Skin Aging in Long-Lived Naked Mole-Rats Is Accompanied by Increased Expression of Longevity-Associated and Tumor Suppressor Genes

Abstract

Naked mole-rats (NMRs) (Heterocephalus glaber) are long-lived mammals that possess a natural resistance to cancer and other age-related pathologies, maintaining a healthy life span >30 years. In this study, using immunohistochemical and RNA-sequencing analyses, we compare skin morphology, cellular composition, and global transcriptome signatures between young and aged (aged 3‒4 vs. 19‒23 years, respectively) NMRs. We show that similar to aging in human skin, aging in NMRs is accompanied by a decrease in epidermal thickness; keratinocyte proliferation; and a decline in the number of Merkel cells, T cells, antigen-presenting cells, and melanocytes. Similar to that in human skin aging, expression levels of dermal collagens are decreased, whereas matrix metalloproteinase 9 and matrix metalloproteinase 11 levels increased in aged versus in young NMR skin. RNA-sequencing analyses reveal that in contrast to human or mouse skin aging, the transcript levels of several longevity-associated (Igfbp3, Igf2bp3, Ing2) and tumor-suppressor (Btg2, Cdkn1a, Cdkn2c, Dnmt3a, Hic1, Socs3, Sfrp1, Sfrp5, Thbs1, Tsc1, Zfp36) genes are increased in aged NMR skin. Overall, these data suggest that specific features in the NMR skin aging transcriptome might contribute to the resistance of NMRs to spontaneous skin carcinogenesis and provide a platform for further investigations of NMRs as a model organism for studying the biology and disease resistance of human skin.

Figure 1.. Visual appearance of the skin and age-related changes in the epidermis of NMRs.

a – Images of the skin in 3- and 23-year-old animals, note the more translucent and less pigmented dorsal skin in aged NMR. b – Hematoxylin/eosin staining of the young and old skin: presence of the dermal pigment (arrow) in young skin and significantly reduced epidermal thickness in old animals. c – Significant decrease in Ki67+ cells in aged versus young NMRs. d-f - Significantly decreased immunofluorescence intensity of K14 in the basal layer (d), K10 in the spinous layer (e), and Loricrin in the granular layer (f) in the epidermis of old NMRs. g - Significant decrease in the immunofluorescence intensity of p63 in epidermal keratinocytes of old versus young NMRs. h – Significant decline in the expression of COL17A1 in basal epidermal keratinocytes and dermal-epidermal basement membrane of old animals. i – Significant decrease in the number of KRT20+ Merkel cells in the epidermis of old NMRs (arrows). (mean ± SD, *p<0.05, Student’s t-test). Scale bars: 1b – 1 cm; 1c – 50 um; 1d-j – 25 um. Y – young animal, O – old animal.

Figure 2.. Age-associated changes in the NMR dermis.

(a) Significantly reduced number of epidermal buds elongating into the dermis (arrows) in the aged NMR skin. (b) Similar distribution of Fibrillin-2+ fibers in young and old NMRs. (c) COL1A1 is broadly expressed in the papillary and reticular dermis of young NMRs, while COL1A1 expression is significantly decreased in the reticular dermis in aged skin. (d) Significant increase in the MMP9 immunofluorescence intensity in the dermis of old NMRs compared to young animals. (e) No differences in the hyaluronan-binding protein (HA-BP) binding pattern or degree of binding between the dermis of young and old NMRs. (f) Similar expression of the HA receptor CD44 in the skin of young and old NMRs. (g) Warthin-Starry stain shows a dramatic decrease in the melanin containing areas in the dermis of old compared to young NMRs, which is accompanied by a significant decrease in the number of gp100+ pigment-producing dermal melanocytes in the dermis of aged animals (h, arrows). Mean ± SD, *p<0.05, Student’s t-test. Scale bars: 25 μm. Y – young animal, O – old animal.

Figure 3.. Aging-associated changes in the number of immune and senescent cells in the NMR skin.

(a) Significant decrease in the number of CD3ε+ T-cells in the epidermis of old NMRs. (b) Significant decrease in the number of MHC II+ cells detected by an anti-27E7 antibody in the epidermis and dermis of old NMRs. (c) No changes in the number of CD8+ cells in the dermis of aged versus young NMRs (arrows). (d) The number of dermal CD11b+ macrophages is similar in the young and old NMRs. (e) Significant increase in the number of senescent SA-β-gal+ cells (arrow) in the epidermis of old NMRs. A tendentious, but not significant, increase in the number of SA-β-gal+ cells (arrows) in the aged dermis (p=0.082). Mean ± SD, *p<0.05, Student’s t-test. Scale bars: 25 μm. Y – young animal, O – old animal.

Figure 4.. RNAseq analyses of the age-associated changes in the cutaneous NMR transcriptome.

(a) Functional annotation of the differentially expressed genes based on QIAGEN Ingenuity Pathway Analysis database and manually curated functional gene sub-categories. (b) A list of five top differentially expressed genes in each functional group (fold-change expression values are indicated by asterisks). (c) GO enrichment analysis showed significant over-representation of the extracellular matrix-associated genes, components of the insulin growth factor (IGF) signaling pathway, regulators of the glucose metabolism and cell proliferation.

Figure 5.. Comparison of age-associated changes in the transcriptome of NMR, human, and mouse skin

(a) The number of differentially expressed genes common between the NMR skin aging transcriptome and the Human Ageing Genome Resource (HAGR) genes (here denoted as aging signature genes) relative to the total number of differentially expressed genes; (b) The number of differentially expressed genes common between the NMR skin aging transcriptome and the Cancer Gene Census (CGC)/Tumor Suppressor Genes (TSG) datasets (here denoted as cancer signature genes) relative to the total number of differentially expressed genes; (c) A Venn diagram shows the differentially expressed genes in the NMR skin aging transcriptome that are shared with the human HAGR and CGC/TSG databases. Comparative analyses of gene expression (old versus young) in the skin of three species: NMR (total skin), human (total skin, epidermis) and mouse (total skin, FACS-sorted basal epidermal keratinocytes). The differences in gene expression are shown as the FPKM fold-change.