APPNL−F/NL−F mice have increased senescent cell accumulation and SASP in visceral white adipose tissue and hippocampus

Relative expression of senescent and SASP markers were assayed in visceral WAT (vWAT) and hippocampus from a separate cohort of 4-month-old C57BL/6 and APPNL−F/NL−F mice. In vWAT, 4-month-old female APPNL−F/NL−F mice had increased expression of p16Ink4α, p21Cip1, IL-6, TNFα, MCP1, IL-10, MIP1α, and CXCL16 compared to age-matched female C57BL/6 mice (Online Resource 1A-H). p21Cip1, IL-6, TNFα, IL-10, and MIP1α were increased in male APPNL−F/NL−F mice compared to 4-month-old male C57BL/6 controls (Online Resource 1B-D, F-G). In the hippocampus, p21Cip1, MCP1, and MIP1α were increased in both sexes of APPNL−F/NL−F mice (Online Resource 1 J, M, O). TNFα was also increased in the hippocampus of 4-month-old female APPNL−F/NL−F mice. (Online Resource 1L). No differences were observed for p16Ink4α, IL-6, IL-10, and CXCL16 (Online Resource 1I, K, N, P). These data indicated knock-in of APP mutations increased senescent and SASP markers by 4 months of age both centrally and periperally, which made this an ideal therapeutic time point to administer senolytic treatment. Also, APPNL−F/NL−F mice at this age do not have overt signs of cognitive deficits making it an optimal period for primary prevention treatment to determine disease-modifying outcomes.

D + Q treatment decreases body weight via reduced adiposity in female APPNL−F/NL−F mice

Metabolic deficits leading to increased oxidative stress has been observed in AD patients and transgenic mouse models prior to plaque and tangle development [30,31,32]. In the APPNL−F/NL−F mice, Aβ deposition begins at 6 months of age with cognitive impairments occurring by 12–18 months [18]. To examine the metabolic changes associated with senolytic administration, body composition, glucose utilization, and energy metabolism were measured. Starting body weight was similar for each treatment group within a sex and monitored weekly thereafter (Online Resource 2A). Chow consumption was monitored weekly and no differences were observed throughout the treatment regimen (Online Resource 2B-C). Immediately before euthanization, mice were weighed to determine body composition. When compared to control treatment, D + Q decreased body weight while an increase was observed in fisetin-treated female APPNL−F/NL−F mice. Male APPNL−F/NL−F mice had no change in body weight with either senolytic treatment (Fig. 1A). We examined body composition of BAT and WAT depots to determine changes from senolytic administration. The percentages of total WAT consisting of subcutaneous WAT, vWAT, and retroperitoneal WAT was reduced in female APPNL−F/NL−F after D + Q treatment, but increased with fisetin. Neither senolytic treatment effected WAT in males (Fig. 1B–E). Fisetin increased interscapular BAT in female APPNL−F/NL−F mice but no changes were observed in the other groups (Fig. 1F). Body weight treatment effects were largely influenced by the changes in adiposity.

Fig. 1

Senolytic intervention reduced adiposity and circulating lipid levels in female APPNL−F/NL−F mice. Mouse body weight (A) along with percentages of total (B), WAT depots (C–E), and BAT (F) in relation to body weight. Plasma triglyceride and cholesterol levels were determined by enzymatic determination (G–H). All tissue was collected at the time of euthanization (14 months of age). Data are represented as means ± SEM (n = 15–21). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05, **p < 0.01, ***p < 0.001 based on a two-tailed Student’s t test

The observed body composition differences could impact circulating lipids promoting dyslipidemia which increases the risk for developing cognitive impairments. Plasma triglyceride levels were decreased solely in females undergoing D + Q treatment but no changes were found in the other groups compared with control treatment (Fig. 1G). Cholesterol levels in plasma did not change with either of the treatment conditions in both the male and female mice (Fig. 1H). In summary, D + Q treatment reduced WAT, body weight, and circulating triglycerides while fisetin treatment increased WAT, BAT, and body weight in female APPNL−F/NL−F mice. Neither D + Q nor fisetin affected these measures in male APPNL−F/NL−F mice.

