Genome-wide assessment of gene expression changes was performed in DBA/2J mice. The ONH and retina from 40 DBA/2J eyes at 10.5 months of age were separately profiled. These eyes were selected because they encompassed a range of glaucoma severity (based on a previously validated method for determining the degree of optic nerve damage; see Methods). Selected eyes had the following degrees of damage: no detectable axon damage compared with controls (no or early [NOE]); 10%–50% of axons lost (moderate [MOD]), and greater than 50% axons lost (severe [SEV]). As the main focus of this study was to identify early molecular events in glaucoma, we included 21 eyes with NOE glaucoma (no detectable damage), 9 eyes with MOD glaucoma, and 10 eyes with SEV glaucoma. To keep numbers of eyes in each group relatively balanced, the 21 NOE eyes were divided into two independent groups (NOE1 n = 10, and NOE2 n = 11). Two control groups were also included: 10 eyes from 10.5-month-old DBA/2J-Gpnmb+/Sj (D2-Gpnmb+) mice (age and strain matched, no-glaucoma control) and 10 eyes from 4.5-month-old DBA/2J mice (young, pre-glaucoma control).
Initially, pairwise comparisons were performed between the 4 morphologically defined groups (NOE1, NOE2, MOD, and SEV groups) and the D2-Gpnmb+ control group (dataset 1, Figure A). For the ONH, 223 and 456 probe sets were DE (q ≤ 0.05) in the NOE1 and NOE2 groups, respectively, compared with control. Importantly, the vast majority of probes DE by at least 2-fold were the same in these independent groups. The two most DE genes were lipocalin 2 (Lcn2; DE 8.2-fold NOE1 and 12.7-fold NOE2) and orosomucoid 1 (Orm1; DE 5.8-fold NOE1 and 6.1-fold NOE2), two immunomodulatory genes. For the retinas of these same eyes, only 7 (NOE1) and 24 (NOE2) probe sets were DE compared with controls, including glial fibrillary acidic protein (Gfap; 2.1-fold NOE2) a marker of reactive astrocytosis. Many more probe sets were DE in the MOD and SEV groups, including decreased detection of genes expressed in RGCs (e.g., Sncg, –1.5-fold MOD and –6.3-fold SEV; Nefl, –1.7-fold MOD and –11.0-fold SEV; and Thy1, –1.3-fold MOD and –3.0-fold SEV).
Early stages of glaucoma identified by hierarchical clustering.
Hierarchical clustering allows early disease stages to be identified.
Comparison of eyes grouped by conventional morphological criteria did not allow for sensitive detection of early gene expression changes. We reasoned that grouping the same set of eyes by molecular clustering of gene expression profiles and then comparing these groups would allow more sensitive detection of early changes in glaucoma. Since a critical insult and early damage are known to occur in the optic nerve, clustering was first used to group eyes based on the similarity of gene expression profiles in the ONH. Unbiased hierarchical clustering of the 40 DBA/2J ONH samples and 10 D2-Gpnmb+
control samples was performed using the gene expression values of 570 disease-relevant probe sets (Methods and Supplemental Figure 1; supplemental material available online with this article; doi:
). Hierarchical clustering first linked the most similar eyes, and continued until all eyes were linked in a dendrogram (Figure B and Methods). As the least similar eyes were linked at the highest branch points, a cutoff (threshold of relatedness) was applied to separate groups of eyes into independent disease stages (Methods). We selected a cutoff that made biological sense based on conventional morphological criteria, e.g., Gpnmb+
controls were grouped into their own stage, and SEV eyes were separate from NOE eyes. We only considered groups containing at least 4 eyes as a stage. Using these criteria, 5 new, molecularly defined, stages of glaucoma were identified (dataset 2, stage 1 to stage 5; Figure , B and C). Not surprisingly, 10 of the 40 eyes did not fit into these 5 stages. This reflects the large number of possible molecular states for such a variable disease.
