Meox2 Haploinsufficiency Accelerates Axonal Degeneration in DBA/2J Glaucoma

Abstract

Purpose: Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma.

Methods: Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches.

Results: URA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age- and disease-specific vascular and myeloid alterations.

Conclusions: Our data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma.

Figure 1

Meox2 is an early upstream regulator of genes that have significant fold changes of expression early in D2 glaucoma. (A) Upstream regulator analysis on DE genes (FDR < 0.05) from stage 1c with a fold change greater than |1.5| was performed. This analysis determined Htatip2 (FC = 2.612, P = 3.65 × 10⁻²), Nfatc1 (FC = 2.577, P = 1.80 × 10⁻¹), Rcan1 (FC = 2.537, P = 1.19 × 10⁻¹), Meox2 (FC = 2.373, P = 1.76 × 10⁻⁶), and Ikzf1 (FC = 2.365, P = 1.08 × 10⁻²) to be the top five regulatory transcription factors (TFs) at this stage. (B) Each of the top five regulatory TFs was further analyzed to determine the predicted number of genes targeted. (34, 30, 18, 7, and 6 genes targeted, respectively). (C) To understand the expression behavior of the top five regulatory TFs through the stages of D2 glaucoma, the fold change of expression over the seven stages was analyzed. Each of the five TFs had a similar expression pattern; all but Meox2 first becomes significant at stage 1b (Meox2 at stage 1c), remaining upregulated through stage 5. (D) Next, the number of mechanistic network associations for each TF was found. Nfatc1 and Meox2 were the only upstream regulators that were associated with mechanistic networks (653 and 364 genes, respectively). (E) KEGG Pathway analysis of genes in the Nfatc1 mechanistic network associations found pathways associated with inflammation, cell–cell interactions, and basement membrane turnover (FDR < 0.05). (F) KEGG Pathway analysis of genes in the Meox2 mechanistic network associations found pathways associated with inflammation, cell–cell interactions, and basement membrane turnover (FDR < 0.05).

Figure 2

Gene ontology (GO) enrichment analysis of microarray data of D2 glaucoma identifies Meox2 in GO terms associated with angiogenesis. (A) Gene ontology enrichment analysis of microarray data obtained by comparing D2 and D2.Gp⁺ controls found the –log(P value) of the three most significant GO terms by Benjamini corrected P < 0.05 were “angiogenesis” (P = 1.35 × 10⁻⁷), “positive regulation of transcription from RNA pol II promoter” (P = 8.97 × 10⁻⁷), and “transcription DNA-templated” (P = 4.26 × 10⁻⁵). The size of the circle associated with the GO term corresponds with the number of genes within that term (angiogenesis = 123 genes, positive regulation of transcription from RNA polymerase II promoter = 399 genes, transcription DNA-templated = 690 genes). (B) The number of differently expressed genes in the GO term “angiogenesis” at each molecularly defined stage of glaucoma that are either up- or downregulated at that stage. (C) Fold change of expression at each molecularly defined state of glaucoma of a selection of genes from the GO term “angiogenesis” and Meox2 was graphed. These genes are related to basement membrane (Col4a1, Col4a2), blood clotting (Fn1), cell proliferation (Pik3cg), breakdown of extracellular matrix (Mmp14), and receptor for vascular endothelial growth factors (Flt4). Col4a1, Fn1, and Pik3cg all become significantly upregulated at stage 1b and continue through stage 5; Col4a2, Meox2, and Mmp14 all become significantly upregulated at stage 1c and continue through stage 5; Vegfc and Flt4 become significantly upregulated in stage 1c; Vegfc remains significant except for stage 4; Flt4 remains significant through stage 4.

