor damage, disorganisation of the photoreceptor layer and gliosis but also retinal ganglion cell loss. Furthermore, we demonstrate in these neonatal rats that taurine supplementation partially prevents these retinal lesions and in particular the retinal ganglion cell loss. These results provide the first evidence of retinal ganglion cell neuroprotection by taurine. They further confirm that taurine supplementation should be administered with the vigabatrin treatment for infantile spasms or epilepsy.
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http://dx.doi.org/10.1016/j.mcn.2010.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864319PMC
April 2010

Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions.

Am J Physiol Gastrointest Liver Physiol 2009 Feb 4;296(2):G235-44. Epub 2008 Dec 4.

Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S872, Paris, France.

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners beta- and alpha-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes.
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http://dx.doi.org/10.1152/ajpgi.90313.2008DOI Listing
February 2009

The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells and in the distribution of junction-associated proteins.

PLoS One 2008 Aug 20;3(8):e3000. Epub 2008 Aug 20.

Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, F-75006 France.

Background: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src.

Methodology/findings: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi.

Conclusions/significance: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2500194PMC
August 2008

E-cadherin-dependent transcriptional control of apolipoprotein A-IV gene expression in intestinal epithelial cells: a role for the hepatic nuclear factor 4.

J Biol Chem 2006 Feb 7;281(6):3560-8. Epub 2005 Dec 7.

Université Pierre et Marie Curie UMRS 505, Paris, F-75006 France, INSERM, UMRS 505, F-75006 Paris, France.

Cell-matrix and cell-cell adhesion play a central role in the control of cell proliferation, differentiation, and gene expression. Integrins and E-cadherin are the key components involved in these processes in epithelial cells. We recently showed that integrin-dependent adhesion to the extracellular matrix reinforces the formation of E-cadherin-actin complexes inducing the polarization of Caco-2 enterocytes and increases the expression of a marker of enterocyte differentiation, the apolipoprotein A-IV (apoA-IV) gene. By impairing or enhancing E-cadherin-dependent cell adhesion, we demonstrate in the present study its involvement in the transcriptional activation of the apoA-IV gene in Caco-2 cells. This control requires the regulatory sequence that we have previously identified as necessary and sufficient to drive and restrict apoA-IV gene expression in enterocytes in vivo. Furthermore, using chimeric E-cadherin-Fc homophilic ligand-coated surfaces, we show that a direct activation of E-cadherin triggers the transcriptional activation of the apoA-IV promoter. Finally, E-cadherin-dependent cell-cell adhesion controls the nuclear abundance of the transcription factor hepatic nuclear factor 4alpha, which is involved in the enterocyte-specific expression of apoA-IV gene. Altogether, our results suggest that E-cadherin controls enterocyte-specific expression of genes, such as the apoA-IV gene, through the control of hepatic nuclear factor 4alpha nuclear abundance.
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http://dx.doi.org/10.1074/jbc.M506360200DOI Listing
February 2006

[Control of the survival/apoptosis balance by E-cadherin: role in enterocyte anoikis].

J Soc Biol 2004 ;198(4):379-83

UMR 505 INSERM-UPMC, Laboratoire de Pharmacologie Cellulaire et Moléculaire de l'Ecole Pratique des Hautes Etudes, 15, rue de l'Ecole de Médecine, 75006 Paris, France.

Cadherins are transmembrane glycoproteins involved in cell-cell adherence. Recent developments indicate that classical cadherins may act as adherence-activated signaling receptors. Here, we review recent data from the literature concerning the role of classical cadherins in the control of cell survival and the signaling pathways involved. We focus on the fate and the role of E-cadherin, the main classical cadherin expressed in epithelial cells, in the cell death program triggered in enterocytes by loss of anchorage from the extracellular matrix (anoikis). These data open new perspectives on the key role of this protein, which is dysregulated in most carcinoma and is considered as a tumour-suppressor.
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September 2005

Early loss of E-cadherin from cell-cell contacts is involved in the onset of Anoikis in enterocytes.

J Biol Chem 2004 Oct 2;279(41):43061-9. Epub 2004 Aug 2.

UMR 505 INSERM-UPMC, Laboratoire de Pharmacologie Cellulaire et Moléculaire de l'EPHE, 75006 Paris, France.

Anoikis, i.e. apoptosis induced by detachment from the extracellular matrix, is thought to be involved in the shedding of enterocytes at the tip of intestinal villi. Mechanisms controlling enterocyte survival are poorly understood. We investigated the role of E-cadherin, a key protein of cell-cell adhesion, in the control of anoikis of normal intestinal epithelial cells, by detaching murine villus epithelial cells from the underlying basement membrane while preserving cell-cell interactions. We show that upon the loss of anchorage, normal enterocytes execute a program of apoptosis within minutes, via a Bcl-2-regulated and caspase-9-dependent pathway. E-cadherin is lost early from cell-cell contacts. This process precedes the execution phase of detachment-induced apoptosis as it is only weakly modulated by Bcl-2 overexpression or caspase inhibition. E-cadherin loss, however, is efficiently prevented by lysosome and proteasome inhibitors. We also found that a blocking anti-E-cadherin antibody increases the rate of anoikis, whereas the activation of E-cadherin using E-cadherin-Fc chimera proteins reduces anoikis. In conclusion, our results stress the striking sensitivity of normal enterocytes to the loss of anchorage and the contribution of E-cadherin to the control of their survival/apoptosis balance. They open new perspectives on the key role of this protein, which is dysregulated in the intestinal epithelium in both inflammatory bowel disease and cancer.
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http://dx.doi.org/10.1074/jbc.M405095200DOI Listing
October 2004

The cellular prion protein PrPc is expressed in human enterocytes in cell-cell junctional domains.

J Biol Chem 2004 Jan 23;279(2):1499-505. Epub 2003 Oct 23.

INSERM U505, Université Pierre et Marie Curie, 15 Rue de l'Ecole de Médecine 75006 Paris.

The physiological function of PrPc, the cellular isoform of prion protein, still remains unclear, although it has been established, in vitro or by using nerve cells, that it can homodimerize, bind copper, or interact with other proteins. Expression of PrPc was demonstrated as necessary for prion infection propagation. Considering the importance of the intestinal barrier in the process of oral prion infectivity, we have analyzed the expression of PrPc in enterocytes, which represent the major cell population of the intestinal epithelium. Our study, conducted both on normal human intestinal tissues and on the enterocytic cell line Caco-2/TC7, shows for the first time that PrPc is present in enterocytes. Interestingly, we found that this glycosylphosphatidylinositol-anchored glycoprotein was localized in cholesterol-dependent raft domains of the upper lateral membranes of enterocytes, beneath tight junctions, in cell-cell junctional domains. We observed that PrPc, E-cadherin, and Src co-localized in adherens junctions and that PrPc was co-immunoprecipitated with Src kinase but not with E-cadherin. Alteration of cell polarity after cholesterol depletion or loosening of the cell-cell junctions after EGTA treatment rapidly impaired membrane targeting of PrPc. Overall, our results point out the signaling of cell-cell contacts as a putative role for PrPc in epithelial cells.
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http://dx.doi.org/10.1074/jbc.M308578200DOI Listing
January 2004