The 5 stages were ordered based on two criteria: (a) Number of DE genes compared with no-glaucoma control: stage 1 had the smallest number of DE genes when stages 1–5 were compared with the Gpnmb+ control group (381 DE genes, Figure C) and was considered to be the earliest molecular stage. (b) Previously determined morphological damage: although stages 2–4 had a similar number of DE genes (approximately 10,000), only stages 1 and 2 contained eyes with no detectable glaucoma (NOE). In comparison, stage 3 contained 3 eyes with NOE glaucoma and 3 eyes with MOD glaucoma. Stage 4 contained 4 eyes with MOD glaucoma. Therefore, stage 3 was determined to be between stage 2 and stage 4. Stage 5 contained only eyes with SEV glaucoma. As expected, axon number decreased with increasing glaucoma stage (Figure A). Also, the number of genes whose expression value was DE by at least 2 SDs from the average of Gpnmb+ controls increased from stages 1 to 5 (Spearman’s correlation 0.9, P < 10–100) (Figure B).
Differences between the molecularly defined ONH stages.
Based on optic nerve damage, 3 of the 6 eyes in stage 3 were indistinguishable from no-glaucoma control eyes. The other 3 eyes had MOD glaucoma (Supplemental Figure 2). This indicates that the molecular changes determining the similarity of eyes in stage 3 must have occurred prior to detectable glaucomatous damage. In addition to RGCs, these early molecular changes may be occurring in a variety of other cell types in the ONH, such as astrocytes, microglia, and endothelial cells.
Principal component analysis (PCA) is a powerful method for reducing the dimensionality of data and is used to facilitate microarray experiments (31
). Here, PCA was used to independently assess whether the 5 identified molecular stages are distinct. The reduced dimensional position of the 40 DBA/2J and 10 control eyes was plotted using the first two principal component vectors calculated for the 570 disease-relevant probe sets (see Methods). As expected, eyes in each of the 5 molecular stages occupied essentially distinct territories (P
< 0.01, Figure C). Therefore, PCA supported the hierarchical clustering.
Molecularly defined ONH stages increase sensitivity to detect DE genes in the ONH.
To identify DE genes, molecularly defined stages 1–5 were compared with the Gpnmb+ control group (Figure ). DE genes were defined as being those with a q value (false discovery rate [FDR]) of 0.05 or less. Significantly more DE genes were detected in the ONH for the molecularly defined stages than for the morphological groups (Figure A). Considering groups with eyes that had no detectable optic nerve damage and only probe sets that were DE at least 2-fold, 1,385 probe sets were DE in molecularly defined stages 1–3 versus 100 in morphologically defined NOE2 eyes (Figure A) and 55 in NOE1 eyes (data not shown). Additionally, many specific genes, such as Timp1, Edn2, Vcan, and Calcb, had a greater fold change in the molecularly as compared with morphologically defined stages (Figure B).
Clustered stages allow sensitive detection of early changes in the ONH.
ECM and immune changes in the ONH.
We used a variety of software-based annotation tools to provide functional insight into the ONH expression differences detected in this study (see Methods). As it is not possible to show all analyses in this article, we highlight a few enriched gene ontology terms, pathways, and networks that are likely to be important during early stages of glaucoma.
Gene ontology (GO) analysis (performed by DAVID; see Methods) revealed that a significant number of genes with the GO terms “immune response” (Gene Ontology Database 0008283), “leukocyte activation” (GO 0045321), and the related term “chemotaxis” (GO 0006935) were DE in the early molecularly defined stages of disease (Figure C). The majority of these changes were not detected in the morphologically based NOE groups. Genes belonging to pathways that are known to affect these same processes were also identified by independent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figure D). Two related KEGG pathways that were significantly overrepresented are “ECM-receptor interactions” (KEGG mmu04512; Figure , A and B) and “focal adhesions” (KEGG mmu04510). Neither of these pathways was found to be important in either of the NOE groups or the MOD group (dataset 1). ECM-receptor interactions are mediated by transmembrane molecules such as integrins and proteoglycans and lead to direct or indirect control of cellular activities that include adhesion, migration, and proliferation. These pathway changes may represent early changes to the glial-vascular unit or altered interactions between glia and neurons in the ONH. One member of this pathway, tenascin C (Tnc
) is 1.9-fold elevated in stage 2, 4.1-fold elevated in stage 3, and 5.7-fold elevated in stage 4 (Figure B) and is localized to the glial lamina region of the ONH (Figure C). Showing the human relevance of our findings, expression of TNC
was increased in glaucomatous compared with normal human ONHs (32
). The importance of ECM-receptor interactions is also supported by computationally distinct k-means clustering, which groups genes that behave similarly across disease stages (Table and Figure ). K-means clustering also suggested that the complement cascade is an important pathway activated early in the ONH. C1qa
is a component of the C1q complex, an early effector in the complement cascade. C1QA is expressed in microglia/macrophages in the ONH during early stages of glaucoma (Figure ).
