Single-Cell RNA Sequencing of Human Lamina Cribrosa Fibroblasts in Glaucoma

The molecular mechanisms that underlie the extracellular matrix (ECM) remodelling of the human lamina cribrosa (LC) in glaucoma remain obscure. This is the first study to characterise human glaucomatous LC cells using single-cell RNA sequencing (scRNA-seq). Our aim is to characterise transcriptional changes in human LC cells from glaucomatous and normal eyes and identify signalling pathways contributing to optic nerve head remodelling. scRNA-seq was performed on LC cells from glaucomatous and normal donor eyes. Differential gene expression was analysed using Seurat. Cell identity was assessed using SingleR and GTEx reference datasets. Pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA), and transcription factor (TF) motif enrichment was evaluated using i-cisTarget. Validation was performed using quantitative PCR, Western blotting, immunohistochemistry, and immunofluorescence. LC cells were treated with ISRIB and 4-PBA. Analysis of 6131 LC cells using shared nearest-neighbour clustering and UMAP visualisation identified four broad transcriptional groups, which were used for subsequent transcriptional characterisation. Cell type mapping identified a fibroblast/mesenchymal cell origin. Glaucomatous LC fibroblasts demonstrated a pro-fibrotic transcriptional profile, with overexpression of multiple pro-fibrotic genes, master TF regulons involved in cell fate decisions, and TF motifs including SRF, as well as key integrated stress response (ISR) genes such as ATF4, ATF3 and CREB3L1. Enriched pathways included E2F, G2M, the Hallmark EMT gene set and MYC, while the p53 pathway was downregulated. Pharmacologic treatment with ISRIB or 4-PBA reduced expression of stress-response and ECM genes, while ISRIB attenuated the increased cellular metabolic activity observed in glaucomatous LC cells. Bioinformatic analysis of scRNA-seq of human glaucoma LC cells identified increased expression of myofibroblast genes, an EMT-associated/profibrotic transcriptional programme, and cell cycle signalling pathways, TF regulatory elements and stress response genes.