Project Summary/Abstract Loss of function and viability of rod photoreceptors is central to the etiology of retinitis pigmentosa (RP) which affects 1 in ~4,000. Our lab has a long-term interest in understanding the endosome's role in membrane trafficking of photoreceptors and much has been learned about the outer segment protein targeting. In contrast, we know very little about how the mistrafficked proteins are degraded, and the consequence(s) of the generation of non-degradable wastes. Emerging studies showed the endo-lysosomal system is a genetic hot spot for several neurological diseases such as Alzheimer's and Parkinson's, whose pathology is contributed by both the primary neuronal lesions and sustained microglial inflammation. During the past grant period, we generated a mouse line with rod-specific deletion of VPS35. The early endosomal protein VPS35 is the hub that centrally controls several interconnected trafficking pathways. VPS35 has been genetically linked to Alzheimer's and Parkinson’s. Our results showed that in these mutant mice several outer segment proteins were mislocalized and underwent proteolytic degradation. Strikingly, VPS35 deficient rod terminals accumulated massive lipid-membrane wastes, which were engulfed by the surrounding microglia, which then migrated away to the subretinal space. The latter expressed the molecular signatures of disease-associated microglia identified in Alzheimer's mouse models. The level of sphingolipid, which has been connected to synaptic membrane integrity and neural inflammation, was also abnormally elevated in mutant mice. The overarching goal here is to test a central hypothesis that the engulfment of the rod-derived sphingolipid-rich wastes activates microglia, leading to several functional deficits (e.g., phagocytosis, clearance) and inflammation. We will mechanistically investigate the pathological contribution by sphingolipids (Aim1) and microglia (Aim2) using interdisciplinary and state-of-the-art techniques (e.g., lipidomics, transcriptomes, 3D electron microscopy, multi-antigen flow cytometry) both in vivo and in vitro. We will also address whether inhibiting any of these pathways can offset the sustained microglial inflammation, and in turn, ameliorate retinal pathology. The proposed studies will provide keen insights into the fundamental understanding of the retina homeostasis harnessed by the photoreceptor-microglia crosstalk. They have a high potential to lead to new strategies for treating RP and potentially other neurological diseases with overlapping etiologies.