Ribotag approach to assess optic nerve astrocyte-specific transcriptional changes following elevated intraocular pressure

Rpl22HA/HACre+ mice underwent unilateral microbead injections to elevate IOP (Fig. 1A-C) and mice were sacrificed at 7 and 30 days post-injection. We purified the astrocyte-specific ribosomal mRNA via immunoprecipitation using an antibody against HA. We previously showed that in the optic nerves of naïve Rpl22HA/HACre+ mice HA expression is specific for astrocytes and that the immunoprecipitated fraction is enriched for astrocyte markers and depleted for markers of microglia, oligodendrocytes, NG2 cells and axons [33]. Microbead injections did not change the specificity of HA expression, which colocalized with astrocyte marker SOX9 and not with markers for microglia (IBA1), oligodendrocytes (OLIG2), or NG2 glial cells (NG2) (Fig. 1D). Likewise, the immunoprecipitated fraction was only enriched for astrocyte markers and not for markers of other cell types (Fig. 1E).

Fig. 1

Characterization of the microbead occlusion model and the ribotag mouse. A Schematic depicting the microbead occlusion model of elevating the IOP followed by IOP measurement. B IOP measures for the two cohorts of animals that were sacrificed at 7 and 30 days. N = 6 for each of the 3 independent groups for each 7 and 30 days; microbead injected mice, saline injected mice, and naïve untreated mice. Mean ± SEM. C Loss of ganglion cell density in the treated eye and loss of ganglion cell in the treated eye as a percentage of the untreated contralateral eye. N = 8 biological replicates. * p < 0.05. Paired students t-test. MB = microbead injected. D Immunostaining for HA and cell-specific markers in the optic nerve of RplHA/HA/Cre+ mice. All images are longitudinal sections of the nerve head region, with the retinal end of the optic nerve on the left. Scale bar in top left panel = 50 µm and applies to all panels. E RT-qPCR of the immunoprecipitate (IP) and input from 7 day microbead injected optic nerve heads showing enrichment of astrocyte ribosomal mRNAs over other cell types. N = 4 per group. Mean ± SEM

Cluster analysis reveals temporally distinct phases of the optic nerve head astrocyte response to elevated intraocular pressure

We began our analysis of the effects of elevated IOP on ONH astrocytes by examining how global gene expression levels vary over time using a likelihood ratio test (LRT). This analysis determines sets of genes that exhibit similar variations in expression over time, and which can then be tested for pathway enrichment. We focused on the ONH region because of its importance as a site of neurodegeneration in glaucoma. A total of 15,218 genes were tested for differential expression across time in the microbead injection condition using the LRT model. Of these, 905 significantly variable genes were identified (adjusted p-value < 0.05) and could be grouped into four statistically unique clusters (Fig. 2 and see Table S2 for genes in each cluster): 1 (466 genes), 2 (266 genes), 3 (77 genes), and 4 (96 genes). The two largest gene clusters (Clusters 1 and 2) demonstrated a temporal pattern reflective of a late response in that there was no significant change between 0 and 7 days—surprising as this coincided with the period of IOP increase in our microbead model—followed by an increase (Cluster 1) or decrease (Cluster 2) towards 30 days, a period when the IOP gradually declined. Over-representation analysis (ORA) against the Reactome database determined Cluster 1 to be primarily enriched in pathways for protein and RNA metabolism (encompassing genes encoding for eukaryotic translation initiation factors and ribosomal proteins), and oxidative phosphorylation (respiratory electron transport, ATP synthesis, Complex I biogenesis, and TCA cycle). Here, most genes encoded for complexes of the respiratory electron transport chain, in particular complex I, the complex producing the most reactive oxygen species (Fig. 2; Table S2) [38]. Cluster 2 was particularly enriched for cholesterol biosynthesis, steroid metabolism, and keratinization and cornification.

