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 (Cep290rd16 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 Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl 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 Cep290rd16 and Mkksko 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.

Figure 1. The CEP290-DSD domain specifically interacts with MKKS.

(A) A schematic of the MKKS protein, with horizontal lines showing variants/mutations identified in this study (right) and previously reported (left). New sequence variants cluster in the same regions as known mutations. (B) Yeast 2-hybrid (Y2H) screen using the Cep290-DSD domain as bait identified MKKS as a specific interactor. p53/T-antigen and laminin/T-antigen combinations were positive and negative controls, respectively. Growth was detected with the MKKS prey only (top panels) and validated by β-galactosidase assay (bottom panels). (C) Interaction of the Cep290-DSD domain with mutant MKKS constructs in yeast 2-hybrid growth assay. The top 3 rows show growth of 10-fold serial cell dilutions on double-selection control media (-U-L); the bottom 3 rows show growth on selective media (-U-L-H). Note equal growth on control media plates (top 3 rows). MKKS mutations at residues 52, 57, 345, and 499 disrupt interaction with CEP290. (D) Yeast 2-hybrid assay using CEP290-DSD (left 4 lanes) or full-length CEP290 (CEP290-FL) (right 3 lanes) as bait with full-length MKKS, BBS10, BBS12, or empty vector as prey. The top 3 rows show growth of 10-fold serial cell dilutions on double-selection control media (-U-L); the bottom 3 rows show growth of 10-fold serial dilutions on selective media (-U-L-H). Only MKKS interacts with Cep290-DSD. The first row of the BBS12 lane on selective medium shows a shadow due to dead cells.

Figure 2. Coimmunoprecipitation of CEP290 and MKKS in transfected cells and from retinal extracts.

(A) Coimmunoprecipitation of CEP290-GFP (full length) and MKKS-myc, expressed in transfected HEK293 cells. Some BBS-associated amino acid substitutions in MKKS disrupt coimmunoprecipitation with CEP290 (top). Expression of mutant MKKS protein is demonstrated by immunoblot analysis of the input for MKKS-Myc constructs (middle). CEP290 expression is shown by bands in input lanes (bottom). (B) Coimmunoprecipitation of WT MKKS-Myc with CEP290-GFP (full length) and negative control plasmids. Sox2 is used as a negative control and BBS2 is used as a positive control. Myc plasmids (middle) and GFP plasmids (bottom) were used as inputs. (C) Coimmunoprecipitation of CEP290 and MKKS from bovine retinal extracts. Immunoblots of proteins immunoprecipitated with anti-MKKS antibody or normal IgG were probed with anti-CEP290 antibody. Note that specific 290-kDa CEP290-immunoreactive protein is pulled down by the anti-MKKS antibody but not by control IgG.

Figure 3. Knockdown by coinjection of subminimal doses of cep290 and mkks morpholinos causes sensory defects in zebrafish embryos.

(A) Whole mount embryos at 72 hours after fertilization. Subminimal doses of morpholinos against cep290 and mkks transcripts and standard negative control (SNC) produced no phenotype, whereas coinjecting subminimal doses of mkks and cep290 resulted in deformed eyes (yellow circles) and ears (red ovals). Original magnification, ×11.5. (B) H&E-stained sections of eyecups and otocysts in control (0.5 ng cep290 plus 1.0 ng SNC) and experimental (0.5 ng cep290 plus 1.0 ng mkks) embryos. Note the lack of normal lens formation and retinal lamination in the double morphant embryos (top panel) and impaired tether cell development in the otocyst (bottom panel). Original magnification, ×60. (C) Quantitation of morphant phenotypes. Subminimal doses of cep290 or mkks morpholino alone resulted in abnormal embryos at a frequency similar to that of uninjected controls (<5%), whereas combining both morpholinos yielded 18% of embryos with ear, eye, and/or axis defects. Higher doses of either morpholino alone revealed a greater percentage of defective embryos, consistent with previous results (27, 43, 44). Eye or ear defects were never observed in uninjected embryos. Error bars are SD. Numbers of embryos examined per condition are 245 (uninjected), 53 (0.5 ng cep290 plus 1.0 ng SNC), 121 (0.5 ng mkks plus 1.0 ng SNC), 208 (0.5 ng cep290 plus 1.0 ng mkks), 98 (1.0 ng cep290), 125 (2.0 ng cep290), and 55 (2.0 ng mkks).

