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. 2021 May 18:15:100341.
doi: 10.1016/j.ynstr.2021.100341. eCollection 2021 Nov.

Cellular senescence as a driver of cognitive decline triggered by chronic unpredictable stress

Yu-Fen Lin  1 , Li-Yun Wang  1 , Chi-Sheng Chen  1 , Chia-Chun Li  1 , Ya-Hsin Hsiao  1   2   3
Affiliations

Cellular senescence as a driver of cognitive decline triggered by chronic unpredictable stress

Yu-Fen Lin et al. Neurobiol Stress. .

Abstract

When an individual is under stress, the undesired effect on the brain often exceeds expectations. Additionally, when stress persists for a long time, it can trigger serious health problems, particularly depression. Recent studies have revealed that depressed patients have a higher rate of brain aging than healthy subjects and that depression increases dementia risk later in life. However, it remains unknown which factors are involved in brain aging triggered by chronic stress. The most critical change during brain aging is the decline in cognitive function. In addition, cellular senescence is a stable state of cell cycle arrest that occurs because of damage and/or stress and is considered a sign of aging. We used the chronic unpredictable stress (CUS) model to mimic stressful life situations and found that, compared with nonstressed control mice, CUS-treated C57BL/6 mice exhibited depression-like behaviors and cognitive decline. Additionally, the protein expression of the senescence marker p16INK4a was increased in the hippocampus, and senescence-associated β-galactosidase (SA-β-gal)-positive cells were found in the hippocampal dentate gyrus (DG) in CUS-treated mice. Furthermore, the levels of SA-β-gal or p16INK4a were strongly correlated with the severity of memory impairment in CUS-treated mice, whereas clearing senescent cells using the pharmacological senolytic cocktail dasatinib plus quercetin (D + Q) alleviated CUS-induced cognitive deficits, suggesting that targeting senescent cells may be a promising candidate approach to study chronic stress-induced cognitive decline. Our findings open new avenues for stress-related research and provide new insight into the association of chronic stress-induced cellular senescence with cognitive deficits.

