The Ercc1-/Δ mouse model of XFE progeroid syndrome undergoes accelerated retinal degeneration

Abstract

Age-related macular degeneration (AMD) is a major cause of vision loss in older adults. AMD is caused by degeneration in the macula of the retina. The retina is the highest oxygen consuming tissue in our body and is prone to oxidative damage. DNA damage is one hallmark of aging implicated in loss of organ function. Genome instability has been associated with several disorders that result in premature vision loss. We hypothesized that endogenous DNA damage plays a causal role in age-related retinal changes. To address this, we used a genetic model of systemic depletion of expression of the DNA repair enzyme ERCC1-XPF. The neural retina and retinal pigment epithelium (RPE) from Ercc1^-/Δ mice, which models a human progeroid syndrome, were compared to age-matched wild-type (WT) and old WT mice. By 3-months-of age, Ercc1^-/Δ mice presented abnormal optokinetic and electroretinogram responses consistent with photoreceptor dysfunction and visual impairment. Ercc1^-/Δ mice shared many ocular characteristics with old WT mice including morphological changes, elevated DNA damage markers (γ-H2AX and 53BP1), and increased cellular senescence in the neural retinal and RPE, as well as pathological angiogenesis. The RPE is essential for the metabolic health of photoreceptors. The RPE from Ercc1^-/Δ mice displayed mitochondrial dysfunction causing a compensatory glycolytic shift, a characteristic feature of aging RPE. Hence, our study suggests spontaneous endogenous DNA damage promotes the hallmarks of age-related retinal degeneration.

Keywords: DNA damage; age‐related retinal degeneration; cellular senescence.

FIGURE 1

Ercc1 ^−/Δ mice show impaired vision. (a) Representative images for optokinetic response (OKR) of an Ercc1 ^−/Δ mouse and WT control at 4‐months‐of‐age. The graphs show eye position in horizontal (blue line) and vertical (red line) components as a function of time for 20 s. Eye movements were analyzed in the absence of stimulus (top) or with a stimulus with a spatial frequency of 0.12 and a contrast of 100 (bottom). (b) Representative scotopic electroretinogram (ERG) using a series of light intensities (−20 dB, −10 dB, 0 dB) on an Ercc1 ^−/Δ mouse and WT control at 3‐months‐of‐age. (c) A‐wave and b‐wave amplitude and implicit time of scotopic ERG (n = 3 mice per genotype). (d) Representative photopic ERG using a series of light intensities (−3 dB, 3 dB, 6 dB, 10 dB) in Ercc1 ^−/Δ and age‐matched WT mice. (e) A‐wave and b‐wave amplitude and implicit time of photopic ERG (n = 3 mice per genotype). In figure c and e, the results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001, ns, not significant.

FIGURE 2

Deficiency of Ercc1 expression affects retinal vasculature. (a) Representative images of retinal vasculature visualized by Isolectin B4 staining of retinal whole mounts from an Ercc1 ^−/Δ mouse and WT control at 4‐months‐of‐age, as well as an aged WT mouse (30‐month‐old). Images were taken in the superficial vascular plexus of the central and peripheral retina. Arrows indicate pathological neovascularization in Ercc1 ^−/Δ and aged WT mouse retina. Scale bar = 150 μm. (b) Graphical representations of the vascular quantification conducted within the central area of the superficial plexus using AngioTool. Vessel area percentage, number of junctions, number of endpoints were measured in n = 4 mice. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, *p < 0.03, **p < 0.002, ns, not significant. (c) Graphical representation of the vascular quantification in the peripheral area of the superficial plexus using AngioTool. Vessel area percentage, number of junctions, number of endpoints were measured in n = 4 mice. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, *p < 0.03, **p < 0.002, ****p < 0.0001, ns, not significant. (d) Retinal cryosections of eyes isolated from 4% PFA perfused animals were stained with stain against Isolectin B4 and antibody against mouse Albumin. Representative images showing sections from WT and Ercc1 ^−/Δ animals where only mutant animals show Isolectin/Albumin patches with visible Albumin spill in the retinal tissue. (e) Quantification of Isolectin/Albumin patches per section in WT and Ercc1 ^−/Δ animals. (f) Expression of genes related to angiogenesis (Pedf, Vegfa, Kdr) in the RPE/choroid and the neural retina from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age, as well as aged WT mice at 30‐months of age (n = 4 per genotype). Gapdh was used as housekeeping gene control. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001, ns, not significant.

