Identification of new membrane receptors for daily retinal phagocytosis – Rephago

At the back of the eye lies a monolayer of cells essential for vision: the retinal pigment epithelium (RPE). Apical microvilli from these polarized cells make close contact with the photosensitive photoreceptor outer segments (POS). POS are constantly renewed to fight the high levels of oxidative damage they are subjected to, and one of RPE cell main roles to maintain lifelong vision is the daily elimination of used POS tips by phagocytosis. Absence or failure to complete this task leads to the development of blinding diseases for which no treatment exists, such as early-onset retinal dystrophies or age-related macular degeneration (AMD).
An important feature of RPE phagocytosis is its rhythmic activity. We showed previously that the alphavbeta5 integrin receptor controls the rhythmic activation of RPE phagocytosis. Subsequently, an intracellular signaling cascade activates the Mer tyrosine kinase (MerTK) internalization receptor. Our recent studies suggest that MerTK also controls the amounts of POS that can be tethered by RPE cells, including via the extracellular cleavage of MerTK.
Interestingly, the known machinery for RPE phagocytosis is close to the clearance of apoptotic cells by macrophages. In macrophages, many molecules intervene in such processes, suggesting that similar intricate protein networks could operate in RPE cells. However, tissue specificity exists, including the permanent contact between photoreceptors and RPE cells, thus regulation of the machinery has to be controlled very tightly via several mechanisms to launch and stop phagocytosis at the proper time. Our recent data on the tissue-specific opposite role of MerTK ligands reinforce this idea. Moreover, several other receptors have been shown to be expressed by RPE cells, but their participation in POS phagocytosis has not been investigated yet.
So far, studies on the phagocytic machinery have been performed on nocturnal rodent species using mostly rod photoreceptors sensitive to dim light. However, central vision in humans is mostly due to cones that give us details resolution and color vision. Rod and cone POS membrane structures are different and they are used for vision almost in exclusion of each other, either at night or during the day, respectively. Therefore, our hypothesis is that the elimination of used cone POS could take place at a different timing and with a different molecular machinery than for rod POS.
For these reasons, with the REPHAGO project we plan on identifying the contribution of new membrane receptors in controlling the daily activation of rod (Aim 1) and cone (Aim 2) POS phagocytosis by RPE cells. Candidate receptors for rod POS phagocytosis will be validated in vitro and then in vivo for interesting candidates according to their implication during the phagocytic process. We will characterize cone-specific molecules for POS clearance using transcriptome studies associated with functional validation assays. For this project, we will use multiple state-of the art approaches (functional phagocytosis assays, in vivo phagocytosis assessment, RNAseq, visual animal phenotyping…) as well as new and innovative animal models (RPE-specific knockout mouse models for rod receptor candidates, cone-rich diurnal rodent model). Identified receptors will be then explored in other phagocytic cells.
Understanding the complexity and specificity of protein networks and interactions to complete daily this crucial task will enlighten us both on normal retinal function and on the consequences of phagocytic defects. This will help us consider new avenues for therapies and therapeutic targets for these pathologies for which no treatment exists. As well, the sequential activation of the RPE machinery can help us decipher molecular pathways that are used in other phagocytic cells, and in particular macrophages. Thus, our results could contribute to the understanding of phagocytic processes occurring in other tissues or pathologies such as atherosclerosis.