Figure 4. Triallelic loss of Mkks and/or the Cep290-DSD domain ameliorate cilia phenotypes in photoreceptors.

(A) Cross sections through the P18 retina in different combinations of Cep290^rd16 and Mkks^ko alleles, as indicated. Note the short, abnormal OSs in Cep290^rd16/rd16 or Mkks^ko/ko genotypes and the more normal OS in the triallelic Cep290^rd16/+;Mkks^ko/ko genotype. Here, the Cep290^rd16/rd16;Mkks^ko/ko genotype looks similar to Cep290^rd16/rd16. The white arrows indicate comparison between 2 similar genotypes that are improved by combining alleles of Cep290 and Mkks. Original magnification, ×40. (B) Quantitation of outer nuclear layer thickness at P18 in the genotypes indicated. Higher variability is noted in double-homozygous mutants (see error bars on Cep290^rd16/rd16 versus Cep290^rd16/rd16;Mkks^ko/ko). Error bars are SD; n = 6 (WT), n = 4 (Cep290^rd16/+;Mkks^ko/ko), n = 3 (Mkks^ko/ko), n = 14 (Cep290^rd16/rd16;Mkks^ko/ko), n = 8 (Cep290^rd16/rd16). (C) Scotopic ERG b-wave amplitudes in the indicated mouse genotypes (at P20). Removing one WT Mkks allele on a Cep290^rd16/rd16 background results in improved responses, as does adding one Cep290^rd16 allele on a Mkks^ko/ko background. Note that single homozygous or double-homozygous genotypes have essentially no ERG b-wave response. Error bars are SD; n = 3 (WT), n = 3 (Cep290^rd16/rd16), n = 6 (Cep290^rd16/rd16;Mkks^ko/+), n = 4 (Mkks^ko/ko), n = 3 (Cep290^rd16/+;Mkks^ko/ko), and n = 3 (Cep290^rd16/rd16;Mkks^ko/ko). (D) Longitudinal EM sections through the OS, connecting cilia, and inner segments in P14 retina show that OS morphology is disrupted in the indicated mutant genotypes. Original magnification, ×3,000. (E) Higher-magnification images (original magnification, ×30,000) of OSs in P14 retina confirm improved OS morphology in Cep290^rd16/+;Mkks^ko/ko and Cep290^rd16/rd16;Mkks^ko/+ triallelic genotypes. OSs of triallelic mice form concentric stacks of discs (parallel orange lines), more similar to WT. The white arrows indicate comparison between 2 similar genotypes that are improved by combining alleles of Cep290 and Mkks. RPE, retinal pigment epithelium; CC, connecting cilia; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; ONH, optic nerve head.

Figure 5. Ultrastructural (EM) analysis of cilia and basal bodies in P14 WT mouse retinal photoreceptors.

(A) Ultrastructural (EM) analysis of cilia and basal bodies in P14 WT mouse retinal photoreceptors. The left panel shows a longitudinal section electron micrograph, with white lines indicating the plane of the cross section electron micrographs (middle panel) through, 1, the basal body; 2, transition zone; 3, connecting cilium; and 4, axoneme of OSs. The right panel shows a representative cross section through inner/OS junction and illustrates each structure in situ. Original magnification, ×20,000. (B) Basal bodies, 1, and connecting cilia, 3, in cross section, showing the normal 9 + 0 arrangement of the microtubule bundles in P14 WT and Cep290^rd16/rd16 animals. In Mkks^ko/ko animals, note the diffuse pericentriolar material surrounding the basal body (white arrows) and flattened cilium (red arrow). Cep290^rd16/+;Mkks^ko/ko mice have improved ciliary and basal body morphology compared with that of single homozygotes. Black arrows indicate comparison between Mkks^ko/ko and Cep290^rd16/+;Mkks^ko/ko genotypes. Additional examples of cilia and basal bodies for each genotype are shown in Supplemental Figure 3. Original magnification, ×50,000. (C) Quantitation of connecting cilium short/long diameter ratio in WT, Mkks^ko/ko, and Cep290^rd16/+;Mkks^ko/ko genotypes, showing rescue of cilia cross-sectional shape in the triallelic genotype. Original magnification, ×50,000. Error bars are SEM; n = 4 (WT), n = 7 (Mkks^ko/ko), and n = 4 (Cep290^rd16/+;Mkks^ko/ko).

