Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis

Abstract

Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290 rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290 rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy BardetBiedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290 rd16 and Mkks ko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.

CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype

“…Perhaps other factors play a role in the pathogenesis of the disease. Indeed, variations in the MKKS and BBS genes have been reported to modify the CEP290 phenotype 32,33 . Nonetheless, demonstrating rescue of ciliogenesis is an important step in treating these and other patients with CEP290 -associated LCA.…”

A CEP290 C-Terminal Domain Complements the Mutant CEP290 of Rd16 Mice In Trans and Rescues Retinal Degeneration

“…Cep290 rd16/rd16 mice also display auditory and olfactory deficits (McEwen et al, 2007; Rachel et al, 2012b). To test whether the myo-tail vector could complement the mutation in cochlear hair cells, which are responsible for auditory sensation, the vector was injected into the posterior semicircular canal at P2 (Isgrig et al, 2017; Suzuki et al, 2017), and the mice were examined for auditory brainstem response (ABR) at 3 months of age.…”

“…AON and CRISPR/ Cas9 genome editing are being evaluated to correct the most common CEP290 -LCA mutation, but in vivo therapeutic effects of these approaches remain unknown. Other treatment strategies, including modulating CEP290 interacting proteins (Rachel et al, 2012b) and inhibiting Raf-1 kinase inhibitory protein (Murga-Zamalloa et al, 2011; Subramanian et al, 2014), have been proposed but not validated through post-natal intervention. Here, we show that a C-terminal CEP290 fragment complements the Cep290 rd16 mutation in trans by preserving photoreceptor cell viability and function and improving visual behavior in the Cep290 rd16/rd16 ;/Nrl — / — mutant mice.…”

“…The Cep290 rd16 mutation, with a deletion of a 299-residue-long coding sequence, is considered a hypomorph because the mutant mice manifest only sensory defects but no other ciliopathy phenotype. In photoreceptors, the CEP290 rd16 protein allows relatively normal ciliogenesis, as evidenced by the formation of CC and rudimentary OS during post-natal development (Rachel et al, 2012b, 2015). However, it is not able to support normal structure and function, leading to rapid photo-receptor dysfunction and death in the Cep290 rd16/rd16 mouse.…”

“…CEP290 directly binds to cellular membranes through its N-terminal domain, which includes a highly conserved amphipathic helix motif, and to micro-tubules through a region located within its myosin-tail homology domain (Drivas et al, 2013). The microtubule-binding domain overlaps with the BBS6 (or MKKS) protein-interacting domain that is deleted in frame (amino acids [aa] 1606–1904, termed deleted in sensory dystrophy [DSD]) in the naturally occurring rd16 allele of mouse Cep290 (Rachel et al, 2012b). In addition, autoinhibitory domains within N and C termini of CEP290 have been identified and are capable of regulating ciliogenesis (Drivas et al, 2013).…”