Amyloid-β-Induced Changes in Molecular Clock Properties and Cellular Bioenergetics

Abstract

Ageing is an inevitable biological process that results in a progressive structural and functional decline, as well as biochemical alterations that altogether lead to reduced ability to adapt to environmental changes. As clock oscillations and clock-controlled rhythms are not resilient to the aging process, aging of the circadian system may also increase susceptibility to age-related pathologies such as Alzheimer's disease (AD). Besides the amyloid-beta protein (Aβ)-induced metabolic decline and neuronal toxicity in AD, numerous studies have demonstrated that the disruption of sleep and circadian rhythms is one of the common and earliest signs of the disease. In this study, we addressed the questions of whether Aβ contributes to an abnormal molecular circadian clock leading to a bioenergetic imbalance. For this purpose, we used different oscillator cellular models: human skin fibroblasts, human glioma cells, as well as mouse primary cortical and hippocampal neurons. We first evaluated the circadian period length, a molecular clock property, in the presence of different Aβ species. We report here that physiologically relevant Aβ_1-42 concentrations ranging from 10 to 500 nM induced an increase of the period length in human skin fibroblasts, human A172 glioma cells as well as in mouse primary neurons whereas the reverse control peptide Aβ_42-1, which is devoid of toxic action, did not influence the circadian period length within the same concentration range. To better understand the underlying mechanisms that are involved in the Aβ-related alterations of the circadian clock, we examined the cellular metabolic state in the human primary skin fibroblast model. Notably, under normal conditions, ATP levels displayed circadian oscillations, which correspond to the respective circadian pattern of mitochondrial respiration. In contrast, Aβ_1-42 treatment provoked a strong dampening in the metabolic oscillations of ATP levels as well as mitochondrial respiration and in addition, induced an increased oxidized state. Overall, we gain here new insights into the deleterious cycle involved in Aβ-induced decay of the circadian rhythms leading to metabolic deficits, which may contribute to the failure in mitochondrial energy metabolism associated with the pathogenesis of AD.

Keywords: Alzheimer's disease; amyloid-β; bioenergetic balance; energetic state; mitochondria.

Figure 1

Amyloid-β increases the circadian period length. (A) Representative luminescence records from human skin fibroblasts transfected with mice Bmal1::luciferase reporter in presence of amyloid-β 1–42 (500 nM) or of the reverse peptide amyloid-β 42-1 (500 nM) compared to the condition in the absence of Aβ species (CRTL). (B, C) Circadian period length determined in synchronized human skin fibroblasts transfected with mice Bmal1::luciferase reporter in presence of amyloid-β 1–42 and 42-1 (B), 1–40, 1–24, 34–42, 25–35, 35-25, and 15–25 (C) compared to the condition in the absence of Aβ species (CRTL) (dashed line). (D) Circadian period length determined in synchronized human glioma A172 cells transfected with Bmal1::luciferase reporter in presence of amyloid-β 1–42 compared to control (CRTL) (dashed line). (E) Circadian period length determined in synchronized cortical primary mouse neurons transfected with Bmal1::luciferase reporter in presence of amyloid-β 1–42 compared to control (CRTL) (dashed line). (F) Circadian period length determined in synchronized hippocampal primary mouse neurons transfected with Bmal1::luciferase reporter in presence of amyloid-β 1–42 compared to control (CRTL) (dashed line). Bars represent the mean of at least three independent experiments ± SD. ^**P < 0.01; ^***P < 0.001 for Student's two-tailed t-test, compared to control.

Figure 2

Aβ induces a decline in circadian bioenergetic homeostasis. (A) Total ATP levels from synchronized human skin fibroblasts treated with Aβ 1–42 compared to non-treated cells (CTRL) measured at the indicated time points (6 time points, n = 6 for each) (Two-way ANOVA; Aβ effect: Df = 1, F = 3890, P < 0.0001; time effect: Df = 6, F = 34.25, P < 0.0001; post hoc Bonferroni, ctrl vs. Aβ: at 16 h, t = 23.43, ^***P < 0.0001, at 28 h, t = 11, ^***P < 0.0001). Rhythmicity of ATP was assessed using the JTK_Cycle algorithm as previously described (Dallmann et al., 2012): JTK_Cycle, P_CTRL = 2.07 × 10⁻¹⁵, P_Aβ = 0.691. (B) Oxygen Consumption Rate (OCR) was evaluated in Aβ-treated human skin fibroblasts compared to untreated control cells at 16 and 28 h after synchronization, respectively. (n = 11 replicates /group, three independent experiments) (Two-way ANOVA, Aβ effect: Df = 1, F = 9.948, P = 0.0021; time effect: Df = 1, F = 44.08, P < 0.0001; post hoc Bonferroni, ctrl vs. Aβ: at 16 h, t = 5.886, ^***P < 0.0001, at 28 h, t = 1.448, P > 0.05). (C) OCR/ATP: The basal OCR corresponding to the ATP peak (at 16 h) and the ATP trough (at 28 h) are plotted against the respective ATP content in control (white symbols) and Aβ conditions (black symbols). Values represent the mean of each group (mean of the ATP levels in abscissa/mean of the OCR in ordinate); n = 11–12 replicates/group of three independent experiments. The red arrows highlight the Aβ-induced metabolic shift at 16 and 28 h after cell synchronization.

Figure 3

Aβ induces a disruption in the cellular redox state. (A) Mitochondrial (mROS) reactive oxygen species levels were evaluated in synchronized human skin fibroblasts treated with Aβ 1–42 compared to non-treated cells (CTRL) measured at the indicated time points (6 time points, n = 4 for each) (Two-way ANOVA; Aβ effect: Df = 1, F = 859, P < 0.0001; time effect: Df = 6, F = 33.16, P < 0.0001; post hoc Bonferroni, ctrl vs. Aβ: at 16 h, t = 9.757, ^***P < 0.0001, at 28 h, t = 9.035, ^***P < 0.0001). Rhythmicity of mROS was assessed using the JTK_Cycle algorithm as previously described (Dallmann et al., 2012): JTK_Cycle, P(CTRL)_mROS = 5.40 × 10⁻²¹, P(Aβ)_mROS = 3.9 × 10⁻¹⁸. (B) The NAD⁺/NADH ratio evaluated in in synchronized Aβ-treated human skin fibroblasts compared to untreated control cells at the indicated time points. Values represent the mean ± SD; n = 9–12 replicates of three independents experiments (Two-way ANOVA; Aβ effect: Df = 1, F = 408.7, P < 0.0001; time effect: Df = 6, F = 1.769, P = 0.1343).