Dulbecco's-modified Eagle medium (DMEM), RPMI-1640 medium, fetal calf serum (FCS), penicillin/streptomycin, NAD⁺, NADH, dihydrorhodamine 123 (DHR), HBSS, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and PES (phenazine ethosulfate) were from Sigma-Aldrich (St. Louis, MO, USA). Glutamax and DPBS were from Life Technologies (Waltham, MA, USA). XF Cell Mitostress kit was from Seahorse Bioscience (North Billerica, MA, USA). Horse serum (HS) was from Amimed, Bioconcept (Allschwil, Switzerland).

Isolated human skin fibroblasts from biopsies and glioma cell line A172 (gift from Dr Steven A. Brown) were cultured in DMEM/1% penicillin-streptomycin (v/v)/1% Glutamax (v/v) [DMEM complete (DMEMc)]/20% heat-inactivated FBS (v/v) (Pagani et al., 2011; Gaspar and Brown, 2015). Confluent cells were infected using mice Bmal1::luciferase lentivirus and were positively selected 3 days after infection. Mouse cortical and hippocampal neurons were prepared from E15 embryos according to the Swiss guidelines and were plated in poly-l-lysine-coated plates according to the instruction of the manufacturer (Lonza, Switzerland). After 7 days at 37°C, 50% of the medium was replaced with fresh medium every third day.

Prior to the assessment of circadian period length, cells were synchronized for 15 min at 37°C with 100 mM dexamethasone as described previously (Pagani et al., 2011), while prior to the measurement of nucleotide levels, mitochondrial reactive oxygen species (mROS) and oxygen consumption rate (OCR) in human primary fibroblast culture, serum shock treatment [DMEMc supplemented with 50% heat-inactivated horse serum (v/v)] was performed for 2 h at 37°C to synchronize the cells accordingly to a previously optimized method for these kind of experiments (Cooper, 2003; Rosner et al., 2013). For measurements of nucleotides levels and mitochondrial reactive oxygen species (mROS) levels, experiments were performed starting from 12 h post-synchronization time point and measured at 4 h intervals. For measurements of oxygen consumption, experiments were performed at 16 and 28 h post-shock.

The different amyloid-beta (Aβ) fragments [Aβ_1–42; reverse peptide (Aβ₄₂₋₁); Aβ_1–40; N-terminal fragment (Aβ_1–28); C-terminal fragment (Aβ_34–42); shortest active peptide fragments (Aβ_25–35, Aβ_15–25)] (Bachem, Bubendorf, Switzerland) were rapidly dissolved in sterile PBS 1x, pH~7.4 (stock concentration 500 μM) and stored as aliquots at −80°C until needed. One day before treatment of cells with Aβ peptide fragments, 50 μM Aβ working solutions were prepared in PBS 1x, pH~7.4. After securing the caps with parafilm, the tubes were incubated on a table-top thermomixer (Eppendorf, Hamburg, Germany) at 1,000 rpm (~250 × g) at 37°C for 24 h in order to induce aggregation of the Aβ peptides into fibrils. For the circadian period length determination, after synchronization with dexamethasone (100 nM, 15 min at 37°C), fibroblasts were treated with 1, 10, 250, and 500 nM of Aβ in DMEM cell culture medium containing 1% penicillin-streptomycin (v/v)/1% Glutamax (v/v) (DMEMc)/10% FBS (v/v) for 5 days until analyzed. This experimental setting requesting a rather long incubation time over several days in aqueous medium, predicting the formation of insoluble fibrillar Aβ aggegrates and fibrils, did not allow to study the effects only of soluble Aβ species. Therefore, also in the following assays, the same Aβ preparations were used for the determination of nucleotides, ROS and oxygen consumption rate to mimic corresponding conditions of Aβ assembly and chronic toxicity. Thus, the cells were pre-treated with 500 nM pre-aggregated Aβ (the most effective concentration on bioenergetics readouts without cell death-inducing properties) in DMEM cell culture medium containing 1% penicillin-streptomycin (v/v)/1% Glutamax (v/v) [DMEM complete (DMEMc)]/20% heat-inactivated FBS (v/v) for 5 days. After the synchronization, cells were treated again with 500 nM Aβ in DMEM/1% penicillin-streptomycin (v/v)/1% Glutamax (v/v) (DMEMc)/2% FBS (v/v) up to 48 h to maintain the Aβ pressure on the synchronized cells.

