Abstract
Purpose :
In glaucoma, RGCs are the initial site of injury, resulting in their death and subsequent vision loss. Currently, there are no FDA-approved drugs or therapies to protect RGCs from death, which is a critical barrier in the field. Here, we Our study leverage the regenerative potential of zebrafish to identify genes and pathways that protect RGCs after axonal injury.
Methods :
After ONT, ~75% of zebrafish RGCs are resilient up to 3 days post injury (dpi) (Chen et al., 2022). Here, we utilized this model and performed bulk and single cell RNA (scRNA) sequencing to determine the subtype diversity of surviving RGCs at 1 and 7dpi and identify genes and pathways that may contribute to their survival. Hybridization chain reaction (HCR) in situ hybridizations were used to validate the results of RNA-sequencing experiments.
Results :
Bulk RNA-seq showed that the number of upregulated differentially expressed genes (DEGs) increased with time; however, only 4 genes were common amongst both timepoints, suggesting that the response to RGC injury occurs in discrete phases, which we categorize into an immediate and a delayed response phase. DEGs included those encoding protein and lipid transport, cell survival, and the regulation of autophagy. scRNA-seq analysis identified clusters that correspond to known zebrafish RGC subtypes (Kölsch et al., 2021). Differential abundance analysis showed that all RGC subtypes remain after injury; however, subtype 3 increased significantly with time after injury. Interestingly, subtype 3 has been reported to have an immature RGC gene expression profile, similar to that in larval zebrafish RGCs, with tubb5, alcamb, and tmsb as marker genes. HCR based RNA-FISH validated that tubb5 and alcamb are upregulated in the injured eye at 7 dpi compared to 1 dpi.
Conclusions :
Taken together, our bulk and scRNA-seq datasets suggest that, unlike mammals, zebrafish RGCs are able to survive ONT because of their distinct early and late injury responses which include cell signaling pathways that regulate protein and lipid transport, cell survival, and autophagy. Validation of candidate genes from our data set using RNA-FISH and CRISPR/Cas9 based knockout fish lines is underway.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.