CEP290 localization in the rod connecting cilium of CEP290^rd16 mice with fluorescence nanoscopy

Abstract

Purpose : Mutations in CEP290 account for roughly 25% of cases of Leber Congenital Amaurosis (LCA). It is associated with the photoreceptor connecting cilia (CC) but its function is not certain. We used an LCA-like mouse model, homozygous for the CEP290^rd16 allele, with an in-frame deletion of the putative microtubule binding domain, to assess the function of this domain. We hypothesized that CEP290 and the CEP290 interacting partner, NPHP5, mislocalize in CEP290^rd16 photoreceptors and that this mislocalization contributes to the rapid retinal degeneration in the CEP290^rd16 animal.

Methods : To obtain sub-diffraction resolution images, we used Structured Illumination Microscopy (SIM) in age-matched wild-type and mutant mice. In order to localize CEP290 prior to retinal degeneration, retinas from 10-day old mice were immunostained with antibodies for CEP290 and antibodies to other CC proteins, including NPHP5. Approximately 15 cilia per experiment were selected to assess and to measure CEP290 and CC protein localization between wild type and CEP290^rd16 cilia.

Results : In SIM reconstructions of 10-day old wild-type rod cilia, we measured that CEP290 and NPHP5 localize throughout the length of the CC (1138.2 nm +/- 170.7 nm and 1217.5 nm +/- 314.9 nm), respectively, and between the microtubule doublets of the axoneme and the ciliary membrane. CEP290 non-uniformly surrounds the axoneme (266.6 nm +/- 50.5 nm in radius), while NPHP5 localizes to one specific side of the axoneme (313.7 nm +/- 50.6 nm in radius). Prior to retinal degeneration in CEP290^rd16 animals, CEP290 and NPHP5 exhibited normal localization. CEP290 measured 1052.9 nm +/- 227.8nm in length and 263.3 nm +/- 73.8 nm in radius, while NPHP5 measured 1730.2 nm +/- 593.3 nm in length and 360.5 nm +/- 53.5 nm in radius. The immunofluorescence signal for both was lower than in WT, suggesting lower protein levels. In contrast, in CEP290^-/- rods, NPHP5 was not observed in the CC at P10.

Conclusions : Our results indicate that the mutant CEP290 does not prevent formation of the CC or the localization of CEP290 or NPHP5 within the CC, and that the retinal degeneration in CEP290^rd16 animals is not due to CEP290 mislocalization. Thus, degeneration may be due to instability of the mutant protein or mislocalization of other proteins.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.