Study on gene knockout mice and human mutant individual reveals absence of CEP78 causes photoreceptor and sperm flagella impairments

BackgroundCone-rod dystrophy (CRD) is a genetically inherited retinal disease characterized by photoreceptor degeneration. In some rare cases, CRD and hearing loss can be associated with male fertility, while the underlying mechanism is not well known. MethodsUsing CRISPR/Cas9 system, we generated Cep78-/- mice. And electroretinogram (ERG), immunofluorescence staining and transmission electron microscopy (TEM) were used to analyze visual function and photoreceptor ciliary structure changes in Cep78-/- mouse. HE/PAS staining, scanning-electron microscopy (SEM) were conducted to Cep78-/- mice and human CRD patient with CEP78 protein loss to illustrate male infertility and multiple morphological abnormalities of the sperm flagella (MMAF) caused by CEP78 deficiency. TEM and immunofluorescence staining were performed to characterize morphological and molecular changes of sperm flagella microtubule arrangement, centriole development and spermatid head shaping in Cep78-/- mice. Mass-spectrometry analyses were conducted to identify protein abnormalities after Cep78 deletion and Cep78 interacting proteins in spermiogenesis. Co-immunoprecipitation was used to show the Cep78-Ift20-Ttc21a trimer. The role of Cep78-Ift20-Ttc21a trimer in cilliogenesis and centriole elongation was assessed by cilia induction assay. ResultsCep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced electroretinogram amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted cilia elongations and structures. Cep78 deletion also caused male infertility and MMAF, with disordered "9 + 2" structure and triplet microtubules in sperm flagella. CEP78 forms a trimer with intraflagellar transport (IFT) proteins IFT20 and TTC21A essential for sperm flagella formation, is essential for their interaction and stability, and recruits IFT20 to centrosome. Insufficiency of any component in the trimer causes centriole elongation and cilia shortening. Additionally, absence of CEP78 protein in human leaded to similar phenotypes in vision and MMAF as Cep78-/- mice. ConclusionsWe found CEP78 as the causative gene of CRD with MMAF in human and mouse. Cep78 forms a trimer with Ift20 and Ttc21a, and regulate the interaction, stability and localization of the trimer proteins, which regulate cilliogenesis, centriole length, and sperm flagella formation. FundingThis work was supported by the National Key R&D Program (2021YFC2700200 to X.G); National Natural Science Foundation of China (82020108006, 81730025 to C.Z, 81971439, 81771641 to X.G, 82070974 to X.C, 82060183 to X.S); Shanghai Outstanding Academic Leaders (2017BR013 to C.Z); and Six Talent Peaks Project in Jiangsu Province (YY-019 to X.G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.