FIGURE 3

Ercc1 deficiency alters retinal structure and increases senescence markers in neural retina. (a) Representative images of color fundus photography of Ercc1 ^−/Δ mouse and WT control at 3‐months‐of‐age to assess retinal integrity. Acquired images show visible changes in retina thickness and lower level of blood‐dependent coloration in the mutant retina. The RPE shows signs of accelerated aging such as degeneration (white arrow) and accumulation of deposits (yellow arrowheads). (b) Representative images of H&E‐stained sections of retinas from an Ercc1 ^−/Δ mouse and WT control at 4‐months‐of‐age, as well as an aged WT mouse at 30‐months‐of‐age demonstrating degeneration of the retina in Ercc1 ^−/Δ mice. Arrows indicate subretinal drusenoid deposits in Ercc1 ^−/Δ and aged WT mouse. Images were taken close to the optic nerve head. Scale bar = 50 μm. (c) Retinal thickness was measured at 6 consecutive points (1000, 2000, 3000 microns to the left and right of the optic nerve head) and plotted as a spider plot. Retinal sections (n = 3 retinal sections per eye) from n = 4 mice for each genotype were measured. The results are represented as mean ± SD and statistical significance was determined by two‐way ANOVA, *p < 0.03, ****p < 0.0001, ns, not significant. (d) Outer nuclear layer thickness was measured at 6 consecutive points (1000, 2000, 3000 microns to the left and right of the optic nerve head) and plotted as a spider plot. Retinal sections (n = 3 retinal sections per eye) from n = 4 mice for each genotype were measured. The results are represented as mean ± SD and statistical significance was determined by two‐way ANOVA, *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001, ns, not significant. (e) Photoreceptor cell numbers were counted per 150 μm² area in the retina. The cells were counted close to the optic nerve head from n = 4 mice for each genotype. Each bar represents mean ± SD statistical significance was determined by one‐way ANOVA, *p < 0.03, ***p < 0.0002. (f) Representative images illustrating the immunostaining of DNA damage markers 53BP1 (green) and γ‐H2Ax (yellow) in retinal sections of Ercc1 ^−/Δ mice and WT controls at 2‐months and 4‐months, as well as aged WT mice at 30‐months‐of‐age (n = 3 per group). The retinal layers are labelled as follows: GCL, Ganglion cell layer; INL, Inner nuclear layer; ONL, Outer nuclear layer. Scale bar = 20 μm. (g) Representative images illustrating the RNAscope detection of senescence markers–p16 (yellow) p21 (cyan) mRNA in retinal sections of Ercc1 ^−/Δ mice and WT controls at 2‐months, 4‐months, as well as aged WT mice at 30‐months‐of‐age (n = 3 per group). The retinal layers are labelled as follows: GCL, Ganglion cell layer; INL, Inner nuclear layer; ONL, Outer nuclear layer. Scale bar = 20 μm. (h) The expression of senescence biomarkers (p16 ^Ink4a and p21 ^Cip1 ) and SASP factors (Tnf, Il6, Mcp1, Pai1) were measured by qRT‐PCR in neural retina of Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age, as well as aged WT mice at 30‐months‐of‐age (n = 4 per group). Gapdh was used as internal control. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001, ns, not significant.

