Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

implicated in the transport and membrane targeting of prenylated proteins destined for photoreceptor OS and shows increased labeling near the connecting cilia of bovine photoreceptors, at the junction of IS and OS (38).

The RPGR transcript that was initially identified is widely expressed and contains 19 exons (RPGRex1-19), encoding a predicted 90 kDa protein (27,28). Exons 1–11 encode the RLD, whereas exons 12–19 encode a C-terminal domain rich in acidic residues and ending in an isoprenylation anchorage signal (Fig. 1A). Subsequently, alternative transcripts were found, the most important of which is RPGRORF15 (29), which shows highest expression in photoreceptors, and is the only transcript known to be involved in retinal disease. Human RPGRORF15 contains exons 1–14 of RPGRex1–19 plus a large alternatively spliced C-terminal exon, ORF15, encoding 567 amino acids (Fig. 1A). The full-length human RPGRORF15 isoform encodes a 1152 amino acid protein. Exon ORF15 encodes a repetitive glycine and glutamic acid-rich domain of unknown function and a basic C-terminal domain (ORF15C2),which is evolutionarily conserved and binds the multifunctional chaperone nucleophosmin (NPM) at centrosomes and mitotic spindle poles (39).

RPGR Function

RPGR is a component of centrioles, ciliary axonemes, and microtubular transport complexes, although its precise function is unknown. It co-localizes with RPGRIP1 at the axonemes of connecting cilia in rod and cone photoreceptors (40) by binding to RPGRIP12 because this localization is lost in Rpgrip1 knockout (KO) mice. Rpgr KO mice develop a slow retinal degeneration, with features resembling a cone-rod degeneration—cone photoreceptors degenerate faster than rods and there is partial mislocalization of cone opsins (41). Some residual RpgrORF15 expression has been reported in this model (42).

RPGR has been shown to co-immunoprecipitate in retinal extracts with a number of different axonemal, basal body and microtubular transport proteins (42). These include nephrocystin-5 and calmodulin, which localize to photoreceptor connecting cilia; the microtubule-based transport proteins, kinesin II (KIF3A, KAP3 subunits), dynein (DIC subunit), SMC1, and SMC3; and two regulators of cytoplasmic dynein, p150Glued and p50dynamitin, which tether cargoes to the dynein motor. Inhibition of dynein by overexpressing p50-dynamitin abrogates the localization of RPGRORF15 to basal bodies. RPGRORF15 can be co-immunoprecipitated from retinal extracts with other basal body proteins, including NPM, IFT88, 14-3-3ε, and γ-tubulin (39,42). RPGRORF15 and RPGRIP1 co-localize at centrosomes in a wide variety of nonciliated cells and at basal bodies in ciliated cells (39). Both proteins are core components of centrioles and basal bodies (39). In summary, RPGRORF15 appears to have a role in microtubule-based transport to and from the basal bodies and within photoreceptor axonemes, perhaps concerned with movement of cargoes between IS and OS.

RPGRORF15 is predominantly expressed in photoreceptor connecting cilia and basal bodies but expression has also been reported in OS in some species (43), although this has been disputed (40). RPGR is also expressed in the transitional zone of motile cilia in the epithelial lining of human bronchi and sinuses (RPGRex1–19 only) and within the human and monkey cochlea (40,44).

Fig. 1. (A) Domain structure of two major RPGR isoforms (RPGRex1–19 and RPGRORF15), as discussed in the text. (B) The distribution of reported mutations in the RPGRORF15 isoform within each exon, showing the large excess of mutations within the C-terminal exon ORF15. The types of mutation are also shown, again indicating the excess of deletions due to the ORF15 mutational “hot spot.”