Keywords: Cellular senescence; Chronic stress; Cognitive decline; Senolytics.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
CUS induces depression-like phenotypes and impairs spatial memory in C57BL/6 mice. (A) Sucrose preference was calculated as a percentage of the volume of sucrose intake over the total volume of fluid intake in each cage. Five mice were housed in one cage (control groups: n = 3 cages; CUS groups: n = 5 cages). Two-way analysis of variance revealed a significant treatment (control vs. CUS-treated mice) × time interaction [F(1,12) = 8.065, p = 0.0149]. *p < 0.05 vs. baseline of the CUS groups as determined by Tukey's post hoc analysis. (B) The total duration of immobility was measured using the forced swim test for 10 min (control groups: n = 30; CUS groups: n = 39) [two-tailed unpaired Student's t-test; t(67) = 8.292; p < 0.0001]. ****p < 0.0001 vs. control groups. (C) The body weights of the control and CUS-treated mice were measured (control groups: n = 14; CUS groups: n = 29 at 8, 9, 10, 11, and 12 weeks). Two-way analysis of variance revealed a significant treatment × time interaction [F(4,203) = 3.153; p = 0.0153]. *p < 0.05 vs. age- and sex-matched controls as determined by Bonferroni's post hoc analysis. (D) Experimental design for the object location test. In the training trial, the objects were placed in the same location. In the test trial, one object was moved and relocated in the opposite direction of another object. (E) A representative heat map and the discrimination index (DI) were used to assess hippocampal-related memory performance (control groups: n = 30; CUS groups: n = 35) [two-tailed unpaired Student's t-test; t(63) = 3.236; p = 0.0019]. **p < 0.01 vs. controls. (F) Schematic drawings of the Y-maze and experimental procedures. (G) Representative heat map and CUS-induced spatial memory deficit in the modified Y-maze test (control groups: n = 14; CUS groups: n = 20) [two-tailed unpaired Student's t-test; t(32) = 3.965; p = 0.0004]. ***p < 0.001 vs. controls. The data are represented as means ± SEM.
Fig. 2
Fig. 2
Markers of senescence are increased in CUS-treated mice. (A) Representative images of the quantitation of SA-β-gal-positive cells, which were increased in the CUS-treated groups (n = 4 in each group). Original magnification, 10 × objective. Scale bar, 100 μm. The inserts show a higher magnification of the boxed area using a 40 × objective. Scale bar, 20 μm [two-tailed unpaired Student's t-test; t(6) = 21.70; p < 0.0001]. ****p < 0.0001 vs. controls. (B) Western blot analysis of p16INK4a expression in control and CUS-treated mice (n = 6 in each group) [two-tailed paired Student's t-test; t(5) = 2.668; p = 0.0444]. *p < 0.05 vs. controls. The data are represented as means ± SEM.
Fig. 3
Fig. 3
Linear regression analysis between senescence markers and memory performance of mice shows a significant negative correlation. Quantitation of SA-β-gal-positive cells vs. the memory score plot of the object location test (A) or Y maze test (B) showed a negative linear relationship in mice (object location test: r = 0.8202, p = 0.0456, n = 6; Y maze test: r = 0.8229, p = 0.0443, n = 6). The percentage of hippocampal p16INK4a levels vs. the memory score plot of the object location test (C) or Y maze test (D) showed a negative linear relationship in mice (object location test: r = 0.6783, p = 0.0108, n = 13; Y maze test: r = 0.6550, p = 0.0151, n = 13).
Fig. 4
Fig. 4
Senolytic drug (D + Q) treatment decreases the number of hippocampal senescent cells. (A) Quantitative analysis of the numbers of SA-β-gal-positive cells in the hippocampus showed a decreased after D + Q treatment (n = 4 in each group). Original magnification, 10 × objective. Scale bar, 100 μm. The inserts show a higher magnification of the boxed area using a 40 × objective. Scale bar, 20 μm. Two-way analysis of variance revealed a significant stress treatment (control vs. CUS-treated mice) × drug administration interaction [F(1,12) = 74.00, p < 0.0001]. ****p < 0.0001 vs. respective control as determined by Tukey's post hoc analysis. (B) Western blot analysis of hippocampal p16INK4a showed decreased expression with D + Q treatment in the CUS group (n = 8 in each group). Two-way analysis of variance revealed a significant stress treatment × drug administration interaction [F(1,28) = 3.698, p = 0.0647]. *p < 0.05 and ***p < 0.001 vs. respective control as determined by Tukey's post hoc analysis. The data are represented as means ± SEM.
Fig. 5
Fig. 5
Treatment with dasatinib plus quercetin rescues memory loss in CUS-treated mice. (A) The total distance traveled and velocity were not significantly different between vehicle- and D + Q-treated control and CUS mice. The data indicate an improvement in memory tests in CUS-treated mice following D + Q administration as determined by the object location (B) and Y maze tests (C) (control and CUS groups: n = 9 for the vehicle group; n = 15 for the D + Q group). Object location test: Two-way analysis of variance revealed a significant stress treatment × drug administration interaction [F(1,44) = 17.29, p = 0.0001]. ***p < 0.001 and ****p < 0.0001 vs. respective controls as determined by Tukey's post hoc analysis. Y maze tests: Two-way analysis of variance revealed a significant stress treatment × drug administration interaction [F(1,44) = 9.097, p = 0.0042]. **p < 0.01 and ***p < 0.001 vs. respective controls as determined by Tukey's post hoc analysis. The data are represented as means ± SEM.
Fig. 6
Fig. 6
D + Q treatment attenuates senescence-associated signatures in CUS-treated mice. (A) Representative heatmaps of multiplex assays indicated fold changes in cytokine/chemokine protein expression in serum from control and CUS mice treated with/without D + Q (n = 11 in each group). Concentrations of the SASP components (B) IL-1β, (C) IL-6, and (D) IL-13 in the serum of control and CUS mice treated with/without D + Q. Two-way analysis of variance revealed a significant stress treatment × drug administration interaction [IL-1β: F(1,40) = 4.222, p = 0.0465; IL-6: F(1,40) = 6.891, p = 0.0122; IL-13: F(1,40) = 5.642, p = 0.0224]. *p < 0.05 vs. respective control as determined by Tukey's post hoc analysis. The data are represented as means ± SEM.
Fig. 7
Fig. 7
SASP levels are correlated with the severity of memory impairment. (A) Correlation between the IL-1β levels and DI values using the object location test (r = 0.4617; p = 0.0046). (B) Correlation between the IL-1β levels and percentage of time spent in the novel arm using the Y maze test (r = 0.6462; p < 0.0001). (C) Correlation between the IL-6 levels and DI values using the object location test (r = 0.5254; p = 0.0007). (D) Correlation between the IL-6 levels and percentage of time spent in the novel arm using the Y maze test (r = 0.5878, p = 0.0002). (E) Correlation between the IL-13 levels and DI values using the object location test (r = 0.4830, p = 0.0028). (F) Correlation between the IL-13 levels and percentage of time spent in the novel arm using the Y maze test (r = 0.5105; p = 0.0015).
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