Male and female C57Bl/6 mice bearing the human Swedish (S:KM670/671NL) and the London (L:V717I) mutations in the 751 amino acid form of human amyloid-beta precursor protein (AβPP) under control of a murine Thy1 promotor were used. All mice were genotyped by tail biopsies and polymerase chain reaction before and after the experiments as previously described (Pohland et al., 2018). Mice were housed in the institute of pharmacology in Frankfurt a. M. according to the German guidelines for animal care with access to water and food ad libitum until they reached the age of 3, 7, and 13 months. Wild-type mice of the same age were used as controls. Experiments were approved by the regional authority (V54-19 c 20/15 – FU/113).

Approximately 1 h before the dark phase, the mouse was moved to a single cage if the mouse was previously housed in a group. If the mouse was housed singly, the test could be performed in the “home” cage. All furnishings except litter were removed from the cage and a nestlet was placed in the cage. After 18 h, the test ended. The nests were photographed and scored by two independent observers. Nests were rated from 1 to 5, with 5 representing a perfect nest and 1 representing an unbuilt nest. The evaluation of the nests was analogous to the protocol described in Deacon (2006).

The test was carried out using a passive avoidance step through system (cat. no. 40533/mice Ugo Basile, Germonio, Italy) and a protocol similar to the protocol published by Shiga et al. (2016). The setup of the passive-avoidance-learning test consisted of two chambers, a light and a dark chamber. The mouse was placed in the light chamber (light intensity 75%). The mouse was acclimated to the light chamber for 30 s before the door opened into the dark chamber. The test was stopped after 180 s or if the mouse entered the dark chamber. In the chamber, the mouse received an electric shock (0.2 mA) for 2 s. After 24 h, the experiment was repeated, with the difference that the acclimation time changed to 5 s and the test was aborted after 300 s. In addition, the electric shock was turned off.

The mouse was put in one arm of a custom-made Y-shaped maze (material: polyvinyl chloride, length of arms: 36 cm, height of arms: 7 cm, width of arms: 5 cm). Afterward, the mouse was able to explore the maze for 5 min. At the end of the experiment, the number of entries was determined as well as the number of alternations. One alternation was defined as the mouse entered all three arms before it entered an already visited arm. The alternation rate was calculated using the formula [(number of alternations/total number of possible alternations) × 100] (Wolf et al., 2016).

Brain samples were homogenized in 10 times the amount of phosphate-buffered saline containing a protease inhibitor cocktail (Roche cOmplete, Mini Protease Inhibitor Cocktail). Afterward, samples were centrifuged (15,000 × g, 30 min, 4°C) and the supernatants were put into a fresh reaction vessel and stored at −80°C until analysis. Aβ_1–40 amounts were measured using a specific solid phase sandwich enzyme-linked immunosorbent assay (ELISA; Life Technologies, Carlsbad, CA, United States).

Dissociated Brain Cells (DBC) were freshly prepared using one hemisphere of mouse brain. The method is described in detail in Reutzel et al. (2020). Briefly, the brain was washed in medium 1 (138 mM NaCl, 5.4 mM KCl, 0.17 mM Na₂HPO₄, 0.22 mM KH₂PO₄, 5.5 mM Glucose × H₂0, 58.4 mM Sucrose, pH = 7.35), cut into small pieces in 2 ml of medium 1 and was pressed through a 200 μm nylon mesh. Afterward, the brain suspension was filtered through a 102 μm nylon mesh. The cell homogenate was centrifuged (2,000 rpm, 5 min, 4°C) and the resulting pellet was dissolved in 20 ml medium 2 (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl₂ × 2 137 H20, 1 mM MgCl₂ × 6 H20, 25 mM Glucose × H20, 70 mM Sucrose, 20 mM HEPES). The centrifugation step was repeated twice and the pellet dissolved in 4.5 ml Dulbecco’s Modified Eagle Medium (DMEM) without supplements. For ATP measurements twelve replicates of 50 μl cell suspension were seeded into a 96 well plate. Respectively 6 wells were incubated with sodium nitroprusside (0.5 mM). For determination of the mitochondrial membrane potential (MMP) 250 μl were seeded into 24 well plate and 6 wells were incubated with 2 mM sodium nitroprusside. Afterward, the plates were incubated for 3 h in a humified incubator (5% CO₂) before measurement of mitochondrial membrane potential or ATP. The remaining suspension was stored for protein determination at −80°C).

For the measurement of mitochondrial membrane potential (MMP) DBC’s were incubated for 15 min in a humified incubator (5% CO₂) with 0.4 μM of the fluorescence dye Rhodamine-123. The reaction was stopped by adding 250 μl of Hank’s balanced salt solution (HBSS) into each well. The plate was centrifuged (914 g, 5 min, room temperature), the medium aspirated and new HBSS was added into the wells. DBC were triturated and MMP was measured by reading the R123 fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 535 nm Victor X3 multilabel counter). The measurement was repeated four times and the values normalized to protein content of the sample.