Involvement of RPGR in Disease

240 different mutations in RPGR have been reported which account for a number of different retinopathies. Numerically, the most important group is XLRP because up to 75% of cases in most series result from mutations in RPGRORF15 (29,45–47). Lower figures are reported in which X-linkage is uncertain. Mutations can occur in any part of the protein but the glycine/glutamic-acid rich domain of exon ORF15 is a mutation “hotspot,” accounting for up to 80% of RPGR mutations (29,45–47) (Fig. 1B). Because of its high-mutation rate, RPGR accounts for disease in up to 20% of all patients with RP (45). The highest frequency of mutations is associated with a central repetitive, purine-rich region of ORF15, which contains only 2–3% pyrimidines. The purine-rich sequences may promote polymerase arrest and slipped strand mispairing because the majority of mutations are out-of-frame deletions of 1–5 bp within short repetitive regions. These are predicted to produce truncated proteins of varying length, and can include novel amino acid sequences that change the charge of the domain from acidic to basic. Severe disease is correlated with longer regions of such abnormal sequence (47). Because ORF15 is a C-terminal exon, it is not subject to nonsense-mediated decay, so that truncated RPGRORF15 may accumulate and cause a gain-of-function or

toxic phenotype (48,49). On the other hand, the majority of missense mutations within the RLD are nonconservative and affect highly conserved residues, suggesting a loss- of-function mechanism. Some of the phenotypic variability found with RPGR mutations (in hemizygotes and heterozygotes) may relate to such differences in the mutant protein.

In addition to unconditional and conditional KO mouse models of XLRP, there are naturally occurring animal models in mouse (rd9) and dogs. Canine X-linked progressive retinal atrophy (XLPRA) is subdivided by mutational type into XLPRA1 (Siberian husky, Samoyed), which have the same 5-bbp deletion in exon ORF15, and XLPRA2, which has a 2-bp deletion in ORF15 (mixed breed) (48). The XLPRA1 mutation results in a frameshift and immediate premature stop codon, and shows normal photoreceptor development and function until about 6 mo of age, after which a slow degeneration of rod and cone photoreceptors occurs, with cones being less severely affected. In contrast, the frameshift in XLPRA2 shows a premature stop codon 71 amino acids downstream, including 34 additional basic residues. The XLPRA2 phenotype is very severe and manifests during retinal development, with disorganized and disoriented rod and cone OS, which is worst in rods, and faster degeneration. The XLPRA2 protein aggregates in the endoplasmic reticulum of transfected cells, in contrast to the normal and XLPRA1 proteins (48).

RPGRORF15 mutations can give rise to central or macular dystrophies, including X- linked forms of CRD (50,51), cone dystrophy (29,52), and atrophic macular degeneration (53). In the X-linked CRD families, a parafoveal ring of fundus autofluorescence, similar to that described in XLRP carriers (24), was evident in younger affected males (but not in carriers). Mutations that are closer to the 3′ end of RPGRORF15 and that lack abnormal (basic) sequence tend to be associated with CRD and milder forms of RP (47). Similarly, loss of the final third (180 amino acids) of RPGRORF15 results in X-linked atrophic macular degeneration (53). A histopathological study of a CRD carrier showed a bull’s eye maculopathy, focally absent macular retinal pigment epithelium and absent perifoveal cones and rods (54). Elsewhere in the macula and in the peripheral retina, cone but not rod photoreceptors were reduced in numbers and both rod and cone OS shortened.

Mutations in RPGR can give rise to a syndrome combining features of a primary ciliary dyskinesia, sensorineural hearing loss, and RP (55–58). This syndrome is most commonly associated with deletion, missense or splice site mutations within the RLD of RPGR and can be confused with Usher syndrome.

RP2-TYPE XLRP AND THE RP2 GENE

The second XLRP locus, which mapped to Xp11.2-p11.3 by linkage studies (25,26,59) was positionally cloned in 1998 and named the RP2 gene (60). RP2 consists of five exons and encompasses 3.8 kb of DNA. Exon 2 encodes a cofactor-C homologous domain (CFCHD) and a microtubule-associated protein homologous domain (MAPHD). Exons 3 and 4 encode a NM23 homologous domain (NM23HD) (61) (Fig. 2A). NM23 belongs to a large family of structurally and functionally conserved proteins, consisting of four to six identically folded subunits of approx 16–20 kDa in size. These oligomeric proteins show nucleoside diphosphate kinase (NDPK) activity