Fig. 2

Cluster analysis reveals temporally distinct phases of the optic nerve head astrocyte response to elevated IOP. Cluster analysis using likelihood ratio test (LRT) on the 15,218 genes found to be differentially expressed across time and condition. This analysis determines sets of genes that exhibit similar variations in expression over time, and which can then be tested for pathway enrichment. 905 significantly variable genes were identified with an adjusted p-value cutoff of 0.05. Over-representation analysis, using the Reactome database was used to determine pathways (q-value < 0.2) enriched for genes from each cluster

In contrast, Clusters 3 and 4 showed an early response, with either a sharp decline (Cluster 3) or increase (Cluster 4) between 0 and 7 days, followed by a return towards baseline. Cluster 3 showed enrichment in the greatest number of pathways, however many contained very few genes and the same set of genes. The most distinguishing were the top 3, cholesterol biosynthesis, and steroid and lipid metabolism. Cluster 4 was enriched in a single pathway with only 4 associated genes (Apol9b, Calr, Hsp90b1, Stab1). For each of the 4 clusters, notable genes associated with glaucoma, neuroinflammation, or reactive astrocytes were: Cluster 1 (Nrf2, Hspb1, S100β), Cluster 2 (Ptgs2, Clcf1), and Cluster 4 (C2, Tlr4, Lcn2, H2-T23, Thbs1, Il6ra) (Table S2).

These results demonstrate important characteristics of the ONH astrocyte transcriptional response: (1) there were at least two temporally distinct phases of the response, (2) there was limited significant early response (e.g., 0 to 7 days); the majority of transcriptional changes happen later in injury (e.g., after 7 days, Clusters 1 and 2), (3) elevations in IOP primarily upregulate oxidative phosphorylation and RNA and protein metabolism, and downregulate cholesterol biosynthesis, and steroid and lipid metabolism, and (4) astrocytes showed a very limited neuroinflammatory response.

Optic nerve head astrocytes upregulate oxidative phosphorylation, proteolysis and antioxidative capacity following elevated intraocular pressure

Having considered the global patterns of variations over time using the LRT analysis, we next examined whether a pairwise time point comparison would demonstrate previously unappreciated differences. We performed a pairwise comparison between time points, beginning with the ONH. The control data (0 days) we use here was generated in [33]. Examination of the 7 vs 0 (early) and 30 vs 7 day (late) datasets explored transcriptional processes during the injury period when IOP is rising and resolving, while the 30 vs 0 dataset explored changes that persisted at 30 days. Principal component analysis using the top 2000 genes with the greatest variance in expression level across all samples showed moderate clustering between pairwise time point comparisons (Fig. 3A). In comparing the ONH 7 vs 0 day samples, a total of 17,554 genes were tested for differential expression and of these only 32 were upregulated and 30 were downregulated, indicating an extremely limited transcriptional response from ONH astrocytes over this time period and confirming the LRT findings (Figs. 3B and C, Table S3). Some notable upregulated genes included (Fig. 3D): reactive astrocyte markers (H2-T23, Lcn2), a transcription factor (STAT1), amino acid transporters (Slc36a2, Slc7a5), apolipoprotein (Apol9b), an anchoring protein important for cell motility, stability and scar formation (Akap12), and heat shock proteins (Hspa1b, Hsph1). With very few differentially expressed genes, gene set enrichment analysis (GSEA) against the Reactome database did not find significantly enriched pathways. This was additionally confirmed against the GO and KEGG database.

Fig. 3

Transcriptional response of optic nerve head astrocytes to elevated IOP. A Principal component analysis based on expression levels of the top 2000 most variable genes across 7 vs 0, 30 vs 7 and 30 vs 0 day ONH samples. B Number of DEGs for each independent time point pairwise comparison. Significance was adjusted p-value < 0.05; no log2 fold change cutoff was applied. C Venn diagram showing the number of unique and overlapping DEGs across the 3 independent time point comparisons (adjusted p-value < 0.05). D Top 30 up and downregulated genes for each of the 3 independent time point comparisons, based on rank from largest to lowest log2 fold change value (absolute value, log2 FC > 0 upregulated, log2 FC < 0 downregulated). E Up and downregulated pathways from the 30 vs 7 day sample. Based on over-representation analysis of the up and down regulated genes using the Reactome database. Listed are all pathways with adjusted p-value < 0.05. Each pathway is color matched for the broader category it belongs to. NES refers to the normalized enrichment score that accounts for differences in gene set size and in correlations between gene sets and the expression dataset. F Up and downregulated pathways from the 30 vs 0 day sample. Based on over-representation analysis of the up and down regulated genes using the Reactome database. Listed are all pathways with adjusted p-value < 0.05. Each pathway is color matched for the broader category it belongs to

