Enhanced Endocytosis, Mitochondrial Stress and NK/NKT Cells Mediated Immune Response Underlie Severe Retinitis Pigmentosa with RHO P347L Mutants

Abstract Purpose: Rhodopsin (RHO) is the initiator of phototransduction and the most frequent cause of autosomal dominant retinitis pigmentosa (adRP). While Class 1 RHO mutations cause a more severe phenotype than Class 2, the underlying pathogenesis remains poorly defined. This study investigates the mechanistic basis for this disparity using cell models and humanized mouse models of Class 1 (P347L) and Class 2 (L125R) mutations. Methods: We first analyzed the localization, degradation and downstream pathway of wild-type (WT) and mutant rhodopsin in 293T cells overexpressing the corresponding plasmids. Then we used humanized mouse models to conduct a side-by-side comparison of mitochondrial dysfunction, photoreceptor death, and neuroinflammation in vivo. Results: Unlike WT rhodopsin, the P347L mutant exhibited plasma membrane localization in a non-polarized manner, whereas the L125R mutant showed partial trafficking to the membrane. Correspondingly, the degradation of P347L was primarily mediated by the lysosomal pathway, while L125R was processed through both proteasomal and lysosomal pathways. The P347L mutant demonstrated a propensity to bind visual arrestin, potentially explaining its observed higher rate of endocytosis. In vivo, the P347L model displayed more severe mitochondrial dysfunction. However, both mutants induced a comparable level of neuroinflammation, characterized by the activation of microglia, natural killer (NK) cells, and natural killer T (NKT) cells. Conclusions: We propose a pathogenic model in which elevated endocytosis and mitochondrial impairment directly underlie the severe RP in P347L mutants, with neuroinflammation serving to amplify the initial insult.