Combination of dasatinib and quercetin improves cognitive abilities in aged male Wistar rats, alleviates inflammation and changes hippocampal synaptic plasticity and histone H3 methylation profile

Abstract

Aging is associated with cognitive decline and accumulation of senescent cells in various tissues and organs. Senolytic agents such as dasatinib and quercetin (D+Q) in combination have been shown to target senescent cells and ameliorate symptoms of aging-related disorders in mouse models. However, the mechanisms by which senolytics improve cognitive impairments have not been fully elucidated particularly in species other than mice. To study the effect of senolytics on aging-related multifactorial cognitive dysfunctions we tested the spatial memory of male Wistar rats in an active allothetic place avoidance task. Here we report that 8 weeks treatment with D+Q alleviated learning deficits and memory impairment observed in aged animals. Furthermore, treatment with D+Q resulted in a reduction of the peripheral inflammation measured by the levels of serum inflammatory mediators (including members of senescent cell secretome) in aged rats. Significant improvements in cognitive abilities observed in aged rats upon treatment with D+Q were associated with changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons and changes in the level of histone H3 trimethylation at lysine 9 and 27 in the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 5 weeks after the cessation of the drugs administration. Our results expand and provide new insights to the existing knowledge associated with effects of senolytics on alleviating age-related associated cognitive dysfunctions.

Keywords: SASP; aging; brain; cognition; hippocampus; memory; plasticity; senescence.

Figure 1

Treatment with D+Q improved cognitive ability in aged rats tested in the AAPAT on spatial memory. (A) Schematic diagram of the experimental design. Experiments consisted of 2 trainings, each with a different position of to-be-avoided place. The number of entries into the shock place (# of entries, B), the number of shocks (# shocks, C) and the number of shocks per one entry ratio (Shocks/entrance, D), together with maximum time avoided (E) and time to the first entry (F) during place avoidance training reflect changes in the learning abilities and memory in aged rats. Performance of place avoidance by young (3-mo old; YOUNG; N = 7) (black - vehicle, orange - D+Q) and aged (22-mo old; OLD; N = 7) (grey - vehicle, red - D+Q) rats in the AAPAT before and after 8 weeks of treatment with D+Q or vehicle (VEH) by oral gavage. Figures labeled with “I” depict comparison between 1st TRAINING and 2nd TRAINING, “II” between VEH and D+Q groups results of the 2nd TRAINING. The results are expressed as mean ± SEM, N = 7, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data obtained for groups at consecutive trials were compared using repeated measures two-way ANOVA (2 groups and 5 days).

Figure 2

D+Q treatment reduces peripheral inflammation in aged rats. The heat map depicts cytokines and growth factor levels in blood serum collected after the final behavioral test from young and aged (6-month-old and 25-month-old respectively at the time of collection) rats treated with D+Q or vehicle (VEH). The results were normalized to the average of the young vehicle group. IL-10 as anti-inflammatory cytokine has been presented separately. The data were analyzed using two-way ANOVA; n = 7–8, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

D+Q treatment changes synaptic plasticity in the apical dendrites of neurons of the CA1 region of the hippocampus. Representative images of DiI stained dendrites (A and B). DiI stained hippocampal slices from aged vehicle (grey bars) or D+Q (red bars) treated rats were used for the analysis of synaptic plasticity. Different parameters of the dendritic spine shape (length, length to width ratio, area, circumference C and D) and spine density (E and F) were analyzed in the CA1 region of the hippocampus. The analysis was done for two dendritic arbours of stratum pyramidale, namely basal (BASAL) (D and F) and apical (APICAL) dendrites (C and E). The data are expressed as mean ± SEM, and analyzed using nested t-test. Dots on the bar plots represent values for single dendritic spines (C and D) or single image (E and F) N_VEH = 3, N_D+Q = 5 animals, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4

Changes in the levels of H3 histones in the hippocampus of aged rats after D+Q treatment. Improved cognitive skills, decreased peripheral inflammation, and changes in neuronal synaptic plasticity are accompanied by changes in H3 methylation profile. Trimethylation of H3K9 (A) and H3K27 (B) relative to the whole H3 in the hippocampus of aged rats after D+Q or VEH treatment measured by Western blot. The data are expressed as means ± SEM normalized to the average of the VEH group, and analyzed using t-test; N = 7, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

Treatment with D+Q significantly improves cognitive abilities in aged rats in spatial memory task in the AAPAT on spatial memory for at least 5 weeks after treatment termination. (A) Schematic diagram of the experimental design in which rats are subjected to the training 3 times, each time with a new position of the to-be-avoided place. The number of entries into the shock place (# of entries, B I–III), the number of shocks (# shocks, C I–III) and number of shocks per one entry (Shocks/entrance, D I–III) together with maximum time avoided (E I–III) and time to the first entry (F I–III) during place avoidance training reflect changes in the learning abilities and memory in aged rats not only early after D+Q treatment (2nd TRAINING) but also after additional 4 weeks (3rd TRAINING). Performance of place avoidance by young (3-month old; YOUNG; orange color) and aged (18-month old; OLD; red color) rats in the AAPAT before and after 8 weeks of treatment with D+Q by oral gavage. Figures labeled with “I” depict comparison between 1st TRAINING and 2nd TRAINING, “II” between 1st TRAINING and 3rd TRAINING, whereas “III” depict comparison between 2nd TRAINING and 3rd TRAINING. The results are expressed as mean ± SEM, N = 7, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data obtained for groups at consecutive trials were compared using repeated measures two-way ANOVA.