Senescent cell transplantation into the skin induces age-related peripheral dysfunction and cognitive decline

Abstract

Cellular senescence is an established cause of cell and tissue aging. Senescent cells have been shown to increase in multiple organs during aging, including the skin. Here we hypothesized that senescent cells residing in the skin can spread senescence to distant organs, thereby accelerating systemic aging processes. To explore this hypothesis, we initially observed an increase in several markers of senescence in the skin of aging mice. Subsequently, we conducted experiments wherein senescent fibroblasts were transplanted into the dermis of young mice and assessed various age-associated parameters. Our findings reveal that the presence of senescent cells in the dermal layer of young mice leads to increased senescence in both proximal and distal host tissues, alongside increased frailty, and impaired musculoskeletal function. Additionally, there was a significant decline in cognitive function, concomitant with increased expression of senescence-associated markers within the hippocampus brain area. These results support the concept that the accumulation of senescent cells in the skin can exert remote effects on other organs including the brain, potentially explaining links between skin and brain disorders and diseases and, contributing to physical and cognitive decline associated with aging.

Keywords: cellular senescence; cognitive decline; paracrine senescence; skin aging.

FIGURE 1

Senescence‐associated markers increase in mouse skin with age. (a) Representative Immuno‐FISH images (using telomere specific nucleic acid probe (CCCTAA) in red and anti‐γH2A.X antibody in green). Arrows indicate co‐localization between telomeres and γH2A.X. (Scale bar 10 μm); (b) Mean number of TAF in skin epidermal layer and (c) percentage of cells presenting more than two TAFs in the epidermis. (d) Representative microscopy images of PCNA (in red) and Lamin‐B1 (in green). (Scale bar 10 μm). (e) Quantification of Lamin‐B1 immunoreactivity and (f) percentage of PCNA positive cells in the epidermis. Data is presented and mean ± S.E.M of 5 animals per group. (g) mRNA levels of p16INK4a; (h) p21 and (i) SASP factors in young and old skin of 6–12 animals. Data are mean ± S.E.M. Statistical significance (*p < 0.05, **p < 0.01) was assessed using Student's t‐test. a.u. Arbitrary units.

FIGURE 2

Transplanting senescent cells intradermally induces paracrine senescence in skin. (a) 5 months after transplantation skin was collected in regions proximal or distal from injection site. (b) Representative images of H&E micrographs from skin located proximally or distally from injection site (scale bar 50 μm). (c) Quantification of subcutaneous far thickness in distal or proximal skin; (d) representative images of HMGB1 immunostaining in proximal (left) and distal (right) transplanted skin (scale bar 20 μm). (e) Quantification of HMGB1 positive nuclei in the epidermis and dermis of proximal skin. (f–h) q‐PCR analysis of p16, p21 and SASP factors in proximal transplanted skin. (i) Quantification of HMGB1 positive nuclei in the epidermis and dermis of distal skin. (j–l) q‐PCR analysis of p16, p21 and SASP factors in distal transplanted skin. Data are expressed as the mean ± SEM. N = 7–10 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 significantly different compared to t‐Prolif mice, as determined by Student's t‐test.

FIGURE 3

Transplanting senescent cells intradermally induces age‐associated parameters in skeletal muscle and bone. (a) 5 months following intradermal transplantation, muscle (quadriceps) and bone were collected. mRNA levels of senescence‐associated markers (b) p16; (c) p21 and (d) several SASP factors were analyzed in skeletal muscle by q‐PCR. (e) representative images of WGA staining in skeletal muscle of mice transplanted with either proliferating or senescent cells (scale bar 60 μm). Yellow arrows denote centrally nucleated fibers. Quantification of (f) of % of centrally nucleated fibers; (g) number of fibers per field and (h) fiber cross‐sectional area (CSA) distribution. (i) Representative reconstructed μCT images of bone microarchitecture of femur cortical bone (diaphysis) 5 months after transplantation of t‐Prolif and t‐Sen. Quantification of (j) femoral diaphysis cortical volumetric bone mineral density (Ct.vBMD) measured in mg.cm‐3 (k and l), Quantification of femoral diaphysis periosteal circumference and endocortical circumference. (m) 4 months after transplantation, mice were assessed for motor coordination using the rotarod test (n) Pole test, (o) forelimb grip strength and (p) multiple parameters of clinical frailty. Data are represented as mean ± SEM (n = 8–15 animals per group). Statistical differences *p < 0.05, **p < 0.01, and ****p < 0.0001 were by Students t‐test and two‐way ANOVA compared with t‐Prolif controls.

FIGURE 4

Transplanting senescent cells intradermally induces paracrine senescence in host hippocampus. (a) Representative images and quantification of RNA‐ISH detection of p21, p16, Il1α and Il6 in the CA3 region of the hippocampus of transplanted mice (Scale Bar 20 μm). Yellow arrows indicate positive cells. (b) Quantification of the percentages of positive cells for p21, p16, Il1α and Il6 in the respective hippocampal region. (c) Representative images of Iba1 immunostaining in the hippocampus of transplanted mice. Yellow arrows indicate activated Iba1+ microglia (Scale Bar 20 μm). Quantification of the number of Iba1 positive cells d) and microglia's soma size (an indication of activation) (e). Data are represented as mean ± SEM (n = 8 animals per group). Statistical differences **p < 0.01, ***p<0.001 and ****p < 0.0001 were by Students t‐test compared with t‐Prolif controls.

FIGURE 5

Transplanting senescent cells intradermally induces cognitive decline in young mice. (a) 2–4 months following intradermal transplantation of proliferative or senescent fibroblasts, mice were evaluated a series of behavioral assessments including the Y‐maze and Stones maze tests, which assess spatial memory, as well as the Elevated Plus Maze (EPM) and Open Field tests, which assess anxiety‐like behavior. Using Y‐maze, transplanted mice were evaluated for (b) Time spent in novel arm and (c) latency to novel arm. Using Stone's maze transplanted mice were assessed for (d) average time to finish the test and (e) average number of errors. Linear regression analysis of time spent exploring Y maze's open arm and (f) p21, (g) IL1α and (h) Il6 positive cells in the hippocampus CA3 region. Data are mean ± S.E.M of n = 15 animals per group. *p < 0.05, **p < 0.01.