Lcn2 is an acute-phase secreted protein, a supposed pan-reactive astrocyte marker, and has diverse functions including immune regulation, neuroinflammation, iron homeostasis, cell proliferation, and differentiation [39, 40]. Astrocytes are thought to be the primary source of Lcn2 in the brain and its up regulation has been noted in the retina secondary to glaucoma, implicating it in the promotion of neuroinflammation in the disease [41,42,43,44,45,46,47]. Lcn2 is localized to optic nerve astrocytes in optic neuritis [48], but it’s localization in the glaucomatous optic nerve is unknown. We found Lcn2 to be upregulated early (Cluster 4, Fig. 2; pairwise comparisons of 7 vs 0 days, ONH log2FC = 0.73 p.adjust value = 0.04, ONP log2FC = 2.33 p.adjust value = 1.01E-09), but surprisingly not significantly expressed at the mRNA and protein levels (Figs. S1A and B). This result highlights the heterogenous nature of the reactive astrocyte response across regions (retina vs optic nerve) and injury models.

Comparing the ONH 30 vs 7 day samples, a total of 16,826 genes were tested and of these 277 were upregulated and 164 were downregulated (Figs. 3B and C, Table S4), a larger number than between 7 vs 0 days but still a relatively small transcriptional response. Prolactin (Prl) was strongly downregulated in all 7 of the 30 day samples except for one, thus giving the appearance of high upregulation on the heatmap (Fig. 3D). Downregulated genes included (Fig. 3D): keratins (Krt5, Krt13, Krt14, Krt17), amino acid transporter (Slc38a5), heat shock protein (Hspa1b), collagen (Col17a1), ATPase transporters (ATP10a, ATP10d) and Pecam1. Pathway analysis showed robust upregulation of oxidative phosphorylation (primarily in the respiratory electron transport chain subunits, as observed in the LRT analysis), and RNA and protein metabolism (Fig. 3E; Table S4). Upregulation of the complex I subunit Ndufc2 and the complex IV subunit Cox5b was confirmed at the protein level using immunohistochemistry (Figs. S1C-H). Many of the downregulated processes relate to astrocyte morphology (keratinization, RHO GTPase), their interaction with the surrounding extracellular matrix (collagens, integrins) and communication with each other (gap junction/connexin regulation) (Fig. 3E). These transcriptomic data are supportive of the known changes in morphology and spatial arrangement that reactive ONH astrocytes undergo in glaucoma, characterized by a general loss of processes and branching, detachment and retraction of distal processes and terminals from the circumferential surface, hypertrophy of remaining processes and in chronic cases becoming amoeboid in shape [17, 19, 49, 50]. These changes would also be expected to break the gap junctional connections between astrocytes. In addition, our data predicts a downregulation in the transport of substrates across astrocyte membranes. Two of the broader categories—vesicle-mediated transport (processes for gap junction/connexin transport and regulation) and transport of small molecules (processes for SLC-mediated, amino acids, cations/anions transport)—were unique to the ONH astrocyte response and not present in the ONP astrocyte response at any time (see below). Some, but not all of these processes recover by 30 days (see 30 vs 0 day comparison below). Subsequent GSEA on the biological process and molecular function subset of the gene ontology (GO) database confirmed these changes, many downregulated processes were associated with cell adhesion and extracellular matrix organization (Fig. S1I). Ribosomal structure was the only GO biological process upregulated (Fig. S1I), in line with the upregulation of oxidative phosphorylation. KEGG pathway analysis predictably showed an upregulation of ribosome and oxidative phosphorylation pathways and downregulation of tight junction, focal adhesion, MAPK and IL-17 signaling pathways (Fig. S1I). Leukocyte transendothelial migration was downregulated, a pathway significantly upregulated in whole ONH tissue gene expression data from the DBA/2 J mouse model of glaucoma [51].

