Ethical use of animals

All animal procedures we performed according to the European Union Directive 86/609/EEC and Recommendation 2007/526/EC, regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish legislation under the directive RD1201/2005. All protocols were approved by the Bioethics Committee of the University of Salamanca and by the Junta de Castilla y Leon (registration number 080).

PFKFB3 Enzymatic assay

Recombinant full length human PFKFB3 protein purified from Sf9 baculoviral system acquired from SignalChem (Cat. #P323-30G). ATP, fructose-6-phosphate (F6P and other chemicals were from Sigma-Aldrich. ADP detection system (ADP-Glo) was purchased from Promega. Inhibitors were synthesized as described by Boyd et al.18. The kinase activity of the PFKFB3 protein was detected by measuring production of ADP from ATP in the presence of F6P. The reactions were assembled in 384 well plates in a total volume of 25 µl. Test compounds were serially diluted in dimethyl sulfoxide (DMSO). Reactions were set up by mixing test compounds with the enzyme and pre-incubating for 15 min. ATP and F6P were next added to initiate the reactions. The final assay composition included: 100 mM Tris-HCl pH 8.0, 4 mM MgCl 2 , 5 mM KH 2 PO 4 , 5 mM DTT, 20 mM KF, 0.02% BSA, 1% DMSO (from the compounds), 15 nM enzyme, 20 µM ATP (Km = 16 µM) and 10 µM F6P (Km = 6 µM). We used a low concentration of PFKFB3 protein (15 nM) since concentrations higher than 50 nM showed to hydrolyze ATP in the absence of F6P. Thus, under the conditions used in our experiments, ADP formation was solely due to F6P phosphorylation by PFKFB3. The kinase reactions were allowed to proceed for 1 hour at room temperature. Aliquots of the reaction mixtures (5 µl) were transferred to fresh white 384 well plates and mixed with 5 µl of the ADP-Glo reagent, followed by incubation for 30 min. The luminescent kinase detection reagent was added (10 µl) and, following additional incubation for 15 min, the plates were read with a luminescence plate reader (Analyst HT). Positive (no enzyme; 100% inhibition) and negative (DMSO instead of AZ67 or PFK158; 0% inhibition) control samples were assembled in each assay plate and were used to calculate percent inhibition values of test compounds.

Fructose-2,6-bisphosphate determinations

For F2,6BP determinations, cells were lysed in 0.1 N NaOH and centrifuged (20,000 × g, 20 min). An aliquot of the homogenate was used for protein determination, and the remaining sample was heated at 80 °C (5 min), centrifuged (20.000 × g, 20 min) and the resulting supernatant used for the determination of F2,6BP concentrations using the coupled enzymatic reaction and F2,6BP standards as described by Van Schaftingen43.

Cell culture

Primary cultures of C57BL/6J mice cortical neurons were prepared from foetal animals of 14.5 days of gestation, seeded at 1.8·105 cells/cm2 in plastic plates coated with poly-D-lysine (10 mg/ml) and incubated in Neurobasal (Life Technologies) supplemented with 2 mM glutamine, 5 mM of glucose, 0.25 mM pyruvate and 2% B27 supplement (Life Technologies). Cells were incubated at 37 °C in a humidified 5% CO 2 -containing atmosphere. At 72 hours after plating, medium was replaced using Neurobasal (Life Technologies) supplemented with 2 mM glutamine, 5 mM glucose, 0.25 mM pyruvate and 2% B27 supplement (Life Technologies) minus antioxidants (MAO; i.e., lacking vitamin E, vitamin E acetate, superoxide dismutase, catalase and glutathione). Six days after plating medium was replaced again. Cells were used at day 9. Primary cultures of brain cortical astrocytes were prepared from either C57BL/6 mice of 0–1 days-old neonates. Cell suspensions were seeded at 2.5 × 105 cells in 175 cm2 plastic flasks and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (BSA). Non-astroglial cells were detached after 7 DIV by shaking the flasks at 200 r.p.m. overnight and discarding the supernatant. The remaining attached, astrocyte-enriched cells were re-seeded in different size plastic plates and further incubated for 7 DIV for the experiments. Adenocarcinomic human alveolar basal epithelial cells (A549 cells) were seeded at 104 cells/cm2 in Dulbecco’s modified Earls Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS).

Cell transfections

Primary neurons were transfected with 1.6 µg/mL of a pIRES2-EGFP plasmid vector (Invitrogen) harbouring the full-length cDNA coding for the human muscle 6-phosphofructo-1-kinase muscle isoform (PFK1-M)28 (accession number, NM_000289.1) using Lipofectamine LTX-PLUS Reagent (Life Technologies) according with manufacturer’s protocol. Transfections were performed 24 hours before cells collection. Control cells were transfected with the empty vector.

