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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.
The cilium is an extension of the cell membrane formed by nucleation of microtubules. The primary cilium is present in almost all cell types and has diverse functions (Pazour and Witman 2003; Davenport and Yoder 2005; Scholey and Anderson 2006); it contains a central core structure (the axoneme), which comprises of nine outer doublet microtubules with no central microtubule pair (9+0) (Pazour and Witman 2003). The ciliary subunits are assembled at the basal body or mother centriole of post-mitotic cells. This process involves coordinated action of centrosomal proteins and small GTPases that control the switch between cytokinesis and ciliogenesis (Doxsey 2001; Spektor et al. 2007; Tsang et al. 2008). During ciliogenesis, protein complexes are transported distally for growth of the axoneme using an elaborate mechanism called intraflagellar transport (IFT) (Rosenbaum et al. 1999). According to the current model of IFT, protein and membrane cargo are transported bidirectionally along the axoneme by coordinated action of kinesin (anterograde; KIF family members) and dynein (retrograde) motors (Besharse et al. 2003; Follit et al. 2006).
Given their near-ubiquitous presence, cilia are involved in diverse cellular processes, including establishment of left–right asymmetry, sonic hedgehog signalling, mechanosensation, olfaction, chemosensation, and photo-transduction (Gerdes et al. 2009). Commensurate with this, defects in primary cilia result in severe developmental and lethal disorders, such as altered embryonic patterning, renal cystic diseases, mental retardation and photoreceptor degeneration (Gerdes et al. 2009).
Photoreceptor inner segment (having the metabolic machinery) and the outer segment (membranous disks containing phototransduction proteins) are linked by a connecting cilium, which is a modified primary cilium (Young 1968) (figure 1). Approximately 10% of outer segments are turned over each day, with new discs being formed proximally and shed distally. About 2000 opsin molecules are transported per minute to maintain the function/integrity of each rod outer segment (Besharse 1986); these molecules are synthesized in the inner segment, sorted at the post-Golgi vesicles and transported to the base of the connecting cilium, where they probably associate with transport proteins for trafficking to the outer segment (Chuang and Sung 1998; Deretic et al. 1998). The polarized post-Golgi trafficking and docking of rhodopsin at the basal bodies involve the activity of small GTPases, including Rab8 and ARF4 (Deretic et al. 1995; Moritz et al. 2001) and is also probably mediated by a FYVE-domain containing protein SARA, phosphatidylinositol 1-phosphate (PI3P), and syntaxin-3 (Chuang et al. 2007; Mazelova et al. 2009). Perturbation of rhodopsin transport leads to RP (Sung et al. 1994; Colley et al. 1995).
The transition zone (TZ) of photoreceptor sensory cilium serves as a ‘transport corridor’ for bidirectional trafficking of macromolecular complexes along the microtubule network (figure 1). IFT particle proteins, including Tg737/Polaris/IFT88, localize at the basal body and the axoneme of photoreceptor cilium (Pazour et al. 2002). Disruption of IFT in Kif3a conditional knockout mice (Marszalek et al. 2000) and in Tg737orpk mice (Pazour et al. 2002) results in opsin accumulation in inner segments and consequently photoreceptor degeneration. The phototransduction proteins, transducin, arrestin, and recoverin undergo lightdependent reversible translocation between outer and inner segments (Sokolov et al. 2002; Strissel et al. 2005, 2006). Transport of arrestin is, at least in part, mediated by simple diffusion and anchoring through protein–protein interactions (Nair et al. 2005).
Retinal degeneration due to ciliary dysfunction appears as part of a spectrum of diseases, where one end are some forms of Leber congenital amaurosis (LCA) (MIM 204000), characterized at birth or during early childhood. On the other side of the spectrum resides RP (MIM 268000), wherein a majority of patients exhibit early signs of night blindness in the early stages (due to rod photoreceptor dysfunction) progressing to decreased visual fields and culminating in complete blindness usually in the later stages of life. Depending upon the gene, age of onset phenotype can vary. RP is inherited in autosomal dominant (~30% of cases), autosomal recessive (~20%) as well as X-linked manner (Daiger et al. 2007).
