Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer's Disease Model

Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative disorder and the most common cause of dementia. Various pathogenic mechanisms have been proposed to contribute to disease progression, and recent research provided evidence linking dysregulated circadian rhythms/sleep and energy metabolism with AD. Previously, we found that the natural compound Nobiletin (NOB) can directly activate circadian cellular oscillators to promote metabolic health in disease models and healthy aging in naturally aged mice. In the current study, using the amyloid-β AD model APP/PS1, we investigated circadian, metabolic and amyloid characteristics of female mice and the effects of NOB. Female APP/PS1 mice showed reduced sleep bout duration, and NOB treatment exhibited a trend to improve it. While glucose tolerance was unchanged, female APP/PS1 mice displayed exaggerated oxygen consumption and CO2 production, which was mitigated by NOB. Likewise, cold tolerance in APP/PS1 was impaired relative to WT, and interestingly was markedly enhanced in NOB-treated APP/PS1 mice. Although circadian behavioral rhythms were largely unchanged, real-time qPCR analysis revealed altered expression of several core clock genes by NOB in the cerebral cortex, notably Bmal1, Npas2, and Rora. Moreover, NOB was also able to activate various clock-controlled metabolic genes involved in insulin signaling and mitochondrial function, including Igf1, Glut1, Insr, Irs1, Ucp2, and Ucp4. Finally, we observed that NOB attenuated the expression of several AD related genes including App, Bace1, and ApoE, reduced APP protein levels, and strongly ameliorated Aβ pathology in the cortex. Collectively, these results reveal novel genotype differences and importantly beneficial effects of a natural clock-enhancing compound in biological rhythms and related pathophysiology, suggesting the circadian clock as a modifiable target for AD.

Keywords: Alzheimer’s disease; Nobiletin (NOB); amyloid beta (Aβ); circadian rhythms; energy metabolism; female APP/PS1 mice; mitochondria; sleep.

Figure 1

Sleep alterations in APP/PS1 mice and effects of Nobiletin (NOB). (A) Average of sleep bout duration (n = 6–10/each group). Data represented by mean ± SEM. * p < 0.05 unpaired Student’s t-test. (B) Histogram of sleep bout duration (n = 6–10/each group). Data represented by mean ± SEM. * p < 0.05 unpaired Student’s t-test. # p < 0.05, ## p < 0.01 one-way ANOVA with Tukey’s multiple comparisons test.

Figure 2

Nobiletin improves systemic metabolism in APP/PS1 mice. (A, B) Total average (left panels) and hourly average (right panels) of carbon dioxide production (VCO2) and oxygen consumption (VO2) using CLAMS metabolic chambers (n = 6–10/each group). Data represented by mean ± SEM. * p < 0.05 t-test. For the hourly average of VCO2: * p < 0.05 at the time points of 19.3 and 22.8 h, two-way ANOVA with Tukey’s multiple comparison test (APP/PS1.Veh vs. APP/PS1.NOB). For hourly average of VO2: * p < 0.05 at the time points of 13.5, 15, and 16.3 h, two-way ANOVA with Tukey’s multiple comparison test (WT.Veh vs. APP/PS1.Veh). (C) Cold tolerance test (n = 6–10/each group). Data represented by mean ± SEM. # p < 0.05, ## p < 0.01 unpaired student t-test (WT.Veh vs WT.NOB). * p < 0.05 unpaired student t-test (APP/PS1.Veh vs APP/PS1.NOB). †† p < 0.01 one-way ANOVA with Tukey’s multiple comparisons test (APPS/PS1.Veh vs. APP/PS1.NOB).

Figure 3

Effects of NOB on expression of core clock genes in WT and APP/PS1 mice. mRNA expression of core clock genes in cortex tissues was measured via real-time qPCR (n ≥ 3/each group). Mice were sacrificed at ZT6 and ZT18 under the LD condition. Data represented by mean ± SEM in bar graph. * p < 0.05, ** p < 0.01 two-way ANOVA with Tukey’s multiple comparisons.

Figure 4

Nobiletin alters metabolic and mitochondrial gene expression in APP/PS1 mice. mRNA expression of (A) insulin signaling and glucose transporter-related genes and (B) mitochondrial thermogenic genes in cortex tissues was measured using real-time qPCR (n ≥ 3/each group). Mice were sacrificed at ZT6 and ZT18 under the LD condition. Data represented by mean ± SEM in bar graph. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons.

Figure 5

NOB modulates AD gene expression and ameliorates Aβ deposits in APP/PS1 mice. (A) mRNA expression of AD-related genes in WT and APP/PS1 mice. mRNA expressions of core clock genes in cortex tissues were measured using real-time qPCR (n ≥ 3/each group). Mice were sacrificed at ZT6 and ZT18 under the LD condition. Data represented by mean ± SEM in bar graph. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (B) Protein levels of APP-FL and CTFs were detected by using human 6E10 and carboxyl-terminal fragments (CTFs)-specific antibodies in cortex lysates at ZT6 and ZT18. GAPDH served as the loading control. Left panels: representative blot images. See Figure S4B for the whole blot images and size markers. Right panels: quantification of APP-FL and CTFs (n = 3/group). Data represented by mean ± SEM. * p < 0.05, unpaired Student’s t-test. (C) Immunohistochemistry of Aβ in APP/PS1 mice using the 4G8 antibody in the cortex (n = 6–11). Right panels: Quantification of amyloid burden (4–6 slices/mouse). Data represented by mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test shows significant statistical difference between APP/PS1.Veh and APP/PS1.NOB (**, p < 0.01).