Cell treatments

For NMDA receptors activation, neurons at 8 days in vitro were incubated with 100 μM glutamate (plus 10 μM glycine) or 100 μM NMDA (plus 10 μM glycine) for 10 minutes. Neurons were then washed and further incubated in culture medium with the PFKFB3 inhibitors for 24 hours. For amyloid-ß treatment, the active truncated amyloid-β peptide Aβ 25–35 (BioNova Cientifica S.L., Madrid, Spain) was used. Aß 25–35 was dissolved in distilled water at a concentration of 1 mg/ml and then incubated at 37 °C for 3 days to induce its oligomerization44. We have shown that Aß 25–35 exerts neurotoxicity following identical mechanism to the full-length Aβ 1–42 peptide21. Neurons were incubated in culture medium containing oligomerized Aβ 25–35 (10 µM) or the corresponding scramble non-aggregable peptide (Aβ 35–25 ) (BioNova Cientifica S.L.), which was used as control. Neurons at 8 days in vitro were incubated with Aß 25–35 plus the PFKFB3 inhibitors for 24 hours. To inhibit the proteasome, neurons were incubated with MG132 (10 µM) for 2 hours. To inhibit cytochrome c oxidase activity, astrocytes at 14 days in vitro were incubated with the nitric oxide donor, DETA-NONOate (0.5 mM)4 for 4 hours.

Oxygen and glucose deprivation (OGD)/reoxygenation protocol

After 8 days in culture, neurons were subjected to oxygen and glucose deprivation (OGD) by incubating cells at 37 °C in an incubator equipped with an air lock and continuously gassed with 95% N 2 /5% CO 2 , for 3 hours. The incubation medium (Neurobasal medium without glucose) was previously gassed with 95% N 2 /5% CO 2 for 5 min. In parallel, neurons were incubated in Neurobasal complete medium (normoxia condition) at 37 °C in a humidified atmosphere of 95% air/5% CO 2 . After OGD, neurons were further incubated for 4 hours with (or not) AZ67 10 nM in Neurobasal medium at 37 °C in a humidified atmosphere of 95% air/5% CO 2 (reoxygenation after OGD)31.

Western blotting

Neurons were lysed in RIPA buffer (2% sodium dodecylsulphate, 2 mM EDTA, 2 mM EGTA and 50 mM Tris pH 7.5), supplemented with protease and phosphatase inhibitor cocktail (100 μM phenylmethylsulfonyl fluoride, 50 μg/ml antipapain, 50 μg/ml pepstatin, 50 μg/ml amastatin, 50 μg/ml leupeptin, 50 μg/ml bestatin, 1 mM o-vanadate, 50 mM NaF, and 50 μg/ml soybean trypsin inhibitor) and boiled for 5 min. Extracts were centrifuged at 13,000 × g for 5 min at 4 °C, and aliquots of lysates (50 μg protein, unless otherwise stated) were subjected to sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) electrophoresis on a 8, 10 or 12% acrylamide gel (MiniProtean, Bio-Rad) including PageRuler Plus Prestained Protein Ladder (Thermo). The resolved proteins were transferred electrophoretically to nitrocellulose membranes (Hybond-ECL, Amersham Bioscience Europe GmbH, Barcelona, Spain). Membranes were blocked with 5% (w/v) low-fat milk in 20 mM Tris, 500 mM NaCl, and 0.1% (w/v) Tween 20, pH 7.5, for 1 h. After blocking, membranes were immunoblotted with primary antibodies at dilutions ranging from 1:500 to 1:40,000 overnight at 4 °C. After incubation with the secondary antibodies (all at 1:10,000 dilution), membranes were immediately incubated with the enhanced chemiluminescence kit WesternBright ECL (Advansta, Menlo Park, California, USA) for 2 min or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Offenbach, Germany) for 5 min, before exposure to Fuji Medical X-Ray film (Fujifilm), and the autoradiograms scanned. Biologically independent replicates were always performed (Supplementary Fig. S5), and a representative western blot is shown.

Primary antibodies for western blotting

Immunoblotting was performed using mouse monoclonal anti-PFKFB3 (1:500) (H0005209-M08, Novus Biologicals), mouse monoclonal anti- glyceraldehyde dehydrogenase (GAPDH; 1:40,000) (AM4300, Ambion) and antiserum against muscle PFK1 isoform (PFK1-M; 1:10)4.

Secondary antibodies for western blotting

Immunoblotting was performed using horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Santa Cruz Biotechnologies).

Mitochondrial ROS

Mitochondrial ROS was detected using the fluorescent probe MitoSox (Life Technologies). Cells were incubated with 2 μM of MitoSox for 30 minutes at 37 °C in a 5% CO 2 atmosphere in Hank’s Balanced Salt Solution (HBSS buffer); (NaCl 134.2 mM; KCl 5.26 mM; KH 2 PO 4 0.43 mM; NaHCO 3 4.09 mM; Na 2 HPO 4 ·2H 2 O 0.33 mM; glucose 5.44 mM; HEPES 20 mM; CaCl 2 ·2H 2 O 4 mM; pH 7.4). Cells were then washed with PBS (phosphate-buffered saline, 0.1 M) and collected by smooth trypsinization. MitoSox fluorescence was assessed by flow cytometry and expressed in arbitrary units (see, also, Supplementary Fig. S2 for the flow cytometry workflow for MitoSox).

H 2 O 2 determination

For H 2 O 2 assessments, AmplexRed (Life Technologies, New York, USA) was used. Cells grown on 96 wells plates were washed with PBS and incubated in KRPG buffer (NaCl 145 mM; Na 2 HPO 4 5.7 mM; KCl 4.86 mM; CaCl 2 0.54 mM; MgSO 4 1.22 mM; glucose 5.5 mM: pH 7.35) in the presence of 9.45 μM AmplexRed containing 0.1 U/ml of horseradish peroxidase. Luminescence was recorded for 2 h at 30 minutes