Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Abstract Background The optic nerve is an important tissue in glaucoma and the unmyelinated nerve head region remains an important site of many early neurodegenerative changes. In both humans and mice, astrocytes constitute the major glial cell type in the region, and in glaucoma they become reactive, influencing the optic nerve head (ONH) microenvironment and disease outcome. Despite recognizing their importance in the progression of the disease, the reactive response of optic nerve head astrocytes remains poorly understood. Methods To determine the global reactive response of ONH astrocytes in glaucoma we studied their transcriptional response to an elevation in IOP induced by the microbead occlusion model. To specifically isolate astrocyte mRNA in vivo from complex tissues, we used the ribotag method to genetically tag ribosomes in astrocytes, restricting analysis to astrocytes and enabling purification of astrocyte-associated mRNA throughout the entire cell, including the fine processes, for bulk RNA-sequencing. We also assessed the response of astrocytes in the more distal myelinated optic nerve proper (ONP) as glaucomatous changes manifest differently between the two regions. Results Astrocytes of the optic nerve exhibited a region-specific and temporally distinct response. Surprisingly, ONH astrocytes showed very few early transcriptional changes and ONP astrocytes demonstrated substantially larger changes over the course of the experimental period. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as highly upregulated processes in both ONH and ONP astrocytes, with the former showing additional upregulation in antioxidative capacity and proteolysis. Interestingly, optic nerve astrocytes demonstrated a limited neuroinflammatory response, even when challenged with a more severe elevation in IOP. Lastly, there were a greater number of downregulated processes in both astrocyte populations compared to upregulated processes. Conclusion Our findings demonstrate an essential role for energy metabolism in the response of optic nerve astrocytes to elevated IOP, and contrary to expectations, neuroinflammation had a limited overall role. The transcriptional response profile is supportive of the notion that optic nerve astrocytes have a beneficial role in glaucoma. These previously uncharacterized transcriptional response of optic nerve astrocytes to injury reveal their functional diversity and a greater heterogeneity than previously appreciated

Rapid isolation of intact retinal astrocytes: a novel approach

Abstract Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA

Additional file 2 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 2: Supplementary Figure 2. (A) Heatmap showing the per-sample activity score for each transcription factor identified as significantly different between ONH 30 vs 0 days.  While there are significant differences based on the log2 fold change of genes as input, the heatmap showed limited clustering by condition. (B) Table summarizing the known motifs found to be enriched in the transcription start site +/- 2kb region of our input gene list, with a p-value < 0.001.  For each known motif, the corresponding transcription factor is specified. (B) Table summarizing the known motifs found to be enriched in the transcription start site +/- 2kb region of our input gene list, with a p-value < 0.001.  For each known motif, the corresponding transcription factor is specified. (D) In-situ hybridization showing the localization of Cartpt mRNA in the optic nerve of a microbead injected mouse at 30 days. (E) Longitudinal sections of the mouse ONH region showing immunostaining for CARTPT and SOX9 in microbead injected mice at 30 days

Additional file 3 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 3: Supplementary Table 1. The quantity and quality of RNA and cDNA measured using the Agilent 2100 Bioanalyzer

Additional file 12 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 12: Supplementary Table 10. Pathways differentiating ONH astrocytes from those in the ONP at 30 days

Additional file 10 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 10: Supplementary Table 8. Common differentially expressed genes in all three timpoint comparisons in the ONP

Additional file 11 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 11: Supplementary Table 9. Differentially expressed genes and pathways identified in ONP 30 vs 0 days

Additional file 6 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 6: Supplementary Table 4. Differentially expressed genes and significant pathways identified in ONH 30 vs 7 days

Additional file 1 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 1: Supplementary Figure 1. (A-B) In-situ hybridization (A, A’) and immunohistochemical staining (B) of the mouse optic nerve for LCN2. We observed very sparse labeling of LCN2 at 7 days after microbead injections (arrowheads). (C-H) Longitudinal sections of the mouse ONH region immunostained for NDUFC2 and COX5B in untreated and microbead injected mice at 30 days. (I, J) Gene set enrichment analysis using the gene ontology (GO) and KEGG database

Additional file 8 of Astrocytes of the optic nerve exhibit a region-specific and temporally distinct response to elevated intraocular pressure

Additional file 8: Supplementary Table 6. Differentially expressed genes and significant pathways identified in ONP 7 vs 0 days