Abstract
Purpose :
Activating transcription factor 6 (ATF6), essential in the unfolded protein response (UPR) and endoplasmic reticulum (ER) function, is linked to achromatopsia (ACHM), a hereditary cone dystrophy. Retinal organoids (RO) developed from ACHM patients reveal that loss of ATF6 leads to severe morphologic and molecular defects in cones, and increased ER stress in cones and Müller cells. Small molecule proteostasis modulators have been discovered that specifically activate or inhibit ATF6 and related UPR and ER stress regulators. We used these small molecule proteostasis modulators to chemically interrogate the role of UPR and ATF6 in developing photoreceptors in ROs.
Methods :
We developed ROs from wild type human embryonic stem cells. We supplemented small molecule ATF6 inhibitor Ceapin-A7 (CA7) or ATF6 activator AA147 to ROs at different ages: Day 230 (D230), followed by analysis after 28 and 56 days; or D35 followed by analysis at D100, D150, and D230. The morphology of photoreceptor cells in growing live ROs was examined through surface-scanning imaging, while the structure of all retinal cells was studied using confocal immunofluorescent microscopy in sectioned ROs. RNA-Seq was performed on ROs, and significant gene expression changes were identified in absence of ATF6.
Results :
Live imaging revealed stunting of the brush border (photoreceptor IS/OS projections) on RO surfaces with CA7 treatment. Fluorescent miroscopy cross-sections of these ROs showed shorter and fewer IS/OS from photoreceptors in CA7-treated ROs. However, no differences in TUNEL staining was seen with CA7 treatment. RNA-Seq revealed significant disruption of retinal and photoreceptor structural integrity genes in ROs lacking ATF6 function.
Conclusions :
We demonstrate that chemical inhibition of ATF6 disrupts RO brush border integrity, similar to the phenotype we previously reported in ATF6 ACHM ROs, with severe stunting of photoreceptor IS/OS in the absence of photoreceptor cell death. We identify transcriptional defects in many retinal and photoreceptor structural integrity genes in absence of ATF6. Based on this, we propose that photoreceptors cannot extend or sustain their IS/OS without ATF6, therefore causing the visual deficits in patients carrying these alleles.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.