Pyruvate

Primary cultures enriched with astrocytes were obtained from the cerebral cortex of newborn Wistar rats as previously described [16 (link)]; this was carried out according to the Brazilian guidelines for the production, maintenance, and use of animals for teaching and scientific research activities and the Ethics Committee for Animal Use and Experimentation (CEUA protocol No. 6731220818). In brief, the cerebral hemispheres were aseptically collected, and meninges were removed. The tissue was mechanically dissociated, and the cell suspension was gently pushed through a sterile 70 µm Nitex mesh. Cells were centrifuged for 10 min at 1000 rpm and 4 °C and placed in 75 cm² culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) (Island Biological Company—Gibco^®, New York, NY, USA), supplemented with streptomycin 100 μg/mL, 100 IU/mL penicillin G (Island Biological Company—Gibco^®, New York, NY, USA), 2 mM L-glutamine, 0.011 g/L of pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 10% Fetal Bovine Serum (FBS) (Island Biological Company—Gibco^®, New York, NY, USA), 10% Horse Serum (HS) (Island Biological Company—Gibco^®, New York, NY, USA), 3.6 mg/L HEPES (Sigma-Aldrich, St. Louis, MO, USA), and 33 mM glucose and cultured in a humid atmosphere with 5% CO₂ at 37 °C. After 10 days in vitro, microglial cells were harvested by shaking at 265 rpm at 37 °C for 3 h, the supernatant was removed, and fresh DMEM with 10% FBS was added to the cultures (containing about 90% astrocytes and ~8% microglia) and maintained for an additional 5 days in vitro. For experiments, after 3 washes with PBS, the cells were detached with trypsin solution at 37 °C (Trypsin/EDTA, Sigma-Aldrich, St. Louis, MO, USA), counted in a Neubauer chamber, and plated at a density of 10⁵ cells/cm² on 96-, 24-, or 6-well plates for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thermo Fisher, Waltham, MA, USA, 0.5 mg MTT per 1 mL), immunofluorescence, Western Blot, and RT-qPCR and maintained in culture for 72 h. Cultures with 18 days in vitro were submitted to treatments.

In vitro tests were performed using taurine (Nacalai Tesque Inc., Kyoto, Japan), 5-oxoproline, N-gamma-ethylglutamine, and homoserine (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 2-hydroxy-4-methylpentanoate (BACHEM AG, Bubendorf, Switzerland); UDP-glucose (MedChemExpress, Monmouth Junction, NJ, USA); Ala-Ala, pyruvate, trehalose 6-phosphate, urocanate, and α-aminoadipate (Sigma-Aldrich, St. Louis, MO, USA); citrulline, propionate, and succinate (Kanto Chemicals Co., Inc., Tokyo, Japan); N1, N12-diacetylspermine (Cayman Chemical, Ann Arbor, MI, USA); 2-aminobutyrate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); 2-hydroxypentanoate (Combi-Blocks Inc., San Diego, CA, USA); 2-hydroxyglutarate (Sigma-Aldrich); glucose-6-phosphate (Oriental Yeast Co., Ltd., Tokyo, Japan).

H1299 cells were seeded in a 6-well dish at an initial density of 2×10⁵ cells per well. The following day, cells were washed twice with PBS and swapped to DMEM without glucose, glutamine, pyruvate, or phenol red (Sigma, D5030) supplemented with 10% dialyzed FBS (Sigma, F0392), 1% penicillin-streptomycin, 25 mM U-¹³C glucose (Cambridge Isotopes Laboratory, CLM-1396), and 4 mM ¹²C glutamine (Sigma, G5792). GSK2194069 treated cells were supplemented with 200 nM of GSK and all plates were placed back in incubator for 24 hours before extraction.