Genes in the ECM-receptor interaction pathway are upregulated in the ONH.
K-means clustering–identified pathways changing in early stages of glaucoma in ONH
K-means clustering identified pathways changing in early stages of glaucoma.
C1qa expression in microglia in the ONH.
Hubs and transcription factors.
Ingenuity pathway analysis (IPA) identifies molecular networks and biological pathways that are overrepresented in a given set of DE genes (see Methods). IPA allows the identification of key genes (or hubs) that lie at the center of any network or subnetwork. Therapeutic modulation of any hub has strong potential to impact a variety of network participants, making hubs attractive targets for future neuroprotective efforts. We separately analyzed the DE genes for stages 1–5 (dataset 2) by IPA. One of the most statistically significant networks included various members of the ECM-receptor interaction pathway and proinflammatory cytokines (Supplemental Figure 3A). Hubs within this network include IL-1β (encoded by Il1b
, shown as IL1 in IPA network) and integrin β3 (Itgb3
), molecules that are reported to affect ECM composition and metabolism (33
). The network also includes various other integrins, fibronectin 1 (Fn1
), matrix metalloproteinase 19 (Mmp19
), tissue inhibitors of matrix metalloproteinases (Timp1
), and a number of collagens. Of interest, caspase-1 (Casp1
) was upregulated in the earliest glaucoma stage and continued to be increased at later stages (Supplemental Figure 3B). CASP1 has interleukin-converting enzyme activity, and its increase preceded the upregulation of Il1b
genes (Supplemental Figure 3B). Thus, proinflammatory responses are among the earliest changes in the ONH in glaucoma.
Clustering identifies early stages of glaucoma in the retina.
Clustering eyes based on morphological criteria (dataset 1) or the similarity of ONH gene expression profiles (dataset 2) was not sensitive for identifying early changes in the retina. No DE genes were identified in the retina in molecular stages 1 and 2, and only 8 DE genes were identified in stage 3 (Figure C). This may be because early changes in the ONH occur prior to changes in the retina, and/or because our sensitivity to detect retinal changes was lower as whole retinas were used (<5% of total retinal cells are RGCs). Alternatively, it is possible that grouping the eyes based on ONH expression patterns is not a sensitive way to determine early glaucoma changes in the retina. It is possible that early expression changes in the retina are completely or partially independent of events in the optic nerve. Thus, we separately clustered the same set of 40 DBA/2J eyes based on the similarity of their retinal gene expression profiles (dataset 3). We used the same methods as were used for the ONH-based clustering and similar criteria for determining stages (see Methods). Retinal clustering divided the eyes into a Gpnmb+ control cluster and 4 glaucoma stages (R1–R4) that made biological sense (dataset 3, Figure , A and B). Stages R1 and R2 contained only eyes that were previously indistinguishable from each other and controls by conventional morphological criteria. Retinal clustering increased the sensitivity of detecting DE genes at early stages of disease (Figure B). Compared with the D2-Gpnmb+ control stage, 48 probe sets were DE in stage R1 and 8,664 in stage R2. Many fewer DE genes were detected in the ONH of the eyes in these retinal clusters (59 DE genes, dataset 3, stage R1, compared with 381 DE genes, dataset 2, stage 1; Figure C). Since clustering using either the ONH or retina tissues is not sensitive at detecting early DE genes in the other tissue, our analyses suggest that events in the retina and ONH occur asynchronously and are not completely interdependent.
Molecular clustering identifies early stages of glaucoma in the retina.
Analyses of the 48 DE probe sets in stage R1 identified the complement cascade as the only significantly overrepresented pathway (P
= 5 × 10–4
). The DE genes in stage R1 included C1qa
, and C1qc
. In stage R2, 12 members of the complement cascade were DE (Figure C). We have found that C1qa
is expressed in RGCs and localizes to synapses in the retina during early stages of glaucoma (36
). The complement cascade was also implicated in the ONH by k-means clustering (Table and Figure , cluster 6), where it was expressed in microglia (Figure ).
