NudC is critical for outer segment disk size and photoreceptor cell viability

Abstract

Purpose : Nuclear distribution protein C (nudC) has established roles in cellular division and neuronal migration. Recently, it has been shown to regulate F-actin dynamics through the regulatory protein cofilin1 in epithelial cells and in zebrafish. Our lab has demonstrated a role for nudC in post-mitotic rods of the vertebrate retina. We have shown that nudC has a direct interaction with rhodopsin and that knockdown of nudC results in rhodopsin mislocalization accompanied by elongated outer segment disks in Xenopus laevis. Here, we investigate the molecular mechanism of nudC in retinal homeostasis and outer segment disk size regulation in mouse rod photoreceptors.

Methods : NudC floxed mice were generated using Nudc^{tm1a(KOMP)Mbp} embryonic stem cells obtained from the UCDavis KOMP repository and bred with rod photoreceptor-specific Cre recombinase (iCre75)-expressing mice to obtain heterozygous or homozygous nudC knock-out (KO) in rod photoreceptors. Transmission electron microscopy (TEM) images of photoreceptor ultrastructure were obtained using ultrathin sections of 3- and 6-week old KO retina. Scotopic and photopic electroretinography (ERG) were performed on 3- and 6-week old KO mice to elucidate the functional phenotype of rods with a lower amount or those lacking nudC. Immunohistochemistry (IHC) and quantitative Western blotting were performed to identify localization and relative amounts of key proteins following nudC knockdown or knockout.

Results : TEM of rod ultrastructure revealed elongated disks at the proximal end of the outer segments similar to rods that have dysregulated F-actin through cytochalasin D treatment. ERG data show that loss of nudC is detrimental to the retinal function; by 6 weeks of age, mice lack a scotopic response with a decreased photopic response. IHC revealed that loss of nudC results in mislocalization of rhodopsin to the inner segment. Quantitative Western blotting of retinal lysate from homozygous nudC KO rods shows an increase in F-actin, and a decrease in cofilin1 levels.

Conclusions : NudC has a crucial function of regulating disk formation in the OS. Our data are consistent with the hypothesis that nudC governs disk dimension by stabilizing the actin-severing protein cofilin1 thus regulating branched F-actin during disk formation at the base of the outer segment. Loss of nudC expression in rod cells ultimately leads to retinal degeneration and blindness in mice.

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