A comparison of the ONH 30 vs 0 day sample showed many persistent transcriptional changes at 30 days, a time when IOP was declining. A total of 13,508 genes were tested and of these 599 were upregulated and 296 were downregulated, the greatest number of DEGs of all three time point comparisons (Fig. 3B, Table S5). The majority of the top 30 upregulated genes encode for ribosomal proteins (Rps23-ps1, Rps29, Rplp2, Rps21, Rps27, Rpl39, Rps28, Rpl37a, Rps12-ps3) and electron transport chain subunits (Cox5b, Ndufc1, Usmg5, mt-Atp6, mt-Nd4l)(Fig. 3D). Two noteworthy genes on the list were mt-Rnr1 (MOTS-c) and mt-Rnr2 (Humanin), both of which have a number of cyto- and metaboloprotective effects [52]. The vast majority of mitochondrial proteins are encoded by nuclear genes, however, both MOTS-c and Humanin are mtDNA-encoded peptides that act as “retrograde signals” relaying activity and stress signals back to the nucleus to enact changes in gene expression of nucleus-encoded mitochondrial proteins and other factors needed to maintain cellular and mitochondrial homeostasis [53, 54]. Some of the stresses known to induce this retrograde signaling include altered mitochondrial ATP production, reactive oxygen species generation and unfolded/improperly imported proteins [55], processes that were significantly upregulated in this study (see below). The top 30 downregulated genes included those for cholesterol/sterol biosynthesis (Cyp51, Acat2, Dhcr7, Sqle, Acss2), fatty acid biosynthesis (Faah), the transcription factor Olig2, purinergic receptor P2ry12 and Pdgfrb (Fig. 3D). Processes for energy metabolism remained upregulated, particularly oxidative phosphorylation, and RNA and protein metabolism (Fig. 3F; Table S5). Other notable upregulated pathways at 30 days included ER-phagosome, endosomal/vacuolar, senescence-associated secretory phenotype (SASP) and cellular response to stimuli. Interestingly, genes encompassed in these pathways are involved in proteolysis—essential for many cellular processes of which a key one is the response to oxidative stress—and include histocompatibility complexes, anaphase-promoting complexes, ubiquitin conjugating enzymes, and proteosome subunits (Table S5). Numerous antioxidant genes were represented within the cellular response to stimuli category, including glutathione peroxidases, peroxiredoxins, superoxide dismutases and thioredoxins (Table S5). Further suggesting an important role of proteolysis, we also observed the upregulation of heat shock proteins early in injury (Hsp90 in Fig. 2, Cluster 4; Hspa1b in Fig. 3D, upregulated in 7 vs 0 days), which identify misfolded or unfolded proteins and target them for proteasomal degradation. Consistent with the LRT analysis, there was persistent downregulation of cholesterol, lipid, and steroid metabolism at 30 days (Fig. 3F). However, additional downregulated pathways emerged in this pairwise comparison, including those for vesicle-mediated transport (gap junction assembly/trafficking, transport of connexons to the plasma membrane), extracellular matrix organization, cell–cell communication (adherens junction interactions) and transport of small molecules (SLC-mediated transmembrane transport). The number of pathways associated with extracellular matrix organization was reduced compared to the 30 vs 7 days comparison (Fig. 3E), suggesting that tissue remodeling programs and morphological changes associated with elevated IOP have normalized by 30 days. Supplementary GSEA on the biological process and molecular function subset of the GO database and KEGG pathway analysis confirmed these findings (Fig. S1J).

We next sought to determine upstream transcription factors associated with the reactive ONH astrocyte response at 30 days. We derived a transcription factor activity enrichment score based on the log2 fold change differential expression analysis of two groups (day 30 vs 0) and observed only limited clustering of differentially expressed transcription factors by condition (Fig. S2A). We performed motif analysis with HOMER to search for existing motifs that were enriched in regions flanking the transcription start site (TSS) of an input list of genes; here we focused on significantly upregulated genes (adjusted p.value < 0.05, log2FC > 0, motifs searched within a region of ± 2 kb around the TSS). Five motifs and their corresponding transcription factors were found to be enriched (ETS, YY1, ELF1, Elk1, Elk4, Fig. S2B).