XLRP is one of the severe forms of RP associated with considerable clinical and genetic heterogeneity and accounts for 10%–20% of inherited nonsyndromic RP (Fishman 1978; Jay 1982; Breuer et al. 2002). Most affected males exhibit early-onset visual symptoms with night blindness in the first decade and rapid progression towards blindness by age 40 (Bird 1975; Fishman et al. 1988; Daiger et al. 2007; Shu et al. 2007). Heterozygous carrier females can show electroretinographic (ERG) abnormalities and tapetal-like reflex (Bird 1975; Fishman et al. 1986; Cideciyan and Jacobson 1994; Sieving 1995). Some XLRP patients have abnormal sperm phenotype (Hunter et al. 1988) or hearing defects (Zito et al. 2003; Iannaccone et al. 2004). To date, six genetic loci have been identified of which, two causative genes RP2 and RP3 (RPGR) have been cloned (Wright et al. 1991; McGuire et al. 1995; Fujita et al. 1996; Meindl et al. 1996; Roepman et al. 1996; Gieser et al. 1998; Schwahn et al. 1998; Hardcastle et al. 2000; Melamud et al. 2006).
Mutations in RP2 account for approximately 10% of XLRP (Hardcastle et al. 1999; Mears et al. 1999; Sharon et al. 2000, 2003; Breuer et al. 2002). The RP2 gene encodes a putative protein of 350 amino acids (Schwahn et al. 1998; Chapple et al. 2000). The crystal structure of the RP2 protein reveals an amino-terminal β-helix that is structurally and functionally homologous to the tubulin-specific chaperone, cofactor C; most disease-causing missense mutations are present in this domain (Bartolini et al. 2002; Grayson et al. 2002; Kuhnel et al. 2006). RP2 appears to be targeted predominantly to the plasma membrane (Chapple et al. 2000; Grayson et al. 2002). It interacts with ADP-ribosylation factor-like 3 (ARL3) (Grayson et al. 2002; Kuhnel et al. 2006), a microtubule-associated small GTP-binding protein (Kahn et al. 2005), which localizes to the connecting cilium of photoreceptors (Grayson et al. 2002; Schrick et al. 2006).
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are associated with more than 70% of the patients with X-linked retinitis pigmentosa (XLRP) and 10%–20% of simplex RP males (Meindl et al. 1996; Roepman et al. 1996; Breuer et al. 2002; Vervoort and Wright 2002; Sharon et al. 2003). These data indicate that RPGR mutations are one of the most common causes of retinal degeneration in addition to rhodopsin. In addition, some patients with mutations in RPGR may also display syndromic features like primary cilia dyskinesia or hearing loss (van Dorp et al. 1992; Iannaccone et al. 2003, 2004; Koenekoop et al. 2003; Zito et al. 2003; Moore et al. 2006). RPGR is predominantly located at the primary cilium of photoreceptors and multiple different isoforms are recognized (Hong et al. 2003; Khanna et al. 2005; He et al. 2008); however two major ones are recognized: RPGRORF15 and RPGREX1−19 (Vervoort et al. 2000; Breuer et al. 2002).
Complex-splicing patterns are reported for RPGR though the physiological relevance of these transcripts is unclear (Yan et al. 1998; Kirschner et al. 1999; Vervoort et al. 2000; Hong and Li 2002; Ferreira 2005). Purine-rich exon splicing enhancers in ORF15 may also modify the efficiency of splicing (Hong and Li 2002). Multiple immunoreactive bands are observed using isoform-specific RPGR antibodies (Yan et al. 1998; Hong and Li 2002; Mavlyutov et al. 2002; He et al. 2008; Khanna et al. 2005; Otto et al. 2005; Shu et al. 2005; Chang et al. 2006). The constitutive RPGREX1−19 isoform is isoprenylated and localizes to golgi in transfected cells (Yan et al. 1998). The amino-terminal region of RPGR encoded by exons 1–15 encompasses a common RCC1-like domain (Meindl et al. 1996; Vervoort et al. 2000) (figure 2). Although RCC1 is a guanine nucleotide exchange factor (GEF) for Ran GTPases (Meindl et al. 1996; Renault et al. 2001; Sazer et al. 2005), no such activity has yet been reported for RPGR. The RPGRORF15 isoforms that include a C-terminal acidic domain rich in Glu–Gly repeats (Vervoort et al. 2000) are reported to have distinct subcellular localizations (Mavlyutov et al. 2002; Hong et al. 2003). In addition, multiple predicted homology domains can also be detected in the primary structure of the RPGR protein. RPGRORF15 localizes predominantly to the cilia and basal bodies of both mouse and human photoreceptors though some additional labelling can be found in the inner and outer segments (Khanna et al. 2005; Shu et al. 2005). In proliferating cells, centrosomes and midbody are labelled with anti-RPGR antibodies (?Chang et al. 2006; Shu et al. 2005). Our analysis of RPGR using isoform-specific antibodies revealed that the RPGREX1−19 and RPGRORF15 isoforms localize to overlapping as well as distinct subcellular compartments in the retina (He et al. 2008). Moreover, the RPGREX1−19 isoforms exist in overlapping protein complexes with RPGRORF15.