FIGURE 4

Genetic depletion of Ercc1 results in cellular senescence of RPE. (a) Expression of senescence biomarkers (p16 ^Ink4a and p21 ^Cip1 ) and SASP factors (Tnf, Il6, Mcp1, Pai1) were measured by qRT‐PCR in RPE isolated from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age, as well as old WT mice at 30‐months‐of‐age (n = 4 per group). Gapdh was used as a housekeeping gene internal control. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001, ns, not significant. (b) Representative images of C₁₂FDG staining to detect SA‐β‐gal activity in primary cultured RPE cells from an Ercc1 ^−/Δ mouse and WT control at 2 and 4‐months‐of‐age. Scale bar = 100 μm. The bar graph represents the percentage of C₁₂FDG positive cells in both 2‐ and 4‐month age groups. The results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, **p < 0.002. (c) Representative images of nuclei staining (Hoechst) in RPE flat mounts from an Ercc1 ^−/Δ mouse and WT control at 4‐months‐of‐age, illustrating enlarged nuclei in the mutant animal. Scale bar = 10 μm. (d) Representative images of F‐actin staining (phalloidin) to detect morphology of RPE cells in flat mounts of an Ercc1 ^−/Δ mouse and WT control at 4‐months‐of‐age as well as an old WT mouse at 30‐months‐of‐age. Scale bar = 50 μm. (e) RPE cell numbers per mm² and cell size (μm²/ cell) were measured in the central to mid‐peripheral area of RPE flat mounts using FIJI software (n = 6 biological replicates). The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, **p < 0.002, ns, not significant.

FIGURE 5

Genetic mutation of Ercc1 leads to glycolytic switch of RPE. (a) Measurement of glycolysis capacity by Seahorse assay using primary mouse RPE cells established from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age (n = 3 biological replicates). (b) Basal glycolysis and compensatory glycolysis calculated from glycolytic rate assay results using Wave software in cultured primary RPE cells from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age (n = 3 biological replicates). The results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, *p < 0.03. (c) The expression of glycolytic genes (Glut1 and Hk) measured using qRT‐PCR in RPE from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age, as well as old WT mice at 30‐months‐of‐age (n = 4 per group). Gapdh was used as housekeeping gene control. The results are represented as mean ± SD and statistical significance was determined by one‐way ANOVA, **p < 0.002, ***p < 0.0002.

FIGURE 6

Genetic mutation of Ercc1 leads to mitochondrial dysfunction in RPE. (a) Mitochondrial function was measured by Mito stress test in primary mouse RPE cultures established from Ercc1 ^−/Δ mice and WT controls at 4‐months‐of‐age (n = 3 mice per genotype) using a Seahorse analyzer. (b) Basal respiration, maximum respiration, ATP production, spare respiratory capacity was calculated from the Mito stress test in panel A using Wave software. The results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, *p < 0.03, **p < 0.002. (c) Representative images of mitochondrial staining using MitoTracker green in cultured primary RPE cells from an Ercc1 ^−/Δ mouse and a WT control at 2 and 4‐months‐of‐age. Scale bar = 20 μm. (d) Mitochondria content measured by fluorescence detection of a mitochondria‐specific dye in primary RPE from Ercc1 ^−/Δ mice and age‐matched WT littermates (data from panel C; n = 3 biological replicates). The results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, *p < 0.03, ns, not significant. (e) Representative images of mitophagy staining using MtPhagy dye in cultured primary RPE cells from an Ercc1 ^−/Δ mouse and a WT control at 2 and 4‐months‐of‐age. Scale bar = 20 μm. (f) Mitophagy was measured by fluorescence detection of mitophagy specific dye in primary RPE from Ercc1 ^−/Δ mice and age‐matched WT littermates (data from panel D; n = 3 biological replicates). The results are represented as mean ± SD and statistical significance was determined by an unpaired two‐tailed Student's t‐test, **p < 0.002; ns, not significant. (g) Expression levels of proteins involved in mitophagy (PINK1 and PARKIN), and protein related to mitochondrial mass (TOMM20) were measured by western blot in RPE from Ercc1 ^−/Δ mice WT animals at 4‐months‐of‐age, as well as old WT mice at 30‐months‐of‐age (n = 3 per group). Tubulin was used as a housekeeping control. The results are represented as mean ± SD and statistical significance was determined by a one‐way ANOVA, *p < 0.03, ***p < 0.0002, ns, not significant.