PROJECT SUMMARY Retinitis pigmentosa (RP) causes irreversible blindness in individuals of all ages. It affects ~1 in 3,500 people worldwide. While RP is initially characterized by night blindness and peripheral vision loss, most RP patients lose their central vision and become legally blind by the age of 40. There is no treatment to slow or stop vision loss. Mislocalization of the light-absorbing protein rhodopsin in the rods is a common hallmark shared by many animal models of RP. The long-term objective of this application is to dissect the molecular pathway that underlies the sorting and delivery of the rhodopsin and determine its disease relevance. This information will accelerate the discovery of new treatments for RP and other retinal degenerative diseases. In the mammalian rods, which are highly compartmentalized, rhodopsin is synthesized in the biosynthetic organelles confined to the inner segment. Rhodopsin is then vectorially delivered to and concentrated in the outer segment (OS). While rhodopsin’s trafficking through the endoplasmic reticulum-Golgi pathway has been investigated, the importance of the endosome in rhodopsin’s OS targeting is unclear. In many other cell types, the endosome s erves as a key sorting station for proteins at the crossroads of multiple intracellular trafficking pathways. Prolonged endosomal accumulation of fly rhodopsin has been shown to lead to light- dependent retinal degeneration. Our preliminary results showed that the newly-synthesized rhodopsin transits through the endosomal compartments in mouse rods in vivo. The OS targeting signal of the rhodopsin binds to an early endosome-specific protein, SARA. SARA deficiency in mouse rods not only causes rhodopsin mislocalization but also several other cellular defects in the endolysosomal system. In this application, we will test the central hypothesis that in mammalian rods the trans-endosomal pathway critically regulates the fidelity and the efficiency of the OS targeting of rhodopsin. First, we will address whether the RP mutant rhodopsins are retained in the endosomes abnormally during their transit to the OS, and whether this defect perturbs the homeostasis of other endomembranes (Aim1). We will generate multiple, complementary mouse models to examine the role of the trans-endosomal pathway in the morphogenesis of the OS and its rhodopsin expression (Aim2). We will also profile the rod proteins that transit through the endosomal system and characterize their interaction with key endosomal trafficking regulators (Aim3). We will achieve these aims by applying state-of-the-art techniques such as rod-specific inducible gene expression and gene deletion, super-resolution confocal microscopy, correlative light-electron microscopy, and 3D scanning electron microscopy. By providing mechanistic insights on photoreceptor protein trafficking and OS biogenesis, this research will contribute to the development of new therapie...