Deoxysphingolipids mediate toxicity though the Unfolded Protein Response (UPR)

Abstract

Purpose : The retinal degenerative disease Macular Telangiectasia (MacTel) is a complex genetic disease with a common metabolic signature, decreased systemic serine and increased deoxysphingolipids (dSLs). Elevated dSLs are toxic to the retina and correlate with disease severity. However, the mechanism of dSL-mediated toxicity is not understood.

Methods : We model dSL toxicity in the retina by adding deoxysphinganine to mature retinal organoids. Toxicity is quantified by assaying cell death in photoreceptors using TUNEL staining. To characterize the retina’s cell stress response to dSL toxicity we perform bulk RNAseq and single-cell RNAseq along with targeted and untargeted pathway analyses. We then utilize selective pharmacological inhibitors and activators of cell stress pathways to assay their functional relevance in the dSL toxicity response.

Results : Through bulk RNAseq we identify the Unfolded Protein Response (UPR) as one of the major stress pathways activated in dSL toxicity, with two of the UPR sub-branches, ATF6 and PERK, comprising the majority of the response. Using single-cell RNAseq we then characterize cell-specific dSL toxicity responses, identifying unique cell-type dSL susceptibility. We next determined the functional role of the UPR pathway sub-branches in dSL toxicity. Inhibition of the UPR PERK pathway prevents cell death indicating that apoptosis from dSL toxicity is mediated through PERK. Conversely, inhibition of the UPR ATF6 pathway exacerbates dSL induced cell death, indicating its role in homeostasis. Next, we screen ATF6 activating drugs to identify multiple compounds that alleviate dSL toxicity through the activation of ATF6.

Conclusions : We provide the first characterization of cell stress responses to dSL toxicity in the retina, identifying the UPR as a major cell stress pathway activated by dSL toxicity. Subsequent single cell RNAseq is used to characterize its unique effect in each cell type providing insight to its pathological mechanism in MacTel. We subsequently identify multiple classes of drugs that can prevent cell death as well as promote compensatory signaling pathways to promote cell health and survival through modification of the UPR.

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