Reduced Retinal Adhesion in Mice Lacking AVB5 Integrin

Abstract

Abstract: : Purpose: Functional apical interactions of the retinal pigment epithelium (RPE) with the interphotoreceptor matrix and photoreceptor outer segments (POS) are critical for retinal adhesion. Permanent failure of retinal adhesion causes photoreceptor degeneration and loss of vision. Despite their importance, the apical surface receptors of RPE mediating these functions are poorly understood. We have recently characterized a knockout model for αvß5 integrin. Mice lacking this apical surface receptor of the RPE exhibit altered diurnal rhythm of POS phagocytosis suggesting different RPE–POS interactions compared to wild–type animals. Age–related blindness in these animals further illustrates the importance of αvß5 receptors for photoreceptor function. Here, we studied the dependence of retinal adhesion on αvß5 integrin. Methods: The identification of apical RPE receptors involved in retinal adhesion requires studying the RPE in situ because no culture model exists fully reconstituting RPE apical interactions. At different times of day, we dissected eyes of age–matched wild–type and ß5 integrin knockout mice from 3 weeks to 18 months of age. We peeled off the retina from the underlying RPE, lysed retinal proteins and quantified RPE melanin pigment attached to the retina. We quantified neural retina yields by immunoblotting with GFAP retinal marker protein. To assess RPE content in extracts of peeled off retinas, we probed immunoblots with RPE–specific proteins. We compared expression of integrin family adhesion proteins using immunoblotting, immunofluorescence and immunohistochemistry. Results: Melanin content in retinas mechanically separated from the RPE varied with age of wild–type mice. This correlated well with content of the RPE–specific marker protein RPE65 in retinal extracts. Interestingly, we found higher levels of melanin in retinal samples from animals sacrificed at 8AM as compared to 2PM. Furthermore, retinas from age–matched ß5 knockout mice contained significantly lower levels of RPE melanin as well as RPE marker proteins. Conclusions: The diurnal variation in strength of retinal adhesion we observed suggests that receptor–ligand interactions that mediate adhesion in the retina are regulated with time of day. The reduction in retinal adhesion in ß5 knockout retina indicates that αvß5 integrin receptors may contribute to retinal adhesion in addition to their role in synchronizing POS phagocytosis.

Keywords: retinal adhesion • retinal pigment epithelium • retinal degenerations: cell biology