Sporothrix schenckii 1099-18 (ATCC MYA 4821) and S. brasiliensis 5110 (ATCC MYA 4823), both clinical isolates (Castro et al., 2013 (link)), were used in this study. Fungal cells were maintained and propagated at 28°C in YPD medium (1% [w/v] yeast extract, 2% [w/v] gelatin peptone, 3% [w/v] dextrose), added with 2% (w/v) agar when required. Conidia were obtained by growing the fungus on YPD, pH 4.5 plates for 6–9 days at 28°C. Then, cells were harvested by placing 5 mL of sterile Phosphate Buffered Saline (PBS) and gently scraping the plate surface with a spreader. Yeast cells were obtained by growing 1 × 10⁶ conidia/mL in 20 mL YPD, pH 7.8, and incubating 18 h at 37°C and shaking (120 rpm). An aliquot of 10 mL was then inoculated in 40 mL of YPD, pH 7.8, and incubated for 4–7 days at 37°C and shaking (120 rpm). Under these conditions, nearly 100% cells displayed a yeast-like morphology. Germlings were obtained incubating conidia for 11–12 h in YPD, pH 4.5 at 28°C and shaking (120 rpm). In all cases, cells were pelleted by centrifuging at 2700 × g for 10 min, and washed twice with PBS. For interaction with human cells, the fungal cell concentration was adjusted at 2 × 10⁶ cell/mL in RPMI 1640 Dutch modification (Sigma) supplemented with 2 mM L-glutamine (Sigma), 100 μM pyruvate (Sigma), and 50 μg/mL gentamicin (Sigma). Cells were immediately used for cytokine stimulation or kept at -20°C until used. To remove cell wall O-linked glycans, cells from the three different morphologies were β-eliminated with 0.1 M NaOH as previously reported (Diaz-Jimenez et al., 2012 (link)), washed twice with sterile PBS and cell density adjusted to 2 × 10⁶ cells/mL. Under these conditions, more than 96% cells kept viability, as tested by CFU/mL before and after treatment with NaOH. Cell heat-killing was achieved by incubating at 60°C for 2 h. Under these conditions, cells lost viability but did not burst, as measured by the amount of 280nm- and 260nm-absorbing material released into the extracellular medium upon incubation (less than 3 ± 0.5% of the total absorbance obtained upon mechanical disruption). Loss of cell viability was confirmed on YPD plates incubated at 28°C for 7 days.