Early DE genes provide molecular markers to assess glaucoma status.
Genes shown to be DE early in glaucoma may allow the development of marker sets to group eyes with no detectable optic nerve damage into those undergoing early glaucoma and those with no glaucoma. Such markers may also be used to determine the degree of glaucoma or to assess the effectiveness of tested treatments. Since many molecular changes occur in glaucoma and specific changes will vary from eye to eye, a reasonable approach is to assess genes from different pathways and to track the number of DE genes for each of these pathways as a measure of glaucoma status.
To assess the feasibility of this approach, we assessed genes from pathways that were activated early in the ONH or retina in the microarray study (Supplemental Tables 2 and 3). These pathways included ECM-receptor interactions, MAPK signaling, and Toll-like receptor signaling for the ONH tissue and the complement cascade for the retina tissue. We assessed completely new sets of 10-month-old DBA/2J and D2-Gpnmb+ control eyes. As we were most interested in eyes with no conventionally detectable glaucoma, we separately assessed the ONH and retina for 21 NOE and 3 MOD DBA/2J eyes, as well as 11 D2-Gpnmb+ control eyes. To further refine the location of retinal changes compared with the microarray study, the retinal tissue was enriched for the RGC layer and lacked the inner nuclear layer and photoreceptor layers (see Methods). For each eye, a gene was classified as DE only if the normalized expression level was at least 2 SDs from the average of the 11 control eyes. This approach ensured that only robust expression changes were regarded as DE. For each eye, we determined the total number of DE genes as well as the number of DE genes in each pathway (Supplemental Figure 5). A pathway was considered “activated” in eyes that had at least 2 times the number of DE genes for that pathway relative to any of the controls.
In both ONH and retina, this analysis clearly distinguished the vast majority of eyes with NOE glaucoma from controls (Supplemental Figure 5A). Individual eyes had differing numbers of DE genes, with eyes classified with MOD glaucoma having the greatest total number of DE genes. For the ONH, ECM-receptor interactions, MAPK signaling, and Toll-like receptor signaling pathways were activated in 18 of the 21 eyes with NOE glaucoma and all 3 eyes with MOD glaucoma. For the retina, the complement cascade was activated in 13 of 21 NOE eyes and 3 of 3 eyes with MOD glaucoma. In the NOE eyes, there was no clear relationship between the number of DE genes in the ONH and retina. Most NOE eyes had high numbers of DE genes in the ONH, with lower numbers activated in the retina (16 of 21 having ≥60% the number of genes activated in moderate for ONH compared with only 3 of 21 for retina). However, the reverse was true in some eyes. Most strikingly, eye number 3 had a large number of DE genes in the retina (similar to eyes with MOD glaucoma), but a low number in the ONH (Supplemental Figure 5). This further suggests that early changes in the retina and ONH are not completely dependent on each other and can occur asynchronously. These marker genes (Supplemental Table 2), along with approaches for assessing glaucoma status in individual eyes, are likely to be valuable for future studies.
C1QA-deficient mice are protected from glaucoma.
Changes in the complement cascade have been reported in animal models and human glaucoma. However, it has not been determined whether these changes occur very early and prior to detectable axon loss, or if they are later and possibly secondary (22
). Our current study demonstrates that complement cascade changes occur very early and prior to detectable glaucoma by conventional assays, with complement genes being among the first genes to change in the retina (Figure C). Given the very early occurrence of these changes and the prior association of complement cascade changes with human glaucoma, we tested the importance of this cascade in DBA/2J mice.
To test the functional importance of the complement cascade in this inherited glaucoma, we generated and analyzed mice with a mutation in the C1qa
gene (Figure ). Clinical examinations of mice from 6.0 to 12.0 months of age assessed the severity of the pigment-dispersing iris disease, which precedes the glaucoma (30
). Both C1QA-deficient and wild-type DBA/2J mice developed prominent pigment dispersion and iris atrophy with a similar age of onset, but progression to the most severe iris stage was slightly delayed in C1QA-deficient mice. Subsequent to the iris disease and despite a modest delay in some C1QA-deficient DBA/2J eyes, IOP became elevated in mice of each genotype, and their IOP distributions overlapped extensively (Figure A). We previously detected an alteration of IOP similar to that in the C1qa
mutants in BAX-deficient DBA/2J mice (39
). In these Bax
mutants, this IOP change did not profoundly alter the degree of optic nerve damage.
