Photoreceptor degeneration is associated with a number of syndromic diseases, such as Senior–Loken syndrome and Bardet–Biedl syndrome (BBS) (4
), as well as with non-syndromic inherited retinal degenerative diseases. Over 100 genetic loci for inherited retinal degenerations have been identified (see RetNet: http://www.sph.uth.tmc.edu/Retnet/
). Mouse models of retinal degeneration provide candidate genes for human retinopathies and offer critical insights into disease pathogenesis. Here, we report that early-onset retinal degeneration in the rd16
mouse is associated with an in-frame deletion in a novel centrosomal protein CEP290. Our data suggest that CEP290 regulates intracellular protein trafficking and that perturbation of its function leads to mislocalization of ciliary and phototransduction proteins resulting in photoreceptor degeneration.
The defects observed in the rd16
retina are consistent with a role of CEP290 in ciliary transport processes. Because the association of ΔCEP290 with microtubule motors (dynein and kinesin-II) remains unaltered, we conclude that abnormal transport of RPGR and opsins in rd16
is not a direct effect of impaired or defective association of ΔCEP290 with the motor proteins. An intriguing finding in this study is that the reduced amount of intact ΔCEP290 is able to associate with considerably higher amounts of specific RPGR-ORF15 isoforms. Such higher association may recruit RPGR from the connecting cilium and redistribute it in the inner segments. Consistent with the involvement of RPGR in intracellular protein trafficking, we observe redistribution of phototransduction proteins, which are potential ‘cargo’ proteins (8
) being transported via the connecting cilium to the outer segments, in the rd16
retina. However, we do not rule out the possibility of a direct association of CEP290 (or ΔCEP290) with rhodopsin and/or arrrestin. Attempts to directly identify associations of cargo proteins with the ciliary transport assemblies have been unsuccessful so far perhaps because of weak or transient interactions (29
Our findings suggest that misrouting of phototransduction proteins is the underlying cause of photoreceptor degeneration in the rd16
retina. Such redistribution of opsins in photoreceptor inner segments owing to impaired ciliary transport has been detected previously in Kif3a
conditional mutants, Tg737orpk
) as well as some other types of retinal degenerations (e.g. rd1
/peripherin and opsin mutants, Q334ter and P347S, but not P23H) (43
). The mislocalization of opsins is possibly due to redistribution of RPGR-ORF15 to the inner segments. The support for this hypothesis comes from a previous observation that the connecting cilium staining of RPGR is impaired in the Rpgrip1−/−
mice, which also develop early-onset photoreceptor degeneration owing to abrogated opsin trafficking (45
). Notably, the Rpgr−/−
mice show considerably slower retinal degeneration with only minor redistribution of rhodopsin (37
) compared with the rd16
mouse. This may be due to the presence of a few RPGR-ORF15 isoforms that are still expressed in the Rpgr−/−
mice and localize to the connecting cilium (29
CEP290 associates predominantly with centrosomes throughout the cell cycle, suggesting its role in microtubule nucleation, originating from the centrioles in dividing cells. However, the delineation of its precise role in cell division will require further investigation. Intriguingly, CEP290 shows homology to and associates with SMC proteins, which in addition to regulating chromosomal dynamics during cell division, also localize to primary cilia of photoreceptors and are postulated to be involved in ciliary transport in photoreceptors by interacting with RPGR (29
). Several ciliary and centrosomal proteins contain SMC-like domains and are involved in microtubule dynamics during retrograde and anterograde protein trafficking (20
). Hence, the SMC-like domains can be hypothesized as signature sequences that are recognized during these transport processes.
While this study was in progress, mutations in the CEP290
gene (also called NPHP6
) were identified in patients with Joubert syndrome, which includes cystic kidney disease (nephronophthisis) and cerebellar defects in addition to early-onset retinal degeneration or coloboma (48
). Extensive evaluation of 6-month-old rd16
homozygotes did not detect any gross brain or kidney pathology (Supplementary Material, Table S2), indicating that the ΔCEP290 protein in rd16
mouse retains at least partial function. Notably, only nonsense or frame-shift mutations (probably, loss of function) in the CEP290
gene have been identified in the Joubert syndrome patients. These observations suggest that the myosin-tail homology domain of CEP290 performs a retina-specific function. However, in the absence of the CEP290 protein, a pleiotropic phenotype is observed in Joubert syndrome patients.
In conclusion, our study shows that CEP290 participates in regulating intracellular protein-trafficking probably by organizing distinct multi-protein complexes for microtubule-based transport. Our findings provide a direct link between microtubule organization, intracellular transport and ciliary function in neurons and may facilitate functional dissection of centrosomal disease-associated proteins using retinal photoreceptors as a paradigm.