Genomic loci modulating retinal ganglion cell death following elevated IOP in the mouse

Abstract

The present study was designed to identify genomic loci modulating the susceptibility of retinal ganglion cells (RGC) to elevated intraocular pressure (IOP) in the BXD recombinant inbred mouse strain set. IOP was elevated by injecting magnetic microspheres into the anterior chamber and blocking the trabecular meshwork using a handheld magnet to impede drainage. The IOP was then measured over the next 21 days. Only animals with IOP greater than 25 mmHg for two consecutive days or an IOP above 30 mmHg on a single day after microsphere-injection were used in this study. On day 21, mice were sacrificed and the optic nerve was processed for histology. Axons were counted for both the injected and the control eye in 49 BXD strains, totaling 181 normal counts and 191 counts associated with elevated IOP. The axon loss for each strain was calculated and the data were entered into genenetwork.org. The average number of normal axons in the optic nerve across all strains was 54,788 ± 16% (SD), which dropped to 49,545 ± 20% in animals with artificially elevated IOP. Interval mapping demonstrated a relatively similar genome-wide map for both conditions with a suggestive Quantitative Trait Locus (QTL) on proximal Chromosome 3. When the relative axon loss was used to generate a genome-wide interval map, we identified one significant QTL (p < 0.05) on Chromosome 18 between 53.6 and 57 Mb. Within this region, the best candidate gene for modulating axon loss was Aldh7a1. Immunohistochemistry demonstrated ALDH7A1 expression in mouse RGCs. ALDH7A1 variants were not significantly associated with glaucoma in the NEIGHBORHOOD GWAS dataset, but this enzyme was identified as part of the butanoate pathway previously associated with glaucoma risk. Our results suggest that genomic background influences susceptibility to RGC degeneration and death in an inducible glaucoma model.

Keywords: Butanoate pathway; GeneNetwork; Glaucoma model; QTL mapping; Systems genetics.

Fig. 1

IOP development over 21 days after bead injection surgery. IOP was measured at the days given on the X-axis and averaged across all strains. IOP rises quickly on the second postoperative day, peaks at 9 days and stays elevated throughout the whole 3 weeks. Error bars show standard error.

Fig. 2

Axon counts for the normal eye (‘control’, grey) and for the eye 21 days after elevation of IOP (‘bead’, red). Error bars show standard error.

Fig. 3

Interval map of axon number across the mouse genome is illustrated for the normal eye (A) and the eye 21 days following elevation of IOP (B). The blue line indicates the total likelihood ratio statistic (LRS) score as specified on the Y-axis. The red line illustrates the contribution from the B6 allele and the green line the contribution from the D2 allele. The location across the genome is indicated on the top from chromosome 1 to chromosome X. Notice one suggestive QTL peak on Chr3 (exceeding the gray line, p < 0.63).

Fig. 4

The average axon loss per strain is given in (A) in decreasing order. The genome-wide interval map is shown in (B). Here, chromosomes are displayed on the X axis, while the LRS score is plotted in blue along the Y-axis. The light red line defines the genome-wide QTL significance interval of p < 0.05, while the light grey line identifies a suggestive QTL. Notice one significant LRS peak on chromosome 18 and one suggestive QTL on chromosome 9.

Fig. 5

Map of gene locations across Chr. 18. The haplotype map for the 47 strains in the axon loss da-taset is shown in the top panel. The haplotype map displays the contribution of each of the two parental strains. The red represents the B6 alleles, green defines the D2 alleles, blue represents regions of the DNA that are heterozygotic and gray is unmapped. At the far right is a list of the specific BXD RI strains with the associated axon loss in decreasing order from top to bottom. The QTL significance is indicated by the blue-line below the haplotype map. A positive additive coefficient (red line) indicates that B6 alleles are associated with higher trait values. The QTL reaches significance from ~56 to ~58 Mb (p < 0.05, light red line).

Fig. 6

A cross section of a C57BL/6J retina stained for nuclei (blue), ALDH7A1 (green), and the neuronal marker TUJ1 (Class III beta-tubulin, red) is shown in (A). Notice the colocalization of ALDH7A1 with RGCs and its stippled distribution in RGC dendrites (white arrows, magnified section). In (B), a flat-mounted retina was stained with ALDH7A1 and RGC marker RBPMS and the ganglion cell layer (GCL) was imaged en face. While all RGCs are double labeled, some cells in the GCL stained exclusively for ALDH7A1 albeit in a less intense manner. (C) is analog to (B) except that it was stained for TUJ1, which also stains RGC axons. ALDH7A1 is most prominently distributed in a perinuclear fashion (arrowhead, magnified section). ALDH7A1 staining is absent in the nucleus, but slightly present in RGC axons. (D) and (E) show optic nerve head cryosections stained for ALDH7A1 and TUJ1 or GFAP, which marks astrocytes and the glial lamina. ALDH7A1 is present throughout the optic nerve and co-localizes with axon bundles rather than astrocytes. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. Low magnification and optic nerve head scale bars = 50 μm. High magnification scale bars = 20 μm.