Figure 3

K-means analysis identifies Meox2 clustered with genes enriched for inflammatory pathways. (A) K-means clustering (k = 6) of genes differentially expressed through stages 2 through 5 identified five groups of genes with similar gene expression patterns across the stages (number of genes: group 1 = 700, group 2 = 931, group 3 = 477, group 4 = 794, group 5 = 455, and group 6 = 780). Meox2 is in group 3. Group 6 contains unclustered genes. (B) KEGG Pathway analysis on Meox2-containing group 3 reported “lysosome,” “phagosome,” “other glycan degradation,” and “osteoclast differentiation” as the most significant pathways by Benjamini corrected P < 0.05 (P = 9.69 × 10⁻⁹, P = 4.24 × 10⁻⁵, P = 2.21 × 10⁻⁴, and P = 5.99 × 10⁻⁴, respectively). These enriched KEGG pathways are correlated with myeloid and inflammatory mechanisms. (C) GO enrichment analysis on group 3 identified “extracellular exosome,” “lysosome,” and “plasma membrane” as most significant (Benjamini corrected P < 0.05). (D) Expression of myeloid cells from enriched pathways matches Meox2 expression patterns. (E) Similarly, genes in glia–vascular-associated pathways also match Meox2 expression patterns.

Figure 4

Meox2 haploinsufficiency does not cause significant differences in blood vessel area or myeloid cell number in the ONH or retinas of young D2 mice. (A) Western blot analysis on 4 months D2 (DBA/2J) and D2.Mx^+/− retinal tissue probing for MEOX2 protein and GAPDH (glyceraldehyde 3-phosphate dehydrogenase, loading control). There was no significant reduction in MEOX2 between young D2 and D2.Mx^+/− mice (P = 0.8847). See Supplementary Figure S3A for full Western blot. (B) Immunofluorescence was performed on 4 months female ONH tissue of both D2 and D2.Mx^+/− labeling for CD31 (endothelial cells) and laminin-111 (LAM, basement membrane). Representative images are shown. There were no observable differences in expression in a sample of n = 6 per group. Scale bar: 50 μm. (C) Immunofluorescence was performed on 4 months female retina tissue of both D2 and D2.Mx^+/− labeling for collagen-4 (COL4, basement membrane) and ionized calcium binding adapter molecule-1 (IBA1, myeloid cells). Representative images are shown. (D) COL4-labeled retinal vessel area was calculated in both 4 months D2 and D2.Mx^+/− mice. There was no significant difference in vessel area in a sample of n = 4 per condition (P = 0.7830). (E) IBA1-positive cells were counted in both 4 months D2 and D2.Mx^+/− mice. There was no significant difference in myeloid cell number in a sample of n = 4 per condition (P = 0.8097). (F) Immunofluorescence was performed on 4 months female ONH tissue of both D2 and D2.Mx^+/− mice labeling for IBA1 and DAPI. (G) IBA1 and DAPI double-positive cells were quantified as a function of area. There was no significant difference between 4 months D2 and D2.Mx^+/− females (P = 0.1782). All P values were calculated using an unpaired t-test.

Figure 5

Meox2 haploinsufficiency does not affect iris pigment dispersion disease progression or intraocular pressure in DBA/2J mice. (A) Representative slit-lamp images from clinical examinations of 9 months mice. Each row contains three images from the same eye (left column, broad beam illumination; middle column, relative dimensions of the anterior chamber; right column, transillumination). There were no observable differences in iris disease progression from D2 and D2.Mx^+/− females (n = 30, 22, respectively) and males (n = 16, 18, respectively) at 9 months. (B) IOP changes relative to age in D2 glaucoma. Here, we analyzed age-related distribution of IOP in D2 and D2.Mx^+/− mice. No significant difference was found between D2 and D2.Mx^+/− mice within sex and age. Dot blots and standard error mean bars were generated using Prism v7.05. Each dot represents the average IOP of a triplicate reading of one eye within one experiment. The dotted line at 21 mm Hg represents a threshold of glaucoma-relevant IOP (8 months: female D2 n = 27, D2.Mx^+/− n = 39; male D2 n = 33, D2.Mx^+/− n = 40) (10.5 months: female D2 n = 24, D2.Mx^+/− n = 30; male D2 n = 20, D2.Mx^+/− n = 46) (12 months: female D2 n = 20, D2.Mx^+/− n = 22; male D2 n = 22, D2.Mx^+/− n = 46). (C) Meox2 haploinsufficiency did not affect blood pressure when compared to wild-type controls; however, there is a significant difference between DBA/2J females and males at 8 months of age (P = 0.0199).