Effects of senolytic drug treatments on glucose metabolism in APPNL−F/NL−F mice

Adipose tissue modulates glucose homeostasis, so we assayed the effects of senolytic intervention on blood glucose levels in APPNL−F/NL−F mice. A GTT for glucose clearance and ITT for insulin sensitivity were performed. GTT and ITT results were unchanged regardless of sex or senolytic treatment compared with control groups (Fig. 2A–F). In female APPNL−F/NL−F mice, D + Q and fisetin treatment reduced fasting glucose, while D + Q treatment lowered fed glucose levels and plasma insulin (Fig. 2G–I). Fisetin and D + Q increased male APPNL−F/NL−F plasma insulin levels (Fig. 2I). D + Q treatment drastically reduced circulating adiponectin levels in male APPNL−F/NL−F mice (Fig. 2J). Despite these changes to plasma insulin and adiponectin in male APPNL−F/NL−F mice, no corresponding blood glucose changes were observed with the GTT or ITT (Fig. 2A, C, E–H).

Fig. 2

Blood glucose and plasma insulin are improved after senolytic treatment in female APPNL−F/NL−F mice. Glucose tolerance test (GTT) and insulin tolerance test (ITT), and their respective area under the curve (AUC) after five treatments (A–F). Fasting (G) and fed (H) blood glucose levels were determined from time = 0 during the GTT and ITT, respectively. Circulating insulin and adiponectin levels were determined by ELISA from plasma collected at the time of euthanization (10 treatments). Data are represented as means ± SEM (n = 13–23). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05, **p < 0.01, ****p < 0.0001 based on a two-tailed Student’s t test

Effects of senolytic drug treatments on energy expenditure in APPNL−F/NL−F mice

Aside from being an important regulator of glucose homeostasis, adipose tissue is also a central metabolic organ in the regulation of energy homeostasis [33]. Since D + Q and fisetin senolytic drug treatments altered adipose tissue composition (Fig. 1A–E), indirect calorimetry was used to assess senolytic treatment effects on energy metabolism in APPNL−F/NL−F mice. Data revealed that D + Q treatment elevated VO2 (Fig. 3B–C) and EE (Fig. 3E–F) without affecting respiratory quotient RQ (Fig. 3H–I) in the female APPNL−F/NL−F mice, while fisetin elicited no effects. In contrast, neither treatment altered the energy metabolism in male APPNL−F/NL−F mice (Fig. 3A, C, D, F, G, I).

Fig. 3

D + Q treatment improves oxygen consumption and energy expenditure in female APPNL−F/NL−F mice. Twenty-four-hour male and female oxygen consumption (A–C), energy expenditure (D–F), and respiratory quotient (G–I; VCO2/VO2) along with their corresponding AUC after 9 treatments were determined by indirect calorimetry. White and black bars along the abscissa indicates the hours of lights on or off, respectively. Data are represented as means ± SEM (n = 15–23). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05 based on a two-tailed Student’s t test

D + Q treatment reduces visceral white adipose tissue and hippocampal senolytic markers in female APPNL−F/NL−F mice

As previously discussed, D + Q and fisetin have been reported to reduce senescent cell burden in numerous models of aging and disease [8]. Adipose tissue, specifically vWAT, contains the highest burden of senescent cells [34] and has a stronger correlation with brain atrophy than age, BMI, hypertension, or type 2 diabetes mellitus [35]. Since D + Q and fisetin treatment altered adiposity in female APPNL−F/NL−F mice (albeit with contrasting effects), we further examined whether senescent cell markers and SASP were affected in vWAT. The results showed that D + Q treatment downregulated gene expression of p16Ink4a, p21Cip1, IL-6, TNFα, MCP1, and IL-10 within female APPNL−F/NL−F mice (Fig. 4A–F). In contrast, D + Q treatment in male APPNL−F/NL−F mice increased gene expression of p21Cip1, IL-6, TNFα, MCP1, IL-10, MIP1α, and CXCL16 (Fig. 4B–H) in vWAT. Fisetin treatment had no effect on vWAT genes in either sex of APPNL−F/NL−F mice. Within the hippocampus, relative expression of p16Ink4a, p21Cip1, and TNFα (Fig. 4I–K) were all decreased in D + Q-treated female APPNL−F/NL−F mice, similar to vWAT. D + Q-treated males also had reduced TNFα hippocampal expression, while no effects were observed in either sex after fisetin treatment. Overall, D + Q treatment reduced central and peripheral senescent and SASP makers in female APPNL−F/NL−F mice.

Fig. 4

D + Q treatment altered senescent cell markers and SASP profile in a sexually dimorphic manner. Gene expression profiles in vWAT (A–H) and the hippocampus (I–K). Data are represented as means ± SEM (n = 8–10). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05, **p < 0.01, ***p < 0.001 based on a two-tailed Student’s t test

Senolytic treatment reduces plasma cytokine levels in female APPNL−F/NL−F mice

Since senescent cell burden is related to secretion of SASP, we examined plasma cytokines by multiplex analysis (Online Resource 3A-E). D + Q treatment in female APPNL−F/NL−F reduced plasma expression of IL-6, TNFα, and IL-1β, while fisetin treatment also reduced these three proinflammatory cytokines and IL-10. Despite elevated vWAT SASP expression in male APPNL−F/NL−F mice, neither senolytic treatment altered plasma cytokine levels.