Adenosine Triphosphate Levels (ATP) concentrations were determined in DBC’s with the ViaLight Plus bioluminescence kit (Lonza, Walkersville, MD, United States). After the incubation, the plate was removed from the incubator and allowed to cool to room temperature for 10 min. All wells were incubated for 10 min with 25 μl lysis buffer in the dark. Next, wells were incubated with 50 μl monitoring reagent. The measurement was conducted according to the manufactures instructions. The emitted light (bioluminescence) was recorded using a Victor X3 multilabel counter and is linearly related to ATP content.

Half a brain hemisphere (the frontal part) was used for the isolation of brain mitochondria for mitochondrial respiration as previously described (Hagl et al., 2013). Briefly, the sample was homogenized using a potter equipped with a Teflon^® pistil in 2 ml mitochondrial respiration medium (MiR05) containing a protease inhibitor cocktail (PI, complete; Roche, Mannheim, Germany). Afterward, the homogenate was centrifuged (1,400 g, 7 min, 4°C). A second centrifugation was conducted with the supernatant for 3 min followed by a centrifugation at 100,000 g for 5 min at 4°C. The resulting pellet containing the mitochondria was dissolved in 250 μl MIRO5 + PI and 80 μl of the solution was used for the measurement of mitochondrial respiration using an Oxygrapph-2k respirometer (Oroboros, Innsbruck, Austria). The remaining mitochondria solution was frozen in liquid nitrogen for citrate synthase activity (120 μl) and protein determination (50 μl).

After injecting 80 μl of the mitochondria suspension into the Oxygraph-2k chamber a protocol elaborated by Prof. Dr. Erich Gnaiger was used including several inhibitors, uncouplers and substrates of the respiratory chain system (Krumschnabel et al., 2015). A detailed description of the different steps of the protocol is described in Hagl et al. (2016). Briefly, the capacity of the oxidative phosphorylation (OXPHOS) was determined using complex-I related substrates pyruvate (5 mM) and malate (2 mM) and ADP (2 mM) followed by the addition of succinate (10 mM). Mitochondrial integrity was measured by addition of cytochrome c (10 μM). Oligomycin (2 μg/ml) was added to determine leak respiration [leak (omy)] and afterward uncoupling was achieved by carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone (FCCP, injected stepwise up to 1–1.5 μM). Complex II respiration was measured after the addition of rotenone (0.5 μM). Complex III inhibition was achieved by the addition of antimycin A (2.5 μM) and was subtracted from all respiratory parameters. COX activity was measured after ROX determination by applying 0.5 mM tetramethylphenylenediamine (TMPD) as an artificial substrate of complex IV and 2 mM ascorbate to keep TMPD in the reduced state. Autoxidation rate was determined after the addition of sodium azide (>100 mM), and COX respiration was additionally corrected for autoxidation.

RNA isolation was conducted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions using ∼ 20 mg RNAlater stabilized brain tissue samples (Qiagen, Hilden, Germany). RNA was quantified measuring the absorbance at 260 and 280 nm using NanoDrop™ 2000c spectrometer (Thermo Fisher Scientific, Waltham, MA, United States). Complementary DNA was synthesized from 250 ng total RNA using the iScript cDNA Synthesis Kit (BioRad, Munich, Germany) according to the manufactuer’s instructions and was stored at −80°C. qRT-PCR was conducted using a CfX 96 Connect™ system (BioRad, Munich Germany) (Reutzel et al., 2020). All primers were received from biomol (Hamburg, Germany) or biomers (Ulm, Germany). cDNA for qRT-PCR was diluted 1:5 with RNase free water (Qiagen, Hilden, Germany) and all samples were performed in triplicates. PCR cycling conditions were an initial denaturation at 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 58°C for 45 s and 72°C for 29 s. Gene expression was analyzed using the –(2ΔΔ C_q) method using BioRad CfX manager software and were normalized to the expression levels of beta 2 microglobulin (B2M) and phosphoglycerate kinase 1 (PGK1). All primer sequences, product sizes and primer concentrations used for quantitative real-time PCR (QRT-PCR) are listed in Table 1.

Citrate synthase (CS) activity in isolated mitochondria was determined photometrically as previously published (Pohland et al., 2018; Reutzel et al., 2020). Briefly, a sample of the isolated mitochondria in MiR05 was thawed on ice and the reaction medium [100 μl of 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 25 μl of 10% Triton X-100, 50 μl of 10 mM oxaloacetate, 25 μl of 12.2 mM acetyl coenzyme A, and 790 μl purified water] was mixed and warmed to 30°C for 5 min. Afterward, 10 μl isolated mitochondria were added to the reaction mixture and transferred into a 10 mm quartz cuvette (Hellma^® Analytics, Müllheim, Germany). CS activity was measured at 412 nm using a GENESYS 5 spectrophotometer (Spectronic via Thermo Fisher Scientific, Waltham, MA, United States). All samples were measured in duplicates.

Protein content was measured with the Pierce™ Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the Manufacturer’s instructions.

Unless otherwise stated, values are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed by applying a two sided, unpaired Student’s t-test and for multiple comparisons a one-way ANOVA with Tukey’s multiple comparison post test (Prism 8.0 GraphPad Software, San Diego, CA, United States). Statistical significance was defined for p*-values of <0.05, p** < 0.01, p*** < 0.001 and p**** < 0.0001.