Proteostasis Modulation through ATF6 Signaling Supports Cone Photoreceptor Differentiation in Retinal Organoids

Abstract

Purpose : The Unfolded Protein Response (UPR) is a conserved regulatory mechanism of the Endoplasmic Reticulum (ER) initiated to maintain protein homeostasis. Dysregulation of the UPR is implicated in the pathology of many human diseases associated with ER stress. Inactivating genetic variants in the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital heritable vision loss, presented as impaired visual acuity and loss of color vision. Cone photoreceptors are dysfunctional in this disease, but the pathomechanims involving the lack of functional ATF6 is unknown. Furthermore, there are no treatments for people carrying these mutations.

Methods : To investigate the functional role of ATF6 during photoreceptor development, we generated retinal organoids from patient iPSCs carrying ATF6 disease-causing variants and ATF6 null hESCs generated by CRISPR. Retinal organoids underwent surface-scanning live imaging to examine morphology of WT and ATF6 mutant rods and cones, and confocal immunofluorescent microscopy of sectioned retinal organoids. In parallel, adaptive optics laser-scanning confocal ophthalmoscopy (AOSLO) was performed to visualize photoreceptor morphology from the ATF6 mutant patient stem cell donors used in this study. RNAseq analysis was performed to establish rod and cone gene expression profiles of WT and ATF6 mutant retinal organoids. Rescue of cone photoreceptor cells in ATF6 mutant retinal organoids was investigated using a small molecule ATF6 agonist.

Results : Cone photoreceptor cells in ATF6 mutant retinal organoids lacked inner and outer segments concomitant with absence of cone phototransduction gene expression; while rod photoreceptors developed normally. Adaptive optics retinal imaging of patients with disease-causing variants in ATF6 also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the phenotypes observed in our retinal organoids. We further show that the ATF6 agonist activates ATF6 to restores the transcriptional activity of ATF6 disease-causing variants and stimulates the growth of cone photoreceptors in patient retinal organoids.

Conclusions : These results reveal that ATF6 is essential for the formation of functional human cone photoreceptors, demonstrating that pharmacologic targeting of ATF6 signaling is a therapeutic strategy that needs to be further explored for blinding retinal diseases.

This is a 2021 ARVO Annual Meeting abstract.