Fig. 2. (A) Domain structure of the RP2 protein, as discussed in the text. CFCHD, cofactor-C homologous domain; MAPHD, microtubule-associated protein homologous domain; NM23HD, NM23 homologous domain. (B) The distribution of reported mutations in RP2 in each exon, showing the excess of mutations within exon 2, which encodes the CFCHD and MAPHD domains. The types of mutation are also shown, indicating a more even distribution than with RPGR.

that catalyzes nonsubstrate specific conversions of nucleoside diphosphates to nucleoside triphosphates. Many NM23 proteins bind DNA. In vivo, NM23-NDPKs regulate a diverse range of cellular events, including growth and development (62).

To date, there are 39 RP2 mutations identified in XLRP families. The distribution and type of mutations are shown in Fig. 2B. Most mutations have been found in exon 2, which encodes the CFCHD and MAPHD domains. In three studies in which both RPGR and RP2 were screened for mutation, RP2 mutations accounted for 7–8% of patients with XLRP whereas RPGR accounted for 55–73% of mutations (45–47).

RP2 protein is widely expressed at low levels in human tissues, and has been reported to be targeted to the plasma membrane by myristoylation and palmitoylation of its N-terminal amino acids (63). There is a potential N-myristoyl transferase recognition site at the N-terminus of RP2. The N-terminal 15 amino acids appear to be sufficient for targeting RP2 to the membrane. A single amino acid deletion of Serine residue 6 in

a patient with RP2 resulted in failure of the protein to associate with the plasma membrane (63). It was therefore proposed that XLRP is prevented from reaching the correct cellular location in sufficient amounts for normal activity (63). C-terminal truncation mutations, which account for two-thirds of pathogenic RP2 variants, led to misfolding and subsequent degradation of the resultant nonfunctional proteins. Loss of the protein and/or its aberrant intracellular distribution might therefore be a common basis for the photoreceptor cell degeneration occurring in patients with RP2 (64).

RP2 and the tubulin-specific chaperone cofactor C share 53% similarity and 29% identity over a domain of approx 200 amino acids. Both proteins stimulate the GTPase activity of native tubulin. Their functions are overlapping but not identical, because only cofactor C participates in the heterodimerization of newly folded tubulin subunits. The adenosine diphosphate (ADP)-ribosylation factor-like 2 (Arl2) protein regulates the tubulin-GTPase activating protein (GAP) activity of cofactor C and D. Arl3 does not affect the tubulin-GAP activity of RP2 or cofactor D. RP2 binds to Arl3 in a nucleotide and myristoylation-dependent manner. An R118H pathogenic mutation, which does not affect the normal plasma membrane localization of RP2 (63), implies that this residue acts as an “arginine finger” to trigger the tubulin-GAP activity (65).

RP2 was localized to the plasma membrane of cells throughout the human retina, whereas Arl3 and cofactor C localized predominantly to the connecting cilium of rod and cone photoreceptor cells. The localization of RP2 and its interacting proteins to this region suggest that RP2 may be involved in vesicular transport (66), providing an intriguing parallel with RPGR. Much remains to be learned about the precise functions of RP2 in the retina but at this stage it is certainly possible that, like RPGR, it has a role in some aspect of microtubular function relating IS and OS of photoreceptors.

X-linked retinitis pigmentosa (RP) is a genetically heterogeneous disorder in which two major genes have been identified (RPGR, RP2) that account for the disease in the majority of patients. RPGR joins a growing number of proteins implicated in ciliary function and may be concerned with microtubule-based transport between inner and outer segments (IS and OS, respectively) of rod and cone photoreceptors. The function of RP2 is less clear but it appears to have a role in microtubular assembly or function in photoreceptor IS.

ACKNOWLEDGMENTS

We would like to acknowledge the financial support of the Medical Research Council, The British Retinitis Pigmentosa Society, the Foundation Fighting Blindness, and EVI-GENORET (European Union) for financial support.

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