We previously showed that cocaine and amphetamine-regulated transcript (Carpt) is an ONH astrocyte specific marker [33]. Here we found that it was not differentially expressed at any time following IOP elevation (7 vs 0 days, log2FC = 0.02, p.adjust 0.95; 30 vs 7 days, log2FC = 0.03, p.adjust 0.92; 30 vs 0 days, log2FC = 0.12, p.adjust 0.54; Tables S3, 4 and 5). It was the top differentially expressed gene in ONH vs ONP comparison at 30 days (Fig. S2C), and its in-situ and immunohistochemical localization remained within ONH astrocytes at 7 and 30 days (Fig. S2D and E). The results indicate that Cartpt remains a specific marker of ONH astrocytes following IOP elevations.

Optic nerve proper astrocytes show a greater transcriptional response to elevated intraocular pressure than optic nerve head astrocytes and they also primarily upregulate oxidative phosphorylation

Optic nerve pathology in glaucoma preferentially affects the nerve head tissue region and we hypothesized that astrocytes in the myelinated ONP region would behave differently to those in the ONH region. Principal component analysis using the top 2000 genes with the greatest variance in expression level across all samples showed clear separation for the 30 vs 7 day samples (Fig. 4A). Based on the number of DEGs between the three independent pairwise time point comparisons, ONP astrocytes showed a greater transcriptional response than ONH astrocytes (Fig. 4B). Although both have approximately the same number of DEGS at 30 days (895 vs 781), ONP astrocytes experienced a greater early change (7 vs 0 days) and during the remainder of the injury period (30 vs 7 days). Comparing the 7 vs 0 day samples there were 310 DEGs (contrasting with only 62 in ONH astrocytes; Figs. 3B and C, Table S6), 143 were upregulated and 167 downregulated. Of the top 30 upregulated genes many were keratins (Krt5, 6a, 13, 14, 17), and several were in common with the top 30 upregulated genes in ONH astrocytes (Lcn2, Apol9b, Hspa1b) (Fig. 4D). Unlike the ONH astrocyte response at this time, pathway analysis showed differential regulation of several processes (Fig. 4E; Table S6). Keratins are intermediate filaments that function as part of the cytoskeleton to mechanically stabilize cells against physical stress. This occurs through connections to desmosomes and hemidesmosomes. Keratinization and cornification increase keratin production, which is in line with the view that astrocytes experience mechanical stress following elevated IOP; however, it was surprising that an early increase was observed in ONP and not ONH astrocytes. Keratinization and cornification were downregulated in both ONH and ONP (see below) astrocytes between 7 and 30 days, indicating that strengthening was only required early when IOP was rising. Genes annotated for cell–cell communication mediate the formation and maintenance of adherens junctions, tight junctions, as well as aspects of cellular interactions with the extracellular matrix and hemidesmosome assembly.

Fig. 4

Transcriptional response of optic nerve proper astrocytes to elevated IOP. A Principal component analysis based on expression levels of the top 2000 most variable genes across 7 vs 0, 30 vs 7 and 30 vs 0 day ONP samples. B Number of DEGs for each independent time point pairwise comparison. Significance was adjusted p-value < 0.05; no log2 fold change cutoff was applied. C Venn diagram showing the number of unique and overlapping DEGs across the 3 independent time point comparisons (adjusted p-value < 0.05). D Top 30 up and downregulated genes for each of the 3 independent time point comparisons, based on rank from largest to lowest log2 fold change value (absolute value, log2 FC > 0 upregulated, log2 FC < 0 downregulated). E Up and downregulated pathways from the 7 vs 0 day sample. Based on over-representation analysis of the up and down regulated genes using the Reactome database. Listed are all pathways with adjusted p-value < 0.05. Each pathway is color matched for the broader category it belongs to. NES refers to the normalized enrichment score that accounts for differences in gene set size and in correlations between gene sets and the expression dataset. F Up and downregulated pathways from the 30 vs 7 day sample. Based on over-representation analysis of the up and down regulated genes using the Reactome database. Listed are all pathways with adjusted p-value < 0.05. Each pathway is color matched for the broader category it belongs to. G Up and downregulated pathways from the 30 vs 0 day sample. Based on over-representation analysis of the up and down regulated genes using the Reactome database. Listed are all pathways with adjusted p-value < 0.05. Each pathway is color matched for the broader category it belongs to

The greatest number of gene expression changes was observed in the 30 vs 7 day comparison where there were 3756 DEGs (2089 upregulated and 1667 downregulated; Figs. 4B and C, Table S7). Optic nerve proper astrocytes demonstrated 22 genes differentially expressed in all three timepoint comparisons suggesting a common set of injury response genes (Fig. 4C; Table S8). The top 30 up and downregulated genes are shown in Fig. 4D. Like ONH astrocytes, oxidative phosphorylation was a key upregulated process, notably the only one (Fig. 4F, Table S7). There were a large number of downregulated processes, and of these, extracellular matrix organization and developmental biology were downregulated at this time point comparison in both ONH and ONP astrocytes (Fig. 4F).