A Rpgr-knockout (ko) mouse with deletion of exons 4–6 of Rpgr is reported to show late-onset cone-rod degeneration (Hong et al. 2000); however, this Rpgr-ko mouse is not a complete null and expresses some RPGRORF15 isoforms that localize to the photoreceptor cilium (Khanna et al. 2005). The phenotype of this mouse can be partially rescued by an ORF15-variant (Hong et al. 2005). However, the same variant in transgenic mice may result in dominant gain-of-function phenotype exhibiting rapid disease progression (Hong et al. 2004). Attempts to generate a complete Rpgr null mutation in mice showing no RPGR isoforms have so far been futile. RPGRORF15 frameshift mutations have been identified in two naturally occurring canine mutants; the XLPRA2 dog exhibits relatively rapid photoreceptor degeneration and severe ERG abnormalities, whereas the XLPRA1 mutant shows a milder phenotype (Zhang et al. 2002; Beltran et al. 2006).
Until 2005, two RPGR-interacting proteins were identified by Y2H analysis using the RLD bait: RPGRIP1, which is localized to the sensory cilium and mutated in patients with LCA (Boylan and Wright 2000;Dryja et al. 2001;Hong et al. 2001); and PDE6d, a prenyl-binding protein involved in the retrieval of PDE from rod outer segmentmembranes by interacting with the GTPase Rab13 (Linari et al. 1999; Zhang et al. 2004). Two chromosome-associated proteins, SMC1 and SMC3 and a centrosomal protein, nucleophosmin (NPM) (Shu et al. 2005) directly interact with RPGR (Khanna et al. 2005; Hirano 2006). Later IP studies identified intraflagellar transport protein, Tg737/Polaris/IFT88, which is involved in ciliogenesis and photoreceptor outer segmentmaintenance in mice (Pazour et al. 2002; Davenport and Yoder 2005) and with several microtubule transport proteins, including cytoplasmic dynein and kinesin-II as part of RPGR containing protein complexes in the retina (Khanna et al. 2005). Studies on the analysis of selected RPGR-interacting ciliary disease proteins are described below.
The RPGR interacting protein 1 (RPGRIP1) was first described as an interactor of RPGR after screening a bovine retina library by yeast two-hybrid analysis (Boylan and Wright 2000; Roepman et al. 2000). Initial analysis showed that RPGR and RPGRIP1 can co-localize from the connecting cilium towards the outer segment of bovine and human photoreceptors, and disease associated mutations in RPGR can disrupt this interaction. Subsequent studies revealed that mutations in RPGRIP1 are associated with LCA (Dryja et al. 2001; Gerber et al. 2001). RPGRIP1 is predominantly localized at the connecting cilium of mouse photoreceptors (Hong et al. 2001), and the Rpgrip1 ko mice features earlyonset retinal degeneration (Zhao et al. 2003). Analysis of the Rpgrip1−/− mice revealed redistribution of RPGR in the inner segments as opposed to the sensory cilium of photoreceptors, suggesting that RPGRIP1 functions as an anchoring protein for RPGR (Hong et al. 2001; Zhao et al. 2003).
Nephronophthisis (NPHP) is characterized by progressive loss of renal function when presented with RP is termed Senior–Loken syndrome (SLSN). Nine NPHP genes (NPHP1–9) have been identified; all are implicated in ciliary function (Hildebrandt and Otto 2005; Chang et al. 2006; Khanna et al. 2009). All patients with NPHP5 mutations develop RP (Otto et al. 2005). We have shown that NPHP5 localizes to photoreceptor cilium and interacts with RPGR, highlighting their functional interaction in ciliary transport (Otto et al. 2005).