C1QA deficiency protects against DBA/2J glaucoma.
mutant mice were protected from glaucomatous damage to the optic nerve and retina at both 10.5 and 12.0 months of age (Figure B), two key ages in this strain (40
). Importantly this protection was very profound at 10.5 months of age, with only 9% of mutant eyes having detectable glaucoma compared with 63% of wild-type eyes (P
= 5 × 10–100
, Figure B). None of the mutant eyes had SEV glaucoma, while 48% of wild-type eyes were severely affected. At 12.0 months of age, only 25% of mutant eyes had SEV glaucoma compared with 48% of wild-type eyes. The percentage of eyes with no detectable glaucoma was more than doubled in C1qa
mutants at 12.0 months (66% versus 30%, P
= 2.2 × 10–9
). Protection in C1qa
mutant mice was confirmed by axon and soma counts (Figure , C–E). Thus, although future experiments are required to completely define the roles of C1QA in ocular drainage and neural tissues during DBA/2J glaucoma, it is likely to have an important role in glaucomatous neurodegeneration. Irrespective of the relative role in different tissues, our findings indicate that complement pathway inhibition has potential as a potent treatment to protect against glaucoma.
Inhibiting the endothelin system protects from glaucoma in DBA/2J mice.
Our current study demonstrates that the Edn2
gene is upregulated in both the retina and the ONH very early in glaucoma (Figure B). The endothelin system was previously found to be upregulated in human and animal models of glaucoma (26
), but our study indicates that the upregulation of Edn2
is a very early event. EDN2 localized to microglia/macrophage-like cells in the nerve fiber layer of DBA/2J retinas (Figure , A–D).
EDN2 is expressed in retinal microglia and may mediate early vascular damage in glaucoma.
Administration of EDN1, which shares receptors with EDN2, is known to kill RGCs. In contrast, EDN2 was suggested to protect photoreceptors from stress (44
). To initially assess whether EDN2 damages RGCs, we injected EDN2 peptide into young DBA/2J eyes and found that it damaged both RGCs and the optic nerve (Figure , E and F). EDN1 is reported to be harmful to neurons by constricting blood vessels, triggering reactive astrocytosis, and/or inhibiting axon transport, but effects on astrocytes and axon transport may be secondary to altered blood flow (45
). Since EDN2 is a potent vasoactive peptide, we hypothesized that EDN2 induces vasoconstriction in the ONH and retina during glaucoma. Even if subtle, this vasoconstriction may metabolically stress RGCs and contribute to their demise in glaucoma. In support of this vasoconstriction hypothesis, we identified a decrease in the ratio of lumen area to total blood vessel area in retinas of DBA/2J eyes at early and later stages of glaucoma (see Methods, Figure , G and H, and Supplemental Figure 6). This decrease was not uniform in any of the eyes investigated, suggesting local differences that may affect some but not all RGCs. The decreased lumen area is likely a result of vasoconstriction, but an EDN2-induced thickening of the vascular smooth muscle (48
) cannot be ruled out. Either way, decreased lumen area is likely to negatively impact perfusion and RGC survival.
To functionally assess the role of the endothelin system in glaucoma, we administered bosentan, an endothelin receptor antagonist, to DBA/2J mice starting at 6.0 months of age (Figure ). We selected bosentan because it antagonizes both types of endothelin receptors and does not alter blood pressure (see Methods). Also, bosentan has been shown to increase ocular blood flow in human glaucoma patients (49
). Administration of bosentan did not alter the onset or progression of either the iris disease or IOP elevation (Figure A). However, bosentan significantly reduced glaucoma at both 10.5 and 12.0 months of age (Figure , B–E). The protection was especially strong at 10.5 months of age, when 80% of treated eyes had no detectable glaucoma, compared with only 39% of untreated eyes (P
= 1.7 × 10–26
). At 12.0 months, 43% of treated eyes had no detectable glaucoma compared with only 31% of untreated eyes (P
= 0.01). These experiments implicate early activation of the endothelin system as pathogenic in this inherited model of glaucoma. Endothelin receptor antagonists offer promise as new treatments to alleviate glaucoma.
Bosentan robustly protects from glaucoma in DBA/2J glaucoma.