Abstract
Purpose :
CEP290 (centrosomal protein 290), a large multi-domain containing protein, is a key component of the transition zone in many primary and motile cilia. Mutations in Cep290 cause several ciliopathies, including Meckel Syndrome (MKS) and retinal specific disorders such as Leber Congenital Amaurosis (LCA). The CEP290 protein has been proposed as a component of the “Y-links” structures, which span from the axoneme microtubule doublets to the ciliary membrane in the connecting cilium (CC) of photoreceptor cells. Our goal is to determine the location of CEP290 protein and the structural effects of CEP290 defects at the nanometer scale.
Methods :
We use a number of advanced microscopies, including transmission electron microscopy and immuno-electron microscopy, super-resolution and expansion fluorescence microscopy, and cryo-electron microscopy to examine ciliary ultrastructure in WT mice and two Cep290 mutant mouse lines with early-onset retinal degeneration. One is virtually devoid of functional CEP290 (near-null, nn), and the other is a complete knockout (KO).
Results :
At post-natal day 10 in both CEP290 mutants, we observe formation of CC, less or no outer segment (OS) discs, and presence of Y-links. Immunogold TEM reveals the ultrastructural location of CEP290 in the area between the microtubules and the ciliary membrane, but not on the Y-links. Immunogold TEM and expansion microscopy also reveal a spiral periodicity in CEP290 staining around the CC. Each mutant displays multiple ciliary protein mislocalization from the CC at P10, preceding photoreceptor degeneration. Each mutant shows slightly different pathogenesis, with extracellular vesicles present instead of OS discs, and more ciliogenesis defects present in the KO.
Conclusions :
These results suggest that CEP290 is not required as a structural component of Y-links in the CC. Its location, the formation of EV’s instead of OS discs, and the mislocalization of multiple ciliary proteins, may facilitate a proposed function as a "gate-keeper" for protein trafficking through the CC. CEP290’s localization throughout the length of the CC, an observation unique to photoreceptors and not ubiquitous to all ciliary transition zone proteins, may help explain the retina-specific symptoms of several CEP290 mutations. The additional photoreceptor phenotypes present in the KO mutants hint at the difference between loss of CEP290 vs presence of mutated CEP290 within photoreceptors.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.