The Profile of Retinal Ganglion Cell Death and Cellular Senescence in Mice with Aging

Abstract

Older age is a risk factor for glaucoma, in which progressive retinal ganglion cell (RGC) loss leads to visual field defects and irreversible visual impairment and even blindness. We recently identified the involvement of cellular senescence in RGC cell death post-optic nerve injury. Here we further aimed to delineate the profile of RGC survival in mice with aging, a physiological process with increasing cellular senescence. The numbers of senescent cells in the ganglion cell layer (GCL) significantly and progressively increased starting at 8 months of age. Yet, significant reduction of ganglion cell complex layer thickness began in the 10-month-old mice, and significant reduction in the number of RGCs began in the 12-month-old mice as compared to the 2-month-old mice. Meanwhile, pyroptosis and ferroptosis markers as well as cellular senescence-related cell cycle arrest proteins p15^Ink4b, p16^Ink4a, p21^Cip1, and p53 were significantly and progressively increased in GCL. In contrast, there were no significant changes in dendritic field, complexity, and branches with increasing ages. Comparing between the 2- and 16-month-old mouse retinas, the differentially expressed genes were involved in the pathways of neurodegeneration, innate immunity, and mitochondrial ATP synthesis. In summary, this study revealed the gradual increase in senescent cells as well as pyroptosis and ferroptosis with progressive RGC reduction in mice with aging. Cellular senescence and the related cell death pathways are potential targets for age-related RGC reduction.

Keywords: aging; cell death; cellular senescence; retinal ganglion cells.

Figure 1

Changes of senescence-associated β-galactosidase expression in mice during aging. Staining and quantification of senescence-associated β-galactosidase (SA-βgal) activity (blue) in ganglion cell layer (GCL) of the 2-to 18-month-old mice. Scale bar: 100 μm. INL: inner nuclear layer; ONL: outer nuclear layer. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. *** p < 0.001 as compared to the 2-month-old mice.

Figure 2

Changes of ganglion cell complex layer thickness in mice during aging. (A) Spectral domain-optical coherence tomography analysis on cross-sectional retina of the 2- to 18-month-old (M) mice in vivo. The thicknesses of (B) whole retina and (C) ganglion cell complex (GCC) layer (composing of retinal nerve fibre layer, ganglion cell layer, and inner plexiform layer) were measured. Vertical and horizontal scale bars: 200 μm. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. ** p < 0.01; *** p < 0.001 as compared to the 2-month-old mice.

Figure 3

Changes of retinal ganglion cell density in mice during aging. Immunofluorescence analysis on the numbers of retinal ganglion cells (RGCs) in the retina of the 2- to 18-month-old mice. Scale bar: 200 μm. Red: β-III tubulin signal. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. * p < 0.05; ** p < 0.01; *** p < 0.001 as compared to the 2-month-old mice.

Figure 4

Changes of retinal ganglion cell dendrites in mice during aging. (A) Confocal microscopy images and dendritic skeleton images of retinal ganglion cell (RGC) dendrites of the 2- to 16-month-old Thy1-YFP-H mice. Scale bar: 200 μm. Green: yellow fluorescence protein signals. (B–D) Quantitative analysis on (B) the area of dendrite field, (C) total dendritic branch length, and (D) branching complexity, comparing to that of the 2-month-old mice. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test.

Figure 5

Changes of cell death marker expression in mice during aging. (A) Images of major time points (2, 8, 12, and 18-months) of the immunofluorescence analysis on cleaved cathepsin B (autolysis), cleaved caspase-3 (apoptosis), cleaved caspase-1 (pyroptosis), and 4-hydroxynonenal (4-HNE; ferroptosis) protein in the retinal sections. Scale bar: 100 μm. Green: Target antibody signal; Blue: DAPI nuclei counter-stain; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. (B–E) Quantification of (B) cleaved cathepsin B-, (C) cleaved caspase-3-, (D) cleaved caspase-1-, and (E) 4-HNE-positive stained cells in GCL of the 2- to 18-month-old mice. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. *** p < 0.001 as compared to the 2-month-old mice.

Figure 6

Changes of cellular senescence-related protein expression in mice during aging. (A) Images of major time points (2, 8, 12, and 18-months) of the immunofluorescence analysis on p15^Ink4b, p16^Ink4a, p21^Cip1, and p53 protein in the retinal sections. Scale bar: 75 μm. Green: target antibody signal; Blue: DAPI nuclei counter-stain; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. (B–E) Quantification of (B) p15^Ink4b-, (C) p16^Ink4a-, (D) p21^Cip1-, and (E) p53-positively stained cells in GCL of the 2- to 18-month-old mice. Data were presented as mean ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. *** p < 0.001 as compared to the 2-month-old mice.

Figure 7

RNA sequencing analysis on the retina of the 2- and 16-month-old mice. (A) Principle component analysis on the whole transcriptome profiles of the retinas of the 2- (blue dots) and 16-month old mice (red squares). (B) Hierarchical clustering analysis of differentially expressed genes in the retinas of the 2- and 16-month old mice. (C) Volcano plots of gene expression changes in the retina of the 16-month-old mice as compared to that of the 2-month-old mice by the independent T-test. Red dots: Significantly upregulated genes; Green dots: Significantly downregulated genes; Blue dots: No significant changes. Significant differential expression was defined as log₂ fold change ≥ 1 and corrected p < 0.05.

Figure 8

Gene expression analysis on the retinas of mice during aging. SYBR green polymerase chain reaction on the expression of significantly (A) upregulated (Csrp3, Glb1l3, Hdc, and Kif4) and (B) downregulated genes (Cfh, Chi3l1, Cntn1, Cp, Cyp1b1, Edn2, Fgf2, Impg2, Jak3, Marcks, Pcdh7, Pmel, Serpina3n, and Sparc) identified by the RNA sequencing analysis in the retina of 2- to 16-month-old mice, comparing to that of the 2-month-old mice. Actb was used as housekeeping gene for normalization. Data presented as mean of relative fold change (2^−ΔΔCt) ± standard deviation and compared by one-way analysis of variance with post hoc LSD test. * p < 0.05; ** p < 0.01; *** p < 0.001.