The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness

Abstract

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel–Gruber syndrome, Joubert syndrome, Senor–Loken syndrome and Bardet–Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290.

The zebrafish eye—a paradigm for investigating human ocular genetics

“…72 Morpholino-induced knockdown of centrosomal protein 290 kDa (cep290) resulted in delayed intracellular transport and reduced visual acuity despite a fully laminated retina, consistent with human LCA patients. 73 Importantly, injection of cep290 mutants with an N-terminal CEP290 construct rescued visual function, supporting a potential treatment The wild-type retina shows characteristic stratification in three nuclear and two plexiform layers. The ome (crb2a 289/289 ) mutant shows widespread retinal degeneration with loss of lamination and irregular patchy islands of plexiform tissue, with absence of large areas of RPE.…”

An ARL3–UNC119–RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium

“…MO-mediated knockdown of genes involved in ciliary function typically result in curved body, defects in the size of the ciliated embryonic structure known as the Kupffer's vesicle (KV), and reduced visual function (Yen et al 2006;Pretorius et al 2010;Baye et al 2011). Zebrafish embryos injected at the one-to four-cell stage with gene-specific MOs were assessed for overall morphology, KV size, and visual function.…”

Small organelle, big responsibility: the role of centrosomes in development and disease

“…Laterality defects are also present owing to shorter motile primary cilia in the Kupffer vesicle, an embryonic organ required for left-right asymmetry [129]. Such defects also occur in CEP290-and BBS4-depleted zebrafish, with the former exhibiting additional retinal anomalies [130,131]. Ofd1 is located on the X chromosome both in mice and humans.…”