Figure 6

Meox2 haploinsufficiency accelerates axon damage in D2 mice. (A) Representative images of DBA/2J optic nerve cross sections stained with paraphenylenediamine (PPD) showing the previously validated morphologic categories of axon damage (NOE, no axonal damage or early molecular changes; MOD, moderate damage; SEV, severe damage). Scale bar: 25 μm. (B) Distributions of axon damage of male and female D2 and D2.Mx^+/− mice aged 4, 8, 10.5, and 12 months. (C) Distributions of optic nerve damage show a significant difference between D2 and D2.Mx^+/− eyes in females at 8 months (χ² values = 4.57928 × 10⁻⁰⁹, 0.015375192, and 6.72681 × 10⁻²⁶, respectively), a significant difference between D2 and D2.Mx^+/− in females and males at 10.5 months (χ² values = 0.000557957, 0.008348159, 0.01096447, and 0.011528155, respectively), and a significant difference between D2 and D2.Mx^+/− in females and males at 12 months (χ² values = 8.02393 × 10⁻⁰⁶, 0.039435742, and 0.000221381, respectively). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Aged Meox2 haploinsufficient mice have reduced vessel density in the retina and increased myeloid cells in the ONH. (A) Western blot analysis on 10.5 months D2 (DBA/2J) and D2.Mx^+/− retinal tissue probing for MEOX2 protein and GAPDH (loading control). There was a 44.4% reduction in MEOX2 protein levels between aged D2 and D2.Mx^+/− mice (P = 0.0026). See Figure S3B for full blot. (B) Immunofluorescence was performed on 10.5 months female ONH tissue of both D2 and D2.Mx^+/− labeling for CD31 (endothelial cells) and laminin-111 (LAM, basement membrane). Representative images are shown. There was no observable difference in expression in a sample of n = 6 per condition. Scale bar: 50 μm. (C) Immunofluorescence was performed on 10.5 months female retina tissue of both D2 and D2.Mx^+/− labeling for collagen-4 (COL4, basement membrane) and IBA1 (myeloid cells). Representative images are shown. (D) COL4-labeled retinal vessel area was calculated in both 4 months and 10.5 months D2 and D2.Mx^+/− mice (young data reproduced from Fig. 4D). There was a significant decrease between 4 months and 10.5 months in both D2 and D2.Mx^+/− mice (P = 0.0005 and P = 0.0244, respectively). However, there was also a significant increase in vessel area between 10.5 months D2.Mx^+/− and D2 mice (P = 0.0133). (E) IBA1-positive cells were counted in the retinas of both 4 months and 10.5 months D2 and D2.Mx^+/− mice. There was no significant difference in myeloid cell number in a sample of n = 7 per condition between 10 months D2 and D2.Mx^+/− mice (P = 0.9615). However, there was significant increase between 4 months and 10.5 months in both D2 and D2.Mx^+/− mice (P = 0.0319 and P = 0.0248, respectively). (F) Immunofluorescence was performed on 10.5 months female ONH tissue of both D2 and D2.Mx^+/− mice labeling for IBA1 and DAPI. (G) IBA1 and DAPI double-positive cells were quantified as a function of area. There was no significant difference between 10.5 months D2 and D2.Mx^+/− females (P = 0.1782). There is a significant increase in IBA1⁺DAPI⁺ cells in D2 and D2.Mx^+/− ONHs from 4 to 10.5 months (P = 0.0017 and P = < 0.0001, respectively). Additionally, there is a significant increase in D2.Mx^+/− ONHs from 8 to 10.5 months (P = 0.0148). All P values were found using a 2-way ANOVA.