CEP290 is a cilia-centrosomal protein that was initially identified as a tumour antigen (Guo et al. 2004). Centrosomes are the major microtubule-organizing centre for the cell (Doxsey et al. 2005) and mutations in centrosomal proteins are associated with syndromic diseases like Alstrom syndrome (MIM 203800) or complex pathological processes like cancer (Andersen et al. 2003; Badano et al. 2005). CEP290 is not the exception; mutations in the CEP290/NPHP6 are associated with syndromic disorders, including Joubert syndrome, Meckel–Gruber syndrome, and BBS (Sayer et al. 2006; Valente et al. 2006; Baala et al. 2007; Leitch et al. 2008). We showed that a hypomorphic allele of the Cep290 gene in the rd16 (retinal degeneration 16) mouse model of autosomal recessive early-onset retinal degeneration and olfactory dysfunction (Chang et al. 2006). The mutant CEP290 protein exhibits increased association with RPGR and likely results in the mislocalization of RPGR in photoreceptors (Chang et al. 2006).
Based on these studies, den Hollander et al. and others screened LCA patients formutations in the CEP290 gene and reported predicted hypomorphic alleles as a frequent cause of LCA (den Hollander et al. 2006; Helou et al. 2007; Perrault et al. 2007). We later showed that hypomorphic CEP290 alleles are also associated with olfactory dysfunction in patients (McEwen et al. 2007). Taken together, we suggest that loss of function mutations in CEP290 can cause pleiotropic and developmental disorders while a moderately functional CEP290 protein primarily results in sensory deficits.
Mutations in RPGR interacting protein 1-like (RP-GRIP1L/NPHP8) are associated with Joubert syndrome, Meckel–Gruber syndrome and Bardet–Biedl syndrome (Arts et al. 2007; Delous et al. 2007; Wolf et al. 2007; Brancati et al. 2008; Doherty et al. 2009). RPGRIP1L regulates sonic hedgehog signalling pathway and mediates left–right asymmetry as well as limb patterning. Interestingly, a missense variant of RPGRIP1L and A229T is frequently associated with retinal degeneration in ciliopathy patients (Khanna et al. 2009). RPGRIP1L can physically interact with RPGR, and the A229T in RPGRIP1L variation severely compromises this interaction (Khanna et al. 2009). These data suggest that variations in NPHP8 can act as modifier alleles that affect the penetrance and expressivity of the retinopathy phenotype, likely through its interaction with RPGR.
Over 200 different RPGR mutations have so far been reported in patients with X-linked retinopathies of diverse clinical phenotypes. A vast majority of these are nonsense mutations or deletions/insertions resulting in frameshift, which are predicted to cause premature truncation of the RPGRORF15 protein. While mutations in the ORF15 exon are generally associated with a milder disease, mutations in RPGR exons 1–14 result in a more severe disease. Initially, most human RPGR mutations were hypothesized to have a null phenotype in males; however, wide variations in clinical phenotype of males and carrier females, and complexities associated with RPGR transcripts and protein isoforms strongly indicate that several disease alleles may in fact be hypomorphs (with partial function).
What is the role of RPGR as part of distinct multiprotein complexes at the cilium and how do mutations in RPGR cause photoreceptor degeneration? Though basic components associated with ciliary transport have been discovered (Rosenbaum et al. 1999; Rosenbaum 2002), the mechanisms of cargo sorting and assembly of protein complexes, and their regulation by signalling pathways in photoreceptors have not been elucidated. Periodic turnover of photoreceptor outer segments demands efficient functioning of the ciliary transport process (Besharse et al. 2003). We hypothesize that RPGR facilitates the assembly of transport protein complexes by interacting with distinct ciliary—basal body— centrosome (CBC) proteins, and that RPGR’s localization in the photoreceptor cilia is necessary for efficient intersegmental transport (figure 3).
Owing to the associated clinical heterogeneity, a detailed genotype–phenotype correlation analysis is critical to understand the progression and pathogenesis of RPGR-associated disease. These studies will benefit from characterization of additional animal model systems representing RPGR mutations as well as identification of components of the RPGR-interactome in photoreceptors. For example, characterization of photoreceptor dysfunction and degeneration in knock-in mouse mutants of Rpgr can assist in understanding associated disease pathogenesis. In addition, functional analysis of the disease-causing mutations of RPGR, such as the effect on RPGR localization, integrity of the interactome and effect on cilia-dependent development are required to delineate the mechanism of heterogenic phenotype observed in patients. These investigations should assist in designing rational therapeutic paradigms for XLRP as well as associated ciliary disorders.
This work is supported by National Eye Institute intramural funds and grants from the National Institutes of Health (EY007961), Foundation Fighting Blindness (FFB), and Midwest Eye Banks and Transplantation Center.