Human skin fibroblasts transfected with the lentiviral circadian reporter mice Bmal1::luciferase (Brown et al., 2005; Gaspar and Brown, 2015) were plated in single culture dishes (35 × 10 mm) at a cell density sufficient to reach 70–80% of confluency on the start day of recording of period length. After synchronization with dexamethasone (100 nM, 15 min at 37°C), cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 10 mM HEPES and 0.1 mM luciferin. For at least 5 days, the amount of produced light that is proportional to Bmal1 gene expression was measured using a Lumicycle instrument (Actimetrics). Data were analyzed with Lumicycle Analysis software (Lumicycle™, version 2.31, Actimetrics Software) and the period of oscillation was calculated by least-mean-squares fitting of dampened sine wave functions to the actual data as described previously (Pagani et al., 2011).

The total ATP content from synchronized human skin fibroblasts was determined using a bioluminescence assay (ViaLighTM HT; Cambrex Bio Science, Walkersville, MD USA) according to the instruction of the manufacturer. Cells were plated in 8 replicates into a 96-wells cell culture plate at a cell density sufficient to reach 40–50% of confluency on the following day. The enzyme luciferase, which catalyzes the formation of light from ATP and luciferin was used. The emitted light is linearly related to the ATP concentration and is measured using a multilabel plate reader VictorX5 (Perkin Elmer).

To measure NAD⁺ and NADH, the two molecules were separately extracted using an acid-base extraction method (HCL 0.1 mol/l–NAOH 0.1 mol/l). To determinate both NAD⁺ and NADH, an assay was used that is based on passing the electron from ethanol through reduced pyridine nucleotides to MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in a PES (phenazine ethosulfate)-coupled reaction resulting in the formation of a purple precipitate (formazan) that, once dissolved, can be quantified at 595 nm (VictorX5, Perkin Elmer).

The OCR was evaluated in synchronized fibroblasts as previously described (Invernizzi et al., 2012). Briefly, human skin fibroblasts were seeded at the density of 3 × 10⁴ cells/100 μl per well on Seahorse Biosciences 24-well culture plates 1 day prior to the serum-shock synchronization. On the next day, cells were synchronized using serum shock (2 h at 37°C). Afterwards, the medium was exchanged to 500 μl of assay medium [glucose-free RPMI-1640 medium containing 2% FBS (v/v), 2 mM sodium pyruvate, pH~ 7.4]. Before to be placed in the Seahorse XF24 Analyzer, the microplates were equilibrated in a CO₂-free incubator at 37°C for 60 min. Then, the OCR allocated to basal respiration at 16 and 28 h after synchronization were recorded in real-time over 30 min.

Total level of mROS was assessed using the fluorescent dye dihydrorhodamine 123 (DHR). Cells were plated in 8 replicates into a black 96-wells cell culture plate at a cell density sufficient to reach 40–50% of confluency on the day of the start of mROS recording. After synchronization, cells were loaded at the indicated time points with 10 μM of DHR for 15 min at room temperature in the dark on an orbital shaker. After washing twice with HBSS, DHR, which is oxidized to cationic rhodamine 123 that localizes within the mitochondria, exhibits a green fluorescence that was detected using the multilabel plate reader VictorX5 at 485 nm (excitation)/538 nm (emission). The intensity of fluorescence was proportional to mROS levels in mitochondria.

Data were presented as mean ± S.D. Statistical analyses were performed using the GraphPad Prism software. The curves were generated by using a standard curve fit function in the GraphPad Prism software. For statistical comparisons, One-way ANOVA followed by Tukey's multiple comparison test or Two-way ANOVA followed by Bonferroni's multiple comparison test was used respectively. P < 0.05 were considered statistically significant. Rhythmicity of ATP, NAD⁺, NADH, and mROS was assessed using a non-parametric algorithm previously described for determination of rhythmic transcripts (Hughes et al., 2010). The JTK-cycle algorithm was used as implemented in R by Kronauer as previously described (Dallmann et al., 2012). A window of 24 h was used for the determination of circadian periodicity and a P-value of < 0.05 was considered as statistically significant.

