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Physiology of the retinal pigment epithelium and associated pathologies

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Team leader : Emeline Nandrot

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Introduction

Our team explores various roles of retinal pigment epithelium cells in the overall homeostasis of the retina. Research projects include the characterization of the rhythmic elimination of aged photoreceptor outer segments by phagocytosis and its control at the molecular level, and the identification of molecular pathways targeted in pathological conditions such as rod-cone dystrophies/retinitis pigmentosa and age-related macular degeneration.
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Presentation

At the back of the eye lie cells from the retinal pigment epithelium (RPE). RPE functions are numerous and all crucial for the homeostasis of the retina and vision: supply in nutrients, ions and oxygen to photoreceptors, elimination of photoreceptor waste, secretion of trophic factors, renewal of photopigments, participation in retinal adhesion, …


Adjacent to RPE cells, photoreceptors initiate the visual signaling reaching the brain in a specific compartment constituted of membranous disks containing photosensitive molecules and named photoreceptor outer segments (POS). POS are submitted to a strong oxidative stress due to their constant exposure to light rays. In order to limit this stress, POS are permanently renewed and their most-aged distal part is shed following a circadian rhythm. POS are phagocytosed by RPE cells at a level of around 25-30 POS per RPE cell. Thus, RPE cells being post-mitotic they represent the busiest phagocytes in the body.

Complete absence or deregulated retinal phagocytosis leads to early or late vision loss respectively, in rodent models as well as in human patients. Depending on the molecular dysfunction, retinal phenotypes observed in rodent models resemble phenotypes such as retinitis pigmentosa or age-related macular degeneration (AMD), first cause of blindness in people over 50 years. AMD is in part caused by the progressive accumulation of poorly digested POS debris that gets oxidized and lead to the slow build up of lipofuscin. Interestingly, human patients carrying mutations in the gene coding the internalization receptor MERTK are affected by rod-cone dystrophies (atypical retinitis pigmentosa) comprising macular lesions, showing an alteration in the phagocytic function of RPE cells.

Projects in the team aim at understanding how deregulation of RPE functions lead to pathologies. Our main interest is the characterization of molecular mechanisms ruling the daily rhythmic disposal of shed POS. Indeed, we previously identified MerTK as the receptor necessary to POS internalization, and then the alphavbeta5 integrin–MFG-E8 couple as the receptor-ligand proteins synchronizing this rhythmic phagocytosis. Indeed, we demonstrated that MerTK can be activated just on time for the phagocytic peak via intracellular signaling pathways ignited via the alphavbeta5 integrin–MFG-E8 couple. However, MerTK also controls the amounts of POS that can be bound by RPE cells as a negative feedback loop. More recently, we showed that MerTK is cleaved from the RPE cell surface and that this mechanism contributes to the regulation of its function with the participation of its ligands that carry tissue-specific opposite roles. Indeed, these molecular mechanisms are closely related to those used by macrophages to discard apoptotic cells. However, in the retina photoreceptors and RPE cells are permanently in close contact and phagocytosis only happens once a day, underlying the strict regulation of this function at the molecular level.

Current projects aim at 1-identifying new receptors present at the RPE cell surface that could intervene in POS elimination, 2-understanding how an ubiquitous defect linked to mutations in genes from the PRPF splicing factors family leads to tissue-specific retinitis pigmentosa phenotypes linked to dysfunctions in RPE cells in collaboration with Prof. Pierce (Boston, USA). In parallel, our team participates to other collaborations in France and abroad on various topics related to RPE cells (RPE defects in pathologies, characterization of RPE derived from induced pluripotent stem cells (iPSCs), …).

For these projects we combine molecular biology, biochemistry and cell biology for in vitro assays. Simultaneously, we determine the in vivo relevance of our results using electrophysiology, histology and biochemistry. We use both traditional and well-established techniques like site-directed mutagenesis and immunoprecipitations, along with novel technologies such as live imaging, assessment of in vivo function during the different phases of POS internalization by RPE cells and high throughput sequencing/screening. Additionally, we take advantage of known (RCS rat, beta5 integrin knockout mouse) and new rodent models to better understand the phagocytic machinery and characterize new phagocytic proteins. In the particular environment of the Institut de la Vision, we benefit from the state-of-the-art core facilities (sequencing, confocal imaging…) and innovative equipments (animal phenotyping, automated fluorescent assay analyzer, high content screening…). As well, our unique knowledge of RPE cells physiology and function in normal and pathologic conditions benefits researchers in the Institut de la Vision that are specialized in different areas of visual function but not covering RPE cells.

Research areas

    • Molecular mechanisms of retinal phagocytosis and associated pathologies: in the past 17 years, tremendous progress has been made in the understanding of the receptors, ligands and associated signaling pathways regulating retinal phagocytosis. However, how all the known partners timely interact is not fully understood. Similarly, the mechanism by which RPE cells sustain such a sharp and time-limited peak of phagocytosis while RPE cells and POS are intimately and permanently in contact is not clear.

    • Identification of new RPE phagocytic receptors: the phagocytic machinery is extremely complex and tightly regulated, thus other receptors could play a role in its course. In addition, the known machinery has been characterized mostly using rodent models. Therefore, the question is open whether the elimination of aged cone POS is similar to the rod one or has some cell type specificity, which could be relevant for the macular area of the human retina.

    • Identifying the molecular targets underlying the RPE phenotype in Prpf mice: we have shown that Prpf mutations impact the rhythm of 2 crucial RPE functions (phagocytosis and adhesion), however we still do not know the molecular targets of the splicing defects. They could directly (molecular machinery) or indirectly (central control) modify these cyclic functions. In addition, how these early deficiencies are converted in an aging phenotype is still unclear. We thus study oxidative stress pathways at different cell levels (mitochondria, endoplasmic reticulum, lipids accumulation...).





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