Retinitis pigmentosa GTPase regulator (RPGRr)-interacting protein is stably associated with the photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium

Retinitis pigmentosa (RP) is a blinding retinal disease in which the photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the photoreceptor connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the cilium. RPGRIP is the only protein known to localize specifically in the photoreceptor connecting cilium. As such, it is a candidate gene for human photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a photoreceptor-specific phenotype.     

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.     

The interplay between RPGR,PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR‐interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo‐loaded PDEδ and exposes the Arl2/Arl3‐binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo‐loaded PDEδ and Arl3 to release lipidated cargo into cilia.     

Polarity proteins control ciliogenesis via kinesin motor interactions

BACKGROUND: Cilia are specialized organelles that play a fundamental role in several mammalian processes including left-right axis determination, sperm motility, and photoreceptor maintenance. Mutations in cilia-localized proteins have been linked to human diseases including cystic kidney disease and retinitis pigmentosa. Retinitis pigmentosa can be caused by loss-of-function mutations in the polarity protein Crumbs1 (CRB1), but the exact role of CRB1 in retinal function is unclear. RESULTS: Here we show that CRB3, a CRB1-related protein found in epithelia, is localized to cilia and required for proper cilia formation. We also find that the Crumbs-associated Par3/Par6/aPKC polarity cassette localizes to cilia and regulates ciliogenesis. In addition, there appears to be an important role for the polarity-regulating 14-3-3 proteins in this process. Finally, we can demonstrate association of these polarity proteins with microtubules and the microtubular motor KIF3/Kinesin-II. CONCLUSIONS: Our findings point to a heretofore unappreciated role for polarity proteins in cilia formation and provide a potentially unique insight into the pathogenesis of human kidney and retinal disease.     

Overexpression of RPGR leads to male infertility in mice due to defects in flagellar assembly

Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.     

Cohesin protein SMC1 is a centrosomal protein

Structural maintenance of chromosome protein 1 (SMC1) is well known for its roles in sister chromatid cohesion and DNA repair. In this study, we report a novel centrosomal localization of SMC1 within the cytoplasm in a variety of mammalian cell lines. We showed that SMC1 localized to centrosomes throughout the cell cycle in a microtubule-independent manner. Biochemically, SMC1 was cofractionated with the centrosomal protein γ-tubulin in centrosomal preparation. Immunohistochemistry and immunoelectron microscopy performed on mouse tissue sections revealed that SMC1 antibody strongly labeled the base of cilia in ciliated epithelia, where basal bodies were located. Furthermore, we showed that SMC1 was associated with both centrioles of a centrosome at G0/G1 stage of the cell cycle. These results demonstrate that SMC1 is a centrosomal protein, suggesting possible involvement of SMC1 in centrosome/basal body-related functions, such as organization of dynamic arrays of microtubules and ciliary formation.     

Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

RPGR-containing protein complexes in syndromic and non-syndromic retinal degeneration due to ciliary dysfunction

Dysfunction of primary cilia due to mutations in cilia-centrosomal proteins is associated with pleiotropic disorders. The primary (or sensory) cilium of photoreceptors mediates polarized trafficking of proteins for efficient phototransduction. Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein mutated in >70% of X-linked RP cases and 10%–20% of simplex RP males. Accumulating evidence indicates that RPGR may facilitate the orchestration of multiple ciliary protein complexes. Disruption of these complexes due to mutations in component proteins is an underlying cause of associated photoreceptor degeneration. Here, we highlight the recent developments in understanding the mechanism of cilia-dependent photoreceptor degeneration due to mutations in RPGR and RPGR-interacting proteins in severe genetic diseases, including retinitis pigmentosa, Leber congenital amaurosis (LCA), Joubert syndrome, and Senior-Loken syndrome, and explore the physiological relevance of photoreceptor ciliary protein complexes.     

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.     