Figure 6. Loss of MKKS in combination with CEP290-DSD rescues hair cell kinocilia and olfactory sensory cilia defects.

(A) Confocal images of stereociliary bundles of hair cells from P0 mice. Minor defects in stereociliary bundle architecture in Cep290^rd16/rd16 mice compared with those in P0 WT mice and marked defects in bundle morphology in Mkks^ko/ko mice (blue circles). Bundle morphology is normal in double mutants (white arrow). Original magnification, ×75.6. (B) Higher-magnification image (original magnification, ×116) of phalloidin-labeled stereocilia bundles (red) and acetyl-α-tubulin–marked kinocilia (green) of outer hair cells in P0 cochlea. In the adjacent monochromatic panel, red lines identify kinocilia. Abnormal bundle rotation and misplaced, malformed, or missing kinocilia in Mkks^ko/ko cells are rescued in the double mutant (white arrows). (C) Quantitation of kinocilium length. Cep290^rd16/rd16 cilia are significantly longer than WT cilia; Mkks^ko/ko kinocilia are highly abnormal or absent; double-mutant kinocilia are indistinguishable from WT kinocilia. ND, not determined. Error bars are SD; kinocilia measured, respectively, are n = 42 (WT), n = 77 (Cep290^rd16/rd16), and n = 35 (Cep290^rd16/rd16;Mkks^ko/ko). (D) ABRs in 3- to 4-month-old mice of indicated genotypes, with mean threshold ± SD. Mkks^ko/ko and Cep290^rd16/rd16 mice show elevated hearing thresholds. Complete rescue of ABR thresholds in animals with triallelic combinations and partial rescue in double homozygotes. Error bars are SD; cochlea examined for each condition, respectively, are n = 8 (WT), n = 8 (Cep290^rd16/rd16), n = 18 (Cep290^rd16/rd16;Mkks^ko/+), n = 6 (Mkks^ko/ko), n = 6 (Cep290^rd16/+;Mkks^ko/ko), and n = 6 (Cep290^rd16/rd16;Mkks^ko/ko). (E) SEM images of olfactory epithelium at 7 to 8 weeks of age, showing loss of cilia in Mkks^ko/ko mice (small white arrow) and their retention in the double-mutant and triple allelic combinations (large white arrow). Original magnification, ×6,500.

Figure 7. CEP290 and MKKS are expressed in adjacent domains in ciliated sensory cells.

(A) CEP290 (red) and rootletin (green) in WT photoreceptors. CEP290 localizes to the connecting cilium, distal to ciliary rootlet. MKKS (red) and rootletin (green); MKKS caps the rootlet in the basal body (BB) region. Original magnification, ×180. (B) CEP290 (green), MKKS (red), and rootletin (rootlet; blue) in photoreceptors. CEP290 and MKKS are expressed in adjacent, nonoverlapping domains corresponding to connecting cilium and basal body. Arrow shows proximal-distal orientation of cilium. Original magnification, ×480. (C) CEP290 (red) continues to be expressed in Cep290^rd16/rd16, Mkks^ko/ko, and in double homozygotes, albeit irregularly and disorderly. Original magnification, ×180. (D) Expression of CEP290 (green) and γ-tubulin (red) in P0 WT cochlea. In hair cells, CEP290 is detected in punctate spots at base of kinocilium (white arrows); γ-tubulin (red) localizes to the basal body. CEP290 is expressed broadly in supporting cells surrounding hair cells. Diagram shows enlargement of the hair cell (purple circle). IHC, inner hair cells; OHC, outer hair cells. Original magnification, ×176.4. (E) CEP290 (green) and γ-tubulin (red) expression in olfactory sensory neurons on the surface of the olfactory epithelium. Olfactory sensory neurons have multiple basal bodies and cilia per cell (schematic on right), with arrow indicating proximal-distal direction. Original magnification, ×60. (F) A possible model depicting interaction of CEP290-DSD in proximal cilia, with MKKS in adjacent basal bodies. The long green cylinder indicates CEP290, with the hook indicating the MKKS-interacting DSD domain. The short green cylinder represents CEP290-ΔDSD, and the red oval represents MKKS. The yellow pentagon and blue diamond represent predicted CEP290- and MKKS-interacting proteins, respectively. Asterisks indicate that the effect of losing both alleles is tissue specific. In the cochlea and olfactory epithelium, improvement is noted with loss of both alleles. In photoreceptors, such improvement does not occur.