A comparison of the 30 vs 0 day sample discovered 781 DEGs (352 upregulated and 429 downregulated; Fig. 4B, Table S9). Among the top 30 upregulated genes were heat shock transcription factor 4 (Hsf4), glutathione peroxidase 3 (Gpx3), neuropeptide Y (Npy), and glucokinase (Gck). Top downregulated genes included: collagen (Col7a1), WNT inhibitory factor 1 (Wif1), RAS protein activator like 1 (Rasal1), arachidonate 5-lipoxygenase (Alox5), C–C motif chemokine receptor (Ccr1), interleukin 23 subunit alpha (Il23a), and solute carrier family 38 member 5 (Slc38a5). The large reduction in DEGs compared to the 30 vs 7 day comparison indicates many processes are likely to have stabilized by 30 days, and this is also reflected in the reduced number of differentially regulated pathways (Fig. 4G). In all, there were several commonalities and differences in the ONH vs ONP astrocyte response to elevated IOP at 30 days, indicating unique functional specializations in the response of different astrocyte populations in the optic nerve. In common, both ONH and ONP astrocytes upregulate oxidative phosphorylation and RNA and protein metabolism (30 vs 0 days), however, ONH astrocytes additionally upregulated antioxidative capacity and proteolysis. Only ONH astrocytes downregulated processes for vesicle-mediated transport (gap junction/connexon trafficking and regulation) and transport of small molecules (SLC-mediated, amino acids, cations/anions). Although both ONH and ONP astrocytes downregulated cholesterol biosynthesis, ONH astrocytes also downregulated other lipid pathways, including steroid, phospholipid and glycerophospholipid metabolism.

Elevated intraocular pressure increases ATP production, mitochondrial biogenesis and shifts the cellular source of ATP

Along with the transcriptional upregulation of oxidative phosphorylation, ONH astrocytes showed an increase in ATP production (Fig. 5A) and mitochondrial biogenesis (Fig. 5B). Using an assay to measure glycolysis and oxidative phosphorylation we observed a metabolic shift in cellular ATP source in response to elevated IOP; ONH astrocytes significantly doubled the proportion of ATP derived from oxidative phosphorylation (from 16 to 32%) and decreased the amount from glycolysis (from 84 to 68%; Fig. 5C), consistent with the transcriptional observations. Interestingly, there was no such shift in ONP astrocytes following elevated IOP (Fig. 5C). Although we use whole tissues for these assays, as the optic nerve head and proper regions are densely populated with astrocytes it is most likely the results reflect changes in astrocyte metabolism. Oxidative phosphorylation also increases the production of reactive oxygen species and ONH astrocytes upregulated defense genes including glutathione peroxidases, peroxiredoxins, superoxide dismutases and thioredoxins (Fig. 3F and Table S5). We used a DHE (dihydroethidium) assay kit to measure reactive oxygen species directly in live unfixed cells at 30 days and indeed found the levels to be significantly lower in all cells of the ONH region compared to the ONP, consistent with an upregulation in the defense gene activity (Fig. 5D).

