Targeting Ca2+-dependent pathways to promote corneal epithelial wound healing induced by CISD2 deficiency

Abstract

Purpose : Chronic epithelial defects of the cornea, which are usually associated with several ocular diseases and are an unmet clinical need. CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928). CISD2 protein is significantly decreased in the corneal epithelium of patients with various corneal epithelial diseases. Here we provide new results describing how targeting Ca2+-dependent pathways can improve corneal epithelial regeneration.

Methods : Cisd2KO mice were generated as previously described. Female mice were employed for all experiments. Mouse corneas were collected, fixed and stained using standard protocols. The human corneal epithelial cells (HCEC) cell line was cultured in HCEC complete medium and maintained. The CISD2 gene was disrupted in HCEC cells by CRISPR/Cas9n method. The differentially expressed genes (DEGs) calculated from the RPKM (reads per kilobase of exon model per million reads) in RNA-Seq data. The Gene Ontology functional characterization was performed by online tools PANTHER and STRING. Heatmaps were graphed by loading log-transformed fold changes into MultiExperiment Viewer 4.9 software.

Results : CISD2 deficiency dysregulates cytosolic Ca2+, impairs cell proliferation and migration, decreases mitochondrial function and increases oxidative stress. As a consequence, these abnormalities bring about poor epithelial wound healing and lead to persistent corneal regeneration and limbal progenitor cell exhaustion. CISD2 deficiency induces three distinct Ca2+-dependent pathways, namely the calcineurin, CaMKII and PKCα signaling pathways. Intriguingly, inhibition of each of the Ca2+-dependent pathways seems to reverse cytosolic Ca2+ dysregulation and restore cell migration during corneal wound healing. Cyclosporin, an inhibitor of calcineurin, appears to have a dual effect on both inflammatory and corneal epithelial cells. Corneal transcriptomic analyses have revealed that there are six major functional groupings of differential expression genes when CISD2 deficiency is present.

Conclusions : CISD2 is important in corneal epithelial regeneration and identifying the potential of repurposing FDA-approved drugs that target Ca2+-dependent pathways manybe applicable for treating chronic epithelial defects of the cornea.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.