Intracellular trafficking, sorting and targeting in health and disease

Background. X-linked retinitis pigmentosa type 3 (XlRP3) is a prevalent form of syndromic retinitis pigmentosa (RP), a photoreceptor neurodegenerative disease, which presents a remarkable degree of allelic and clinical heterogeneity, such as association with partial deafness and increase susceptibility of the upper respiratory tract to infections. The XlRP3 gene encodes the retinitis pigmentosa GTPase regulator (RPGR), whose N-terminal domain shares homology to RCC1 (A), a nuclear guanine-nucleotide exchange factor (GEF) for Ran GTPase. Even though RPGR is expressed ubiquitously, its role is selectively critical to photoreceptor neurons (Ba). To gain insights into the role of RPGR in phototoreceptor function and disease, our laboratory discovered a protein, the RPGR-interacting protein-1 (RPGRIP1), which interacts specifically with RPGR and nephrocystein-4 (NPHP4) (Bb). Mutations affecting distinct domains of RPGRIP1 cause its uncoupling from RPGR or NPHP4. NPHP4 cause nephronophthisis type 4, an end-stage renal failure disorder affecting adolescents, or Senior-Løken syndrome, a retinal-renal disease causing blindness with an early onset. NPHP4 variants have been also linked recently to cardiac malformations. Among the RPGRIP1 isoforms our laboratory identified, the RPGRIP1 isoform is expressed selectively in mouse photoreceptor neurons, where it localizes to their ciliary region (Bc). The lack of RPGRIP1 expression in a mouse model of RPGRIP1 blunts the targeting of RPGR, NPHP4 and other critical ciliary proteins, such as the serologically defined colon cancer antigen-8 (SDCCAG8, also called NPHP10/BBS16), to the connecting cilium of photoreceptors, but not of various cell types of the kidney (C). Impairment of SDCCAG8 in the human causes also Senior-Løken syndrome, nephronophthisis type 10 (NPHP10) or Bardet-Biedl (BBS) syndrome. In cell lines, RPGR prevents the aggregation of RPGRIP1 and it promotes RPGRIP1 localization to or retrieval from the ER (D). The ultimate physiological effect of the impairment of RPGRIP1 is the lack of outer segment formation in photoreceptors followed by their rampant degeneration (D). In the human, mutations in RPGRIP1 cause Leber congenital amaurosis (LCA), the leading cause of blindness in children, and variants of RPGRIP1 have been associated to several forms of glaucoma (e.g. POAG and NTG). Current projects. An emerging model supports RPGRIP1 is crucial to the ER-to-ciliary targeting of its direct partners, RPGR and NPHP4, and of other higher-order assembly components, such as SDCCAG8. However, the dynamic composition of this complex, the biological and functional relationships between their components, and the molecular bases for their cell-context-dependent functions and clinical manifestations and how these processes contribute to or determine the morphogenesis of subcellular structures (e.g. outer segments) are elusive. Current studies are directed at tailoring the RPGRIP1 interactome to probe routes, components and mechanisms underlying polarized trafficking and targeting and to re-examine prior models of non-selective bulk flow or cargo-capture transport of substrates from the ER. Further, disease mutations affecting all components of the RPGRIP1 interactome will aid to the understanding of the biological roles and physiological relevance of such factors in ciliary targeting, morphogenesis of subcellular structures and disease pathogenesis. This overall goal is achieved by expanding and modifying cell-based assays we developed to probe specific steps of protein routing and by examining mouse models of RPGRIP1 and its partners where such steps may be impaired. These approaches will lead to a broader understanding of protein intracellular trafficking and how its impairment promotes neurodegenerative and other diseases which were once thought to be etiologically distinct.

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