Investigating the role of Cwc27, a splicing factor, in retinal degeneration

Abstract

Purpose : The mechanism of degeneration due to mutations in splicing factors has not been well studied due to limitations of past animal models. The Cwc27^{K338frameshift} mouse model has been shown to be an autosomal recessive retinal degeneration model caused by mutations in a splicing factor, however the mechanism behind this degeneration has yet to be elucidated. We hypothesize that mutations in splicing factors cause an accumulation of double-stranded RNA (dsRNA) that initiates an immune response and results in cell dysfunction and retinal degeneration.

Methods : The retinal function of live mice was assessed at 3 (n=3), 4 (n=4), and 6 (n=6) months-of-age via electroretinography. The retinal pigmented epithelium (RPE) function was assessed at 3 months-of-age by assessing rhodopsin digestion 3 hours post-light exposure. RNA-Sequencing was conducted on the RPE and retina independently (n=3) to assess gene expression and intron retention. Upregulated genes and intron retention were validated using RT-PCR and specific protein markers were assessed via western blot and/or immunofluorescent staining on primary tissues.

Results : Cwc27^{K338frameshift} mice exhibit retinal dysfunction by 3 months-of-age and morphological changes to the outer nuclear layer (ONL) are present by 4 months-of-age. The RPE shows rhodopsin digestion dysfunction by 3 months-of-age, indicating that the RPE and photoreceptors are both affected in this model. RNA-Sequencing reveals significant upregulation of the unfolded protein response as well as the immune system, specifically of interest is upregulation of the interferon response in the RPE. Intron retention in a set of genes was also observed by RNA-Sequencing and validated by RT-PCR in the retina and RPE.

Conclusions : The Cwc27^{K338frameshift} mice are observed to have significantly increased intron retention in both the retina and RPE. We anticipate that the retained introns can be recognized as viral RNA and results in cell death. The RNA-Sequencing shows significantly increased expression of anti-viral pathway markers in the mutant RPE. Upon knockdown of Cwc27 in RPE cells increased expression of the interferon pathway is observed, indicating that an immune response, potentially triggered by dsRNA, is activated. We propose the first model of retinal degeneration due to anti-viral pathway activation induced by dsRNA as a direct result of a mutation in a splicing factor, Cwc27.

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