Fig. 5

Expression of various astrocyte genes and signaling pathways. A Functional ATP assay measuring ATP levels in optic nerve head tissues from control and 30 days microbead injected animals. MB = microbead injected. N = 4 for each group. Mean ± SEM. * p < 0.05. B Mitochondrial biogenesis in optic nerve head tissues from control and 30 days microbead injected animals. MB = microbead injected. N = 4 for each group. Mean ± SEM. ** p < 0.01. C Changes in the proportion of cellular ATP derived from glycolysis versus oxidative phosphorylation in response to elevated IOP within optic nerve head tissues. Oligomycin was used to inhibit ATP synthesis by oxidative phosphorylation. N = 4 for each group. Mean ± SEM. *** p < 0.01. D Reactive oxygen species was measured using DHE (dihydroethidium) labeling of live unfixed longitudinal sections of the optic nerve. E Log2 fold change values for various astrocyte genes, as determined in the differential expression analysis of the 3 independent time point pairwise comparisons. Astericks indicate significant differences between the 2 groups (adjusted p-value < 0.05). F Log2 fold change values for reactive astrocytes genes, as determined in the differential expression analysis of the 3 independent time point pairwise comparisons. Astericks indicate significant differences between the 2 groups (adjusted p-value < 0.05). G Gene set enrichment analysis on the 3 time point pairwise comparisons against a user defined subset of the GO, KEGG and Reactome pathway, focusing on key reactivity and injury signaling pathways: nuclear factor kappa B (NFkB), calcineurin-nuclear factor activating of T-cells (CaN-NFAT), mitogen-activated protein kinase (MAPK), janus kinases/signal transducer and activator of transcription (JAK/STAT), Wnt/b-catenin, and sonic hedgehog (adjusted p-value < 0.05). NES = normalized enrichment score

Genes and pathways that differentiate the optic nerve head astrocytes from those in the optic nerve proper are largely retained following elevated intraocular pressure

We wanted to determine whether the transcriptional profile that differentiated ONH astrocytes vs those in the ONP was retained following elevations in IOP. We compared 30 day ONH samples with matching ONP samples and found that genes and pathways distinguishing ONH vs ONP astrocytes at 0 day [33] continued to be high differentially expressedø in the 30 day comparison. Numerous top 50 genes in the ONH vs ONP comparison at 0 days were also found at 30 days, including: Cartpt, Mgarp, Dct, Tes, Clec18a, Cntnap2, Tmem37, Bok, Ankrd33b, Col8a1 (compare Fig. S2C with Fig. 3B in [33]). Similarly, oxidative phosphorylation, metabolism of protein, RNA and extracellular matrix organization differentiated ONH vs ONP astrocytes in both 0 and 30 days (Table S10). This indicates that regional astrocyte identity is largely maintained in elevated IOP.

Optic nerve head astrocytes show a limited neuroinflammatory transcriptional response following elevated intraocular pressure

Having examined genes and pathways differentially expressed using a global, unbiased approach, we next looked at genes of interest related to general astrocyte biology, including canonical astrocyte markers and genes involved in astrocyte homeostasis. We looked at the cytoskeletal markers Gfap, Vim and Nes, the cytosolic markers Aldh1l1, Aldoc, S100β, the lipoprotein Apoe, and the water channel Aqp4 (Fig. 5E). All were not significantly differentially expressed at any time point comparison except for S100 β which showed upregulation at 30 days (30 vs 7; log2FC 0.53, p.adjust 0.03; 30 vs 0; log2FC 0.42, p.adjust 0.03). Similarly, very few genes associated with the general function of astrocytes (homeostasis, phagocytosis, Ca2+ flux, connexins/ATP channels, receptors) were differentially expressed (Fig. 5E). Abca1 was upregulated at 30 days (log2FC 0.48, p.adjust 0.02) and is associated with numerous functions such as (1) the efflux of cholesterol and phospholipids to lipid-free apolipoproteins, (2) phagocytosis, although its pathway molecules Megf10 and Gulp1 were not upregulated, (3) immune cell regulation and (4) anti-inflammation. Lamp1 is a lysosomal membrane protein and was downregulated at 30 days (log2FC -0.44, p.adjust 0.03). Atp2b4 is a plasma membrane Ca2+-transporting ATPase and was upregulated at both 7 (log2FC 0.82, p.adjust 0.04) and 30 days (log2FC 0.51, p.adjust 0.02). The ONH is densely populated with astrocytes and is a site of neuroinflammatory changes in glaucoma. Of the genes related to complement, neuroinflammation and MHC I very few were upregulated (Fig. 5E). C2 was upregulated at day 30 (log2FC 0.53, p.adjust 0.0004) and C1ra downregulated (log2FC -0.58, p.adjust 0.04). C3, a general marker of astrocyte reactivity [