Achromatopsia Retinal Organoid Transcriptomes Reveal Increased Gliosis and Dysregulation of Unfolded Protein Response

Abstract

Purpose : Variants of Activating Transcription Factor 6 (ATF6), a key regulator of the Unfolded Protein Response (UPR), cause severe morphologic and molecular defects in cone photoreceptors leading to achromatopsia (ACHM). ATF6 is expressed in all retinal cell types, and it is unclear how ATF6 disease variants affect retinal cells other than cones. Here, we analyzed the transcriptomes of major retinal cell types in retinal organoids created from induced pluripotent stem cells (iPSC) of ACHM patients carrying biallelic ATF6 disease variants.

Methods : Retinal organoids were generated using iPSCs from ACHM patients with biallelic ATF6[Y567N] variant and from asymptomatic heterozygous family members as controls. Bulk RNAseq was performed on individual organoids at day 290 (n=3 each for patients and controls). Transcriptomic gene sets specifically associated with cones, rods, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and Müller cells (MC) were extracted from 3 RNAseq analyses of non-diseased human retina and retinal organoids (2019 Liang PMID: 31848347; 2019 Menon PMID: 31653841, 2020 Cowan PMID: 32946783). Using these normal human retinal cell type transcriptome profiles, we evaluated expression levels of these retinal cell specific gene-sets in ATF6 mutant ACHM retinal organoids.

Results : Cone photoreceptor-specific genes were significantly down-regulated in ATF6-ACHM retinal organoids (RO) compared to controls (Wilcoxon, P <0.001). In contrast, Muller cell gene expression was increased in ATF6-RO versus controls (Wilcoxon, P <0.01). No significant changes in gene expression were observed in all other retinal cell types in ATF6-ACHM retinal organoids. Last, we identified dysregulation of UPR specifically a significant increase of IRE1-XBP1-regulated genes (Wilcoxon, P=0.02) in the ATF6-RO.

Conclusions : Consistent with prior study (2021 Kroeger PMID: 34561305), ATF6-RO showed severely impaired cone photoreceptor-specific gene expression when compared with the 3 new wild-type human retina/retinal organoid transcriptome datasets. Interestingly, Muller cell-specific gene expression was increased in ATF6-RO. The IRE1-XBP1 signaling pathway of the UPR was also upregulated in the ATF6-RO. Our data suggest 2 new potential retinal pathomechanisms that may contribute to ATF6-associated ACHM – Muller gliosis and abnormal activation of the IRE1-XBP1 branch of the UPR.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.