Abstract
Purpose :
Retinal astrocytes are a small percentage of all cells in the eye but a key component of the glial network that supports neural function throughout the retina and optic nerve. Despite being potential therapeutic targets in glaucoma, our understanding of their contributions is limited by their sparseness. By combining a specialized microdissection approach with Ribotag immunoprecipitation and bulk RNA-sequencing, we have generated the most comprehensive transcriptional profile yet of retinal astrocytes.
Methods :
Our combined approach allowed us to isolate purified retinal astrocyte RNA from adult mice (n=3) for sequencing and transcriptional profiling. We performed pairwise comparisons with retinal input samples, and with purified astrocyte RNA samples from optic nerve head (ONH, n=6) and myelinated optic nerve proper (ONP, n=6), to detect genes and pathways enriched in retinal astrocytes. We additionally investigated potential specific markers of retinal astrocytes identified by these pairwise comparisons.
Results :
Pairwise comparison of retinal astrocytes and input controls reveal a distinct transcriptional profile, with 743 differentially expressed genes (DEGs) significantly enriched (p-adj < 0.01), and 19 Reactome pathways identified by over-representation analysis (ORA) (p-adj < 0.05). We also found substantial heterogeneity between populations and identified 853 DEGs enriched in retinal astrocytes versus those of the ONH, and 1544 DEGs relative to those of the ONP. A total of 197 DEGs were enriched across all 3 comparisons, indicating that they are highly retinal astrocyte specific. Pathways identified by ORA link retinal astrocytes to energy provisioning, lipid-signaling, and secretion, alongside core astrocyte functions such as structural support and buffering of ions and neurotransmitters. We additionally identified a highly specific marker of retinal astrocytes, and an existing Cre-driver mouse line suitable to target these cells.
Conclusions :
Prior work on retinal astrocytes has largely relied on inferences from other astrocyte populations or disruptive cell culture models, but a paucity of transcriptional data has hampered investigation. Our study establishes a clear transcriptional identity for retinal astrocytes, reveals heterogeneity between these glia and their counterparts in the ONH and ONP, and enabled us to identify a retinal astrocyte specific marker.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.