D + Q treatment reduces hippocampal plaque burden in female APPNL−F/NL−F mice

Higher senescence and SASP markers in adipose tissue has been linked to cognitive deficits and increased dementia risk [36,37,38]. Accordingly, it is important to know whether the senolytic drug treatments mitigate senescence and amyloid plaques in the brain of APPNL−F/NL−F mice. SA-β-gal staining has been extensively used as a marker of cellular senescence in vivo in both whole-mount and cryosections [39]. To examine the treatment effects on cell senescence and amyloid plaques in the hippocampus, tissue was co-stained with SA-β-gal for senescence (blue) and congo red for amyloid plaques (red). The representative stained images (Fig. 5A) and quantified data showed that D + Q treatment in the female APPNL−F/NL−F mice reduced the percentage of SA-β-gal staining (Fig. 5B) and the plaque number, size, density, and burden throughout the hippocampus (Fig. 5C–F). Since soluble Aβ42 is considered the neurotoxic species associated with AD development and its aggregation results in insoluble plaque deposition, we also assayed its hippocampal levels. Hippocampal soluble Aβ42 was reduced in D + Q-treated female APPNL−F/NL−F mice (Fig. 5G). D + Q treatment in male APPNL−F/NL−F mice as well as fisetin treatment in both sexes did not drastically alter hippocampal soluble or insoluble amyloid levels (Fig. 5C–G). Furthermore, a sex effect was discovered with respect to males portraying a smaller plaque size than females within the control condition (Fig. 5D), which is consistent with our previous observations [28]. However, this sex effect did not carry over into any of the treatment groups.

Fig. 5

APPNL−F/NL−F mice have reduced hippocampal senescent cell burden as well as insoluble and soluble Aβ42 after D + Q treatment. Representative × 4 and × 40 hippocampal images (A) of SA-β-gal staining (blue) and plaque accumulation (red) for male (left) and female (right) after 10 treatments of vehicle (top), D + Q (middle), or fisetin (bottom). Arrows indicate magnified image area shown to the right of each whole hippocampal image. Percentage of SA-β-gal staining (B) along with insoluble (C–F) and soluble Aβ42 (G) levels are shown. Data are represented as means ± SEM (n = 8–10). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05, **p < 0.01 based on a two-tailed Student’s t test

D + Q treatment improved spatial learning and memory in female APPNL−F/NL−F mice

To examine whether senolytic treatment improves spatial learning and memory recall in APPNL−F/NL−F mice, the MWM test was performed after 9 months of treatments at 13 months of age. The MWM data demonstrated that D + Q-treated female APPNL−F/NL−F mice improved spatial learning during the 5-day training period by reducing cumulative distance (Fig. 6B–C), decreasing corrected integrated path length (CIPL; Fig. 6E–F), and increasing path efficiency (Fig. 6H–I). D + Q treatment also improved spatial memory recall by significantly increasing platform and annulus 40 entries during the probe challenge (Fig. 6J–K). Fisetin treatment in female APPNL−F/NL−F mice had minimal effects on spatial navigation, though an increase in path efficiency during the training days was observed (Fig. 6H–I). MWM spatial learning and memory recall were not altered in male APPNL−F/NL−F mice receiving either D + Q or fisetin (Fig. 4A, C, D, F, G, I–K). We did not observe sex or treatment effects with novel object recognition memory (data not shown). The improved spatial recognition learning and memory observed in female APPNL−F/NL−F may be due to the reduced SASP profile as well as soluble and insoluble amyloid accumulation.

Fig. 6

Spatial learning and memory recall was improved in female APPNL−F/NL−F mice receiving D + Q treatment. An eight-day MWM paradigm consisting of 5 training days followed by a single probe challenge 48 h after was used to assess spatial cognition after the ninth treatment (13 months of age). The cumulative distance (A–C), corrected integrated path length (CIPL; D–F), and path efficiency (G–I) along with their corresponding AUC are shown for each training day. The number of platform crosses into the former location of the hidden escape platform (J) and annulus 40 (K) are reported for the probe challenge. Data are represented as means ± SEM (n = 20–25). Results of a two factorial analysis are shown above each bar graph for the Sex (S) and Treatment (T) categorial variables and their interaction (S × T). *p < 0.05 based on a two-tailed Student’s t test