Reagents: Isolation Medium Buffer (225 mM sucrose, 75 mM mannitol, 1 mM EGTA, 5 mM HEPES, pH 7.4). Percoll (GE LIFESCIENCES 17-5445-02), Bovine Serum Albumin (1120180100, Merck Millipore), respiration Buffer (125 mM KCl; 0.1% BSA; 20 mM HEPES; 2 mM MgCl2; 2.5 mM KH2PO4), Pyruvate (P2256, Sigma Aldrich), BCA Protein Assay Kit (23227, Thermo Fisher Scientific), Malate (M6413, Sigma Aldrich), CM-H2DCFDA (C6827, Thermo Fisher Scientific), ATP determination kit (A22066, Invitrogen), CaCl2 (7521789, Merck), Curcumin (C7727, Sigma Aldrich), MitoQ (Mitoquinol-Mesylate, 01ATP04C-02-13, MitoQ Ltd).
Animals: C57BL/6 mice male and female from 3, 6, 12 and 18mo were handled according to the guidelines of the National Institute of Health (NIH, Baltimore, MD). Animals were housed in cages at controlled temperature (24 °C), in a 12-h light/dark cycle with food and water ad-libitum. Experimental procedures were approved by the Bioethical and Biosafety Committee of the University San Sebastian, Chile. After the behavior test, the animals were anesthetized with isoflurane and killed by decapitation. Then, the hippocampus was removed for biochemical analysis. For the first part of this study, to determine the age-related cognitive and mitochondrial differences, each group was formed by an n = 8 animals. In the second part of the study, the control, MitoQ and Curcumin groups were formed by n = 6 different animals to perform the cognitive and biochemical assays.
Mice treatment: Mice of 3, 12 and 18mo were subjected to MitoQ or Curcumin treatment by 5 weeks. Control group was injected with saline solution and controlled water volume. Curcumin was injected intraperitoneally (25 mg/kg) 3-times/week. MitoQ was administrated in a 250 μM water solution. These doses were used because the oral administration of 250 μM MitoQ in drinking water has been demonstrated to be safe, tolerable and beneficial to aged mice, without secondary effects, after 4 weeks of administration [38 (link)]; whereas i.p. injection of Curcumin (Cc) is one of the most common methods of administration in mice for several weeks, where 25 mg/Kg showed positive results in diverse mouse pathological models [39 (link),40 (link)]. MitoQ drinking water was measured (Supplementary Figure 2). The MitoQ consumption was 1.520 ± 0.3598, 1.787 ± 0.3891 and 1.743 ± 0.6061 mol/MitoQ/day/mouse by the group of 3, 12 and 18mo respectively. We not registered the body weight during both treatments, due to no apparent differences were observed with a naked eye. This observation is consistent with previous reports that indicate that neither MitoQ nor Curcumin generates changes in body mass and weight [[41] (link), [42] (link), [43] , [44] (link)].
Behavioral test: All behavioral tests were monitored by Any-MAZE Behavioral software (Stoelting Co), using the chambers and instruments manufactured or recommended by the manufacturer. All behavioral tests were performed in the 12 h light phase of the animals light/dark cycle.
Novel object localization (NOL) test: NOL test was performed in a 40 × 40 × 32 cm box [45 (link)], chamber provided by Stoelting Co. The software register both the head and the body of the animal. The animals were exposed to a habituation phase without objects for one day. The next day, for testing each animal was exposed to 2 identical objects for 10 min. 2 h later, the animal was exposed to an old and a new object localization. Recognition index was calculated dividing the time that the animals spend exploring the new localization by the time exploring both localizations. After each test, the box chamber was cleaned with ethanol previous to a different mouse is tested.
Novel object recognition (NOR) test: 2 h later NOL test, the animals were exposed to an old and a new object. Recognition index was calculated dividing the time that the animals spend exploring the new object by the time exploring both objects. After each test, the box chamber was cleaned with ethanol previous to a different mouse is tested.
Barnes Maze (BM) test: The mice were accustomed to a circular platform containing 20 holes where one of them is the escape chamber [46 (link)]. Four visual signals were placed around the platform. The mice were exposed to a habituation phase followed by 2 days of training, in presence of white noise. The animals learn the location of a dark escape chamber under the platform. 48 h after, the time to find the escape chamber was evaluated. After each test, the chamber was cleaned with ethanol previous to a different mouse is tested.
Morris Water Maze (MWM) test: The MWM task was performed as previously described [47 (link)]. The mice were trained in a circular pool (24 °C). Each animal was trained for the location of the platform. Test was performed for 10 consecutive days, with 3 trials per day, with exception of the days 6 and 7 (training off). A submerged 9 cm platform was used, with a maximum trial duration of 60 s, where each mouse was introduced in the pool from the opposite quadrant of the platform. The test was performed with 3 trials per day and the escape latency was measured. 24 h after training, the platform was removed, and we evaluate the time in which each animal remained in the platform area for 1 min.
Extraction of an enriched fraction of hippocampal synaptosomes (containing synaptic mitochondria) and non-synaptic mitochondria. Mitochondrial populations were obtained using a Percoll gradient [28 (link)]. The hippocampus (both hemispheres) was homogenized in Isolation Medium and centrifuged at 1300 g for 3 min (4 °C). The pellet was homogenized in Isolation Medium and centrifuged at 1300 g for 3 min (4 °C). The supernatants were centrifuged at 21200 g for 10 min (4 °C). To separate synaptosomes and non-synaptic mitochondria, a Percoll gradient was used (15%–24% - 40%) and centrifuged at 30700 g for 8 min (4 °C). Synaptosomes (containing synaptic mitochondria) were obtained between the 15% and 24% phase, while non-synaptic mitochondria between 24% and 40% phase of the gradient. Both fractions were suspended in Isolation Medium and centrifuged at 16700 g for 10 min (4 °C). BSA (10 mg/ml) in isolation Medium was added to the pellet and was centrifuged at 6900 g for 10 min (4 °C). Finally, the mitochondria were suspended in Respiration Buffer.
Measurement of mitochondrial ROS: ROS production was measured using 25 μM DCF (485 nm, 530 nm) [48 (link)], in the Biotek Synergy HT plate reader. 25 μg of mitochondrial protein were added to respiration buffer containing Pyruvate (5 mM) and Malate (2.5 mM) and incubated at 37 °C for 30 min. The maximum fluorescence of each sample minus the blank sample (in the absence of mitochondrial proteins) was analyzed.
Measurement of ATP concentration: ATP was measured in the supernatant of 25 μg of mitochondria after incubation with oxidative substrates, using an ATP bioluminescence assay kit, as previously described [49 (link)].
Measurement of the calcium response: The mitochondrial response to calcium was measured by absorbance to 540 nm (30 °C) [50 (link)] during 3 min (basal), then we added 20 μM CaCl2 and evaluated the response during 15 min. Finally, we added 200 μM CaCl2 and measured for 15 min to evaluate mitochondrial swelling.
Transmission Electron Microscopy (TEM). Hippocampal samples were used according to standard procedures of the Electron Microscope Facility of the Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile. For the analysis of mitochondrial membrane integrity, we consider one intact mitochondria when these mitochondria present an intact double-membrane across their whole perimeter. We count the number of total intact mitochondria per each image obtained (26,1 μm²), in a total of 35 images per each experimental group, and then we graph the mean ± standard error.
Statistical Analysis. The data were presented as graphs indicating the mean ± standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni's post-test. p-values ≤ 0.05 were considered statistically significant. In the figures, p-values between 0.01 and 0.05 are marked with one significance mark (* or #), p-values between 0.001 and 0.01 with two significance markers (** or ##) and p-values less than 0.001 are shown with three significance markers (*** or ###). * indicates significant differences with the 3mo control group. # indicates significant differences between control and treated-mice of the same age. All statistical analyses were performed using Prism software (GraphPad Software, Inc.). Pearson's correlation analysis was used to examine the relationship between ATP or ROS produced by synaptic mitochondria and recognition index of NOR test or escape latency of the Morris Water Maze in the day 10.