A ketone ester diet exhibits anxiolytic and cognition-sparing properties, and lessens amyloid and tau pathologies in a mouse model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) involves progressive accumulation of amyloid β-peptide (Aβ) and neurofibrillary pathologies, and glucose hypometabolism in brain regions critical for memory. The 3xTgAD mouse model was used to test the hypothesis that a ketone ester-based diet can ameliorate AD pathogenesis. Beginning at a presymptomatic age, 2 groups of male 3xTgAD mice were fed a diet containing a physiological enantiomeric precursor of ketone bodies (KET) or an isocaloric carbohydrate diet. The results of behavioral tests performed at 4 and 7 months after diet initiation revealed that KET-fed mice exhibited significantly less anxiety in 2 different tests. 3xTgAD mice on the KET diet also exhibited significant, albeit relatively subtle, improvements in performance on learning and memory tests. Immunohistochemical analyses revealed that KET-fed mice exhibited decreased Aβ deposition in the subiculum, CA1 and CA3 regions of the hippocampus, and the amygdala. KET-fed mice exhibited reduced levels of hyperphosphorylated tau deposition in the same regions of the hippocampus, amygdala, and cortex. Thus, a novel ketone ester can ameliorate proteopathic and behavioral deficits in a mouse AD model.

Fig. 1

Experimental design and body weight of mice during the course of the dietary intervention. (A) To characterize the behavioral effects of the ketone ester on 3xTgAD mice, we developed a strategy to analyze the cognitive performance of the mice at 12 months (phase 1) and at 15 months (phase 2). The dietary intervention was started at 8.5 months with initiation of either a carbohydrate-enriched (CHO) or a ketone ester (KET) diet. The mice were euthanized at the age of 16.5 months after completion of all behavioral testing for phase 2. (B) Body weight of 3xTgAD mice in the CHO and KET diet groups during the course of the study. Values are the mean and SEM (n = 11–15 mice per group).

Fig. 2

Ketone ester feeding exerts an anxiolytic action in 3xTgAD mice. Results of both phase 1 and phase 2 elevated plus maze testing of 3xTgAD mice in the carbohydrate-enriched diet (CHO) and ketone ester (KET) diet groups. (A and B) Results for time spent in the open arms (A) and the closed arms plus the center arm of the maze (B). (C and D) Heat maps for representative 3xTgAD mice from the CHO and KET diet groups from phase 1 (C) and phase 2 (D) tests showing the cumulative amounts of time that the mice spent in the different regions of the elevated plus maze. Green, yellow, and red colors correspond to low, medium, and high percentage of spent time in the indicated area, respectively. Values are the mean and SEM (n = 10–14 mice per group) *p < 0.05 by t test. #p < 0.05 by two-way ANOVARM.

Fig. 3

Ketone ester-fed 3xTgAD mice exhibit greater exploratory activity in the open field test. (A) Total distance traveled (cm) in phases 1 and 2. (B) Total ambulatory time (seconds) in phases 1 and 2. (C) Total ambulatory counts in phases 1 and 2. (D, E, and F) Distance traveled (D), ambulatory time (E), and ambulatory counts (F) in the open area. (G) Examples of the paths of individual mice in the open field in phase 2 tests. Values are the means and SEM (n = 10–14 mice per group) *p < 0.05 by t test. #p < 0.05 by two-way ANOVARM. CHO, carbohydrate-enriched diet; KET, ketone ester diet.

Fig. 4

A ketone ester (KET) diet improves the performance of 3xTgAD mice in hippocampus-dependent water maze tests of spatial memory. (A, B) Results for goal latencies for memory acquisition trials (hidden platform) and reversal trials in phase 1 (12 months) and phase 2 (15 month) tests. The results of the working memory tests in phase 2 are also shown. Performance in the KET group proved significant using two-way ANOVARM measures analysis for phase 1 in Hidden and Reverse (p = 0.040) and Reverse alone (p = 0.026). For phase 2, significance was demonstrated for Hidden, Reverse, and Working (p = 0.018). Values are mean and SEM (n = 10–14).

Fig. 5

Fear conditioning tests demonstrated that ketone ester (KET) fed 3xTgAD mice exhibit a stronger context-dependent fear response related to hippocampal memory and a more rapid extinction of amygdala-dependent tone-related conditioned fear compared with 3xTgAD mice fed the carbohydrate-enriched (CHO) control diet. (A) Freezing counts on days 2, 7, and 15 in a contextual conditioning test. (B) Freezing times on days 2, 7, and 15 in a contextual conditioning test. (C) Freezing counts on days 2, 7, and 15 in a cued tone conditioning test. (D) Freezing times on days 2, 7, and 15 in a cued tone conditioning test. *p < 0.05 by t test. Values are means and SEM (n = 10–13 mice per group). Two-way ANOVARM analysis (days 2, 7, and 15) did not show a significant difference between the KET and CHO groups.

Fig. 6

Ketone ester feeding reduces intracellular accumulations of amyloid β (Aβ) and phosphorylated tau (pTau) in the subiculum, CA1 and CA3 area of hippocampus, amygdala, and cerebral cortex of 3xTgAD mice. (A) Aβ immunoreactivity in brain sections from mice in the carbohydrate-enriched (CHO; left) and ketone ester (KET; right) diet groups. The upper panels are low magnification images of the regions of the hippocampus and the lower panels are high magnification images of the regions of the subiculum, CA1, cerebral cortex, and amygdala. The graph on the right shows the results of counts of Aβ immunoreactive cells in the indicated brain regions. (B) pTau immunoreactivity in brain sections from mice in the CHO (left) and KET (right) diet groups. The upper panels are low magnification images of the regions of the hippocampus and the lower panels are high magnification images of the regions of the subiculum, CA1, cerebral cortex, and amygdala respectively. The graph on the right shows the results of counts of pTau immunoreactive cells in the indicated brain regions. Scale bars: lower magnification images, 200 μm; high magnification images, 100 μm. Values are the mean ± SEM (n = 6–9 mice per group). *p < 0.05 and **p < 0.001 by the Student t test.