RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP).

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

Prenylated retinal ciliopathy protein RPGR interacts with PDE6δ and regulates ciliary localization of Joubert syndrome-associated protein INPP5E.

Ciliary trafficking defects underlie the pathogenesis of severe human ciliopathies, including Joubert Syndrome (JBTS), Bardet-Biedl Syndrome, and some forms of retinitis pigmentosa (RP). Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are common causes of RP-associated photoreceptor degeneration worldwide. While previous work has suggested that the localization of RPGR to cilia is critical to its functions, the mechanism by which RPGR and its associated cargo are trafficked to the cilia is unclear. Using proteomic and biochemical approaches, we show that RPGR interacts with two JBTS-associated ciliary proteins: PDE6δ (delta subunit of phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E). We find that PDE6δ binds selectively to the C-terminus of RPGR and that this interaction is critical for RPGR's localization to cilia. Furthermore, we show that INPP5E associates with the N-terminus of RPGR and trafficking of INPP5E to cilia is dependent upon the ciliary localization of RPGR. These results implicate prenylation of RPGR as a critical modification for its localization to cilia and, in turn suggest that trafficking of INPP5E to cilia depends upon the interaction of RPGR with PDE6δ. Finally, our results implicate INPP5E, a novel RPGR-interacting protein, in the pathogenesis of RPGR-associated ciliopathies.

Variegated yet non-random rod and cone photoreceptor disease patterns in RPGR-ORF15-associated retinal degeneration.

Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients.

Ocular Ciliopathies: Genetic and Mechanistic Insights into Developing Therapies.

Developing suitable medicines for genetic diseases requires a detailed understanding of not only the pathways that cause the disease, but also the identification of the genetic components involved in disease manifestation. This article focuses on the complexities associated with ocular ciliopathies - a class of debilitating disorders of the eye caused by ciliary dysfunction. Ciliated cell types have been identified in both the anterior and posterior segments of the eye. Photoreceptors (rods and cones) are the most studied ciliated neurons in the retina, which is located in the posterior eye. The photoreceptors contain a specialized lightsensing outer segment, or cilium. Any defects in the development or maintenance of the outer segment can result in severe retinal ciliopathies, such as retinitis pigmentosa and Leber congenital amaurosis. A role of cilia in the cell types involved in regulating aqueous fluid outflow in the anterior segment of the eye has also been recognized. Defects in these cell types are frequently associated with some forms of glaucoma. Here, we will discuss the significance of understanding the genetic heterogeneity and the pathogenesis of ocular ciliopathies to develop suitable treatment strategies for these blinding disorders.

The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo.

Mutations inRPGR(ORF15)(retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGR(ORF15)(1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15)is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2)domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15)is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants ofRpgr, which do not express RPGR(ORF15)due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15)is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15)in photoreceptor cilia.

Cilia in photoreceptors.

Retina is a neurosensory tissue lining the back of the eye and is responsible for light detection and relaying the signal to the visual cortex in the brain. Mammalian retina consists of six major types of neurons (including photoreceptors; rods and cones) and one type of glial cells arranged in distinct layers. Photoreceptors are the most abundant cell types accounting for approximately 60% of all cells in the retina. Owing to their unique structure and function as ciliated neurons and their vast majority, dysfunction and degeneration of photoreceptors is associated with several inherited blindness disorders, such as retinitis pigmentosa, cone-rod degeneration, and age-related macular degeneration. Therefore, it is imperative to examine the structure and function of photoreceptors so that better understanding of the pathogenesis of associated diseases can be obtained for designing therapeutic modalities. In this chapter, we will provide detailed methods for analyzing photoreceptor function (electroretinography), structure, and biochemical analysis of sensory cilia of photoreceptors using mammalian retina as model system. These methods are widely used to assess photoreceptor development and degeneration during disease.

Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition.

Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgr(ko) retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgr(ko) PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgr(ko) PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.

Loss of Raf-1 kinase inhibitory protein delays early-onset severe retinal ciliopathy in Cep290rd16 mouse.

PURPOSE: Mutations in the cilia-centrosomal protein of centrosomal protein of 290 kDa (CEP290) result in severe ciliopathies, including autosomal recessive early onset childhood blindness disorder Leber congenital amaurosis (LCA). The Cep290(rd16) (retinal degeneration 16) mouse model of CEP290-LCA exhibits accumulation of CEP290-interacting protein Raf-1 kinase inhibitory protein (RKIP) prior to onset of retinal degeneration (by postnatal day P14). We hypothesized that reducing RKIP levels in the Cep290(rd16) mouse will delay or improve retinal phenotype. METHODS: We generated double mutant mice by combining the Cep290(rd16) and Rkip(ko) alleles (Cep290(rd16):Rkip(+/ko) and Cep290(rd16):Rkip(ko/ko)). Retinal function was assessed by ERG and retinal morphology and protein trafficking were assessed by histology, transmission electron microscopy (TEM), and immunofluorescence analysis. Cell death was examined by apoptosis. RESULTS: Prior to testing our hypothesis, we examined ERG and retinal morphology of Rkip(ko/ko) mice and did not find any detectable differences compared with wild-type mice. The Cep290(rd16):Rkip(+/ko) mice exhibited similar retinopathy as Cep290(rd16); however, Cep290(rd16): Rkip(ko/ko) double knockout mice demonstrated a substantial improvement (>9-fold) in photoreceptor function and structure at P18 as of Cep290(rd16) mice. We consistently detected transient preservation of photoreceptors at P18 and polarized trafficking of opsins to sensory cilia in the double mutant mice; however, retinal degeneration ensued by P30. CONCLUSIONS: Our studies implicate CEP290-RKIP pathway in CEP290-retinal degeneration and suggest that targeting RKIP levels can delay photoreceptor degeneration, assisting in extending the time-window for treating such rapidly progressing blindness disorder.

Ciliary transition zone (TZ) proteins RPGR and CEP290: role in photoreceptor cilia and degenerative diseases.

INTRODUCTION: Primary cilia are microtubule-based extensions of the plasma membrane in nearly all cell types. In vertebrate photoreceptors, the sensory cilium develops as outer segment (OS) that contains the photopigment rhodopsin and other proteins necessary for phototransduction. The distinct composition of proteins and lipids in the OS membrane is maintained by the selective barrier located at the border between the basal body and the ciliary compartment, called the transition zone (TZ). AREAS COVERED: In this review, we will discuss the identification and function of two ciliary TZ proteins, RPGR (retinitis pigmentosa GTPase regulator) and CEP290. Mutations in these proteins account for a majority of retinopathies due to ciliary dysfunction. We will also discuss the potential of such information in designing therapeutic approaches to treat cilia-dependent photoreceptor degenerative diseases. EXPERT OPINION: RPGR and CEP290 perform overlapping yet distinct functions in regulating trafficking of cargo via the TZ of photoreceptors. While RPGR modulates the trafficking by acting as a GEF for the small GTPase RAB8A, CEP290 may be involved in maintaining the polarized distribution of proteins in the OS by modulating intracellular levels of selected proteins involved in inhibiting OS formation.