X 性连锁视网膜色素变性中的RP G R 基因的研究进展

视网膜色素变性是一组常见的遗传性致盲眼病,患病率约为1／3500。X染色体连锁遗传RP作为其中的一种类型,具有发病早,损害最为严重等特点。而在XLRP的相关基因中RPGR有着重要的意义。本文就RPGR的定位克隆、结构、功能及其突变谱予以综述,并对该基因的突变研究的临床意义作出了相关阐述。     

A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP. Mutations in an alternatively spliced RPGR exon, ORF15, have recently been shown to account for 60% of XLRP in a European cohort of 47 families. We have performed, in a North American cohort of 234 families with RP, a comprehensive screen of the RP2 and RPGR (including ORF15) genes and their 5' upstream regions. Of these families, 91 (39%) show definitive X-linked inheritance, an additional 88 (38%) reveal a pattern consistent with X-linked disease, and the remaining 55 (23%) are simplex male patients with RP who had an early onset and/or severe disease. In agreement with the previous studies, we show that mutations in the RP2 gene and in the original 19 RPGR exons are detected in <10% and approximately 20% of XLRP probands, respectively. Our studies have revealed RPGR-ORF15 mutations in an additional 30% of 91 well-documented families with X-linked recessive inheritance and in 22% of the total 234 probands analyzed. We suggest that mutations in an as-yet-uncharacterized RPGR exon(s), intronic changes, or another gene in the region might be responsible for the disease in the remainder of this North American cohort. We also discuss the implications of our studies for genetic diagnosis, genotype-phenotype correlations, and gene-based therapy.     

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.     

Phosphorylation by casein kinase 2 induces PACS-1 binding of nephrocystin and targeting to cilia

Mutations in proteins localized to cilia and basal bodies have been implicated in a growing number of human diseases. Access of these proteins to the ciliary compartment requires targeting to the base of the cilia. However, the mechanisms involved in transport of cilia proteins to this transitional zone are elusive. Here we show that nephrocystin, a ciliary protein mutated in the most prevalent form of cystic kidney disease in childhood, is expressed in respiratory epithelial cells and accumulates at the base of cilia, overlapping with markers of the basal body area and the transition zone. Nephrocystin interacts with the phosphofurin acidic cluster sorting protein (PACS)-1. Casein kinase 2 (CK2)-mediated phosphorylation of three critical serine residues within a cluster of acidic amino acids in nephrocystin mediates PACS-1 binding, and is essential for colocalization of nephrocystin with PACS-1 at the base of cilia. Inhibition of CK2 activity abrogates this interaction and results in the loss of correct nephrocystin targeting. These data suggest that CK2-dependent transport processes represent a novel pathway of targeting proteins to the cilia.     

Selective loss of RPGRIP1-dependent ciliary targeting of NPHP4, RPGR and SDCCAG8 underlies the degeneration of photoreceptor neurons

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 Rpgrip1^nmf247 photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1^nmf247 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 Rpgrip1^nmf247, 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.     

Different effects of Tetrahymena IFT172 domains on anterograde and retrograde intraflagellar transport

Intraflagellar transport (IFT) particles are multiprotein complexes that move bidirectionally along the cilium/flagellum. The Tetrahymena IFT172 gene encodes a protein with an N-terminal WD domain (WDD) and a C-terminal repeat domain (RPD). Epitope-tagged Ift172p localized to the basal body and in cilia along the axoneme, and IFT172 knockout cells lost cilia and motility. Using serial deletion constructs to rescue the knockout cells, we found that neither the WDD nor the RPD alone is sufficient to assemble cilia. Ift172p containing only the WDD or the RPD failed to enter cilia. Constructs with a partial truncation of the RPD still rescued although cilia were assembled less efficiently, indicating that the WDD and a part of the RPD are sufficient for anterograde transport. Partial truncation of the RPD caused the accumulation of truncated Ift172p itself and of Ift88p at ciliary tips, suggesting that IFT turnaround or retrograde transport was affected. These results implicate different regions of Ift172p in different steps of the IFT process.     

Characterization of MPP4, a gene highly expressed in photoreceptor cells,and mutation analysis in retinitis pigmentosa

Membrane-associated guanylate kinase (MAGUK) proteins are cell-cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein-protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31-33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa.     

Independent origin and restricted distribution of RPGR deletions causing XLPRA

Canine X-linked progressive retinal atrophy (XLPRA) is an inherited blinding disorder caused by mutations in the ORF15 of the RPGR gene and homolog to human retinitis pigmentosa 3 (RP3). The disease is observed in 2 variations, XLPRA1 in Siberian husky and samoyed and XLPRA2 derived from mongrel dogs. A third, neutral, deletion has been described in red wolves. Haplotype analysis of the 633-kbp RP3 interval in 6 different canidae confirmed the same decent for the XLPRA1 mutation in both affected breeds but suggests a recent and independent origin for both forms of XLPRA. The RP3 interval was excluded from causative associations with blindness in the red wolf and akita, a breed closely related to Nordic sled dogs. Overall, these data suggest a limited distribution of the affected haplotypes and indicate that mutations in the ORF15 are likely to be limited to the described dog breeds.