To address the question whether Aβ is able to disturb the circadian function, we first characterized the circadian period length of synchronized fibroblasts in the presence of different Aβ fragments (Figures 1A–C). For that purpose, we infected cells with a circadian reporter construct (Bmal1 promoter-driven expression of firefly luciferase-harboring lentivirus). First, the effect of Aβ_1–42, the major component of amyloid plaques in AD brains that is more aggregation prone and exhibits higher neurotoxicity than Aβ_1–40, was investigated. Notably, cells had a significantly longer period length in the presence of aggregated Aβ_1–42 at concentrations from 10 up to 500 nM compared to controls (Figures 1A,B), while 1 nM was not yet effective. At a concentration of 250 nM the maximum effect on prolongation of period length was already reached. However, amplitude (the difference between peak and nadir expression values) was unchanged (data not shown). Notably, up to a concentration of 500 nM, cell viability was not significantly impaired in these experimental settings (data not shown). Consistent with findings showing that the reverse control peptide Aβ_42-1 is inactive (Zhang et al., 2002), Aβ_42-1 did not disturb the cellular circadian properties. Similarly, Aβ_1–42 treatment also induced an increase in the period length in synchronized glioma A172 cells as well as in cortical and hippocampal primary mouse neurons (Figures 1D–F). In order to identify the specific Aβ fragment/s responsible for the increase in the period length, we then treated the synchronized fibroblasts with different Aβ species (Aβ_1–40, the most abundant Aβ isoform in the brain, Aβ_1–28, Aβ_34–42, Aβ_15–25, and Aβ_25–35) within the same range of concentrations (Figure 1C). Surprisingly, all the fragments induced a significant alteration of the circadian function to a similar extent as Aβ_1–42 did itself. Together, these results suggest that the aggregated nature of the Aβ species seem to represent an important factor for the AD-related disruption of the circadian rhythm.

Since mitochondria were found to be a target of Aβ (Schmitt et al., 2012), leading to mitochondrial dysfunction including a decline in OxPhos and ultimately in ATP content, we investigated the impact of Aβ on ATP content and on mitochondrial oxidative metabolism with regard to rhythmic changes due to the the tight relationship between the circadian clock and metabolism (Brown, 2016; Panda, 2016). As ATP is primarily generated via mitochondrial oxidative phosphorylation (OxPhos), we first monitored whole cell ATP content as readout of OxPhos in Aβ_1–42-treated (500 nM) compared with untreated synchronized human skin fibroblasts. Under normal conditions, total ATP levels displayed a circadian rhythmicity in control cells (with the peak at 16 h and the trough at 28 h), whereas ATP levels in the Aβ-treated cells did not oscillate and were, in addition, significantly reduced (Figure 2A).

We then monitored the real–time oxygen consumption rate (OCR), an indicator of mitochondrial respiration in Aβ-treated human skin fibroblasts compared to untreated cells at 16 h corresponding to the ATP peak and at 28 h related to the ATP trough using the Seahorse Bioscience XF24 Flux Analyzer (Figure 2B). In untreated control cells, the OCR was significantly decreased at the time point of the ATP trough (at 28 h) compared to that of the ATP peak (at 16 h) (Figure 2B). Thus, the circadian oscillations of OCR and ATP appear to be in the same phase and fit well together in the way that at the time point of high energy production, the oxidative phosphorylation system provides the maximum performance. In contrast, Aβ_1–42 treatment completely dampened the variation in the OCR and no differences were found at 16 and 28 h after synchronization (Figure 2B). Overall, these observations support the hypothesis that circadian control of the mitochondrial function is strongly disturbed by Aβ which may lead to a mitochondrial inability to respond to an increasing metabolic demand. In addition, we characterized the cellular bioenergetic profile of Aβ-treated human skin fibroblasts compared to untreated cells, by mapping OCR (basal respiration) vs. ATP for the indicated time points (Figure 2C). Remarkably, untreated cells switched between a metabolically active state corresponding to the high ATP (16 h) and a metabolically resting state corresponding to low ATP levels (28 h), while Aβ-treated cells remained in the metabolically inactive state at both time points.

To further investigate the Aβ-induced imbalance in mitochondrial homeostasis, we next evaluated the mitochondrial ROS levels as well as the NAD⁺/NADH ratio as oxidative stress readout in synchronized human skin fibroblasts in the presence or absence of Aβ (Figure 3). Again under normal conditions, we found circadian oscillations for mitochondrial ROS (mROS) levels exhibiting a peak at 16 h and a trough at 28 h after synchronization consistent with our findings on ATP oscillations. Interestingly, Aβ_1–42 (500 nM) increased globally the mROS levels in the Aβ-treated cells, but it did not induce a dampening of the circadian rhythmicity compared to the untreated cells as found for bioenergetic readouts (Figure 3A). In contrast, the NAD⁺/NADH ratio did not exhibit a circadian pattern in control cells (Figure 3B). However, the ratio was significantly augmented by Aβ_1–42 treatment (about 10 times) compared to untreated cells (Figure 3B). Together, our findings suggest that Aβ triggers a decline of mitochondrial bioenergetics including an abolishment of variations in ATP levels coupled to a shift in the redox homeostasis toward a highly oxidized state.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.