Abstract
The retinitis pigmentosa GTPase regulator (RPGR) and nephrocystin-4 (NPHP4) comprise two key partners of the assembly complex of the RPGR-interacting protein 1 (RPGRIP1). Mutations in RPGR and NPHP4 are linked to severe multisystemic diseases with strong retinal involvement of photoreceptor neurons, whereas those in RPGRIP1 cause the fulminant photoreceptor dystrophy, Leber congenital amaurosis (LCA). Further, mutations in Rpgrip1 and Nphp4 suppress the elaboration of the outer segment compartment of photoreceptor neurons by elusive mechanisms, the understanding of which has critical implications in uncovering the pathogenesis of syndromic retinal dystrophies. Here we show RPGRIP1 localizes to the photoreceptor connecting cilium (CC) distally to the centriole/basal body marker, centrin-2 and the ciliary marker, acetylated-α-tubulin. NPHP4 abuts proximally RPGRIP1, RPGR and the serologically defined colon cancer antigen-8 (SDCCAG8), a protein thought to partake in the RPGRIP1 interactome and implicated also in retinal–renal ciliopathies. Ultrastructurally, RPGRIP1 localizes exclusively throughout the photoreceptor CC and Rpgrip1nmf247 photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1nmf247 mice without RPGRIP1 expression lack NPHP4 and RPGR in photoreceptor cilia, whereas the SDCCAG8 and acetylated-α-tubulin ciliary localizations are strongly decreased, even though the NPHP4 and SDCCAG8 expression levels are unaffected and those of acetylated-α-tubulin and γ-tubulin are upregulated. Further, RPGRIP1 loss in photoreceptors shifts the subcellular partitioning of SDCCAG8 and NPHP4 to the membrane fraction associated to the endoplasmic reticulum. Conversely, the ciliary localization of these proteins is unaffected in glomeruli or tubular kidney cells of Rpgrip1nmf247, but NPHP4 is downregulated developmentally and selectively in kidney cortex. Hence, RPGRIP1 presents cell type-dependent pathological effects crucial to the ciliary targeting and subcellular partitioning of NPHP4, RPGR and SDCCAG8, and acetylation of ciliary α-tubulin or its ciliary targeting, selectively in photoreceptors, but not kidney cells, and these pathological effects underlie photoreceptor degeneration and LCA.
Highlights
The retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) interactome was found to be comprised of at least three proteins, RPGR, nephrocystin-4/nephroretinin (NPHP4) and RPGRIP1 itself.[12,13,14,15,16] human-recessive
The localizations of RPGRIP1a and its assembly components at various ciliary regions of photoreceptors (Figures 1a and b) were compared between wild-type and Rpgrip1nmf[247] mice at P12 of age (Figures 1c–g), when the outer segments begin to develop in wild-type mice and importantly, before pathomorphological changes in the inner segments and cell death ensue in Rpgrip1nmf[247] photoreceptors,[35] because the outcome of these manifestations may lead to the expression of confounding phenotypes
RPGRIP1a localized to the connecting cilium (CC) distally to the centriole/basal body (BB) marker, centrin-2,42 with which it colocalizes partially (Figure 1c)
Summary
RPGRIP1a determines distinct ciliary localizations of NPHP4, RPGR and SDCCAG8 in photoreceptors. RPGRIP1a abuts distally another ciliary marker, acetylated a-tubulin, but its immunostaining was strongly decreased in Rpgrip1nmf[247] photoreceptors (Figure 1d). (c–g) Left and right panels, respectively, are low- and high-magnification images of distal regions of P12 retinas showing: localization of RPGRIP1a in the connecting cilium and juxtaposed distally to the centriolar/basal body marker, centrin-2, whose localization in the basal body is not affected by loss of RPGRIP1a in Rpgrip1nmf[247] (c); ciliary localization of RPGRIP1a distal of and abutting acetylated a-tubulin and whose ciliary signal is strongly decreased in Rpgrip1nmf[247] (d); ciliary localization of RPGRIP1a distal to NPHP4, whose ciliary localization is abolished in Rpgrip1nmf[247] (e); localization of RPGR at the connecting cilium distal to NPHP4, the ciliary localization of both proteins are abolished in Rpgrip1nmf[247] (f); NPHP4 ciliary localization abutting proximally SDCCAG8, whose ciliary localization is strongly reduced in Rpgrip1nmf[247] (g). Despite the strong decrease and lack of ciliary staining of SDCCAG8 and NPHP4, respectively, in Rpgrip1nmf[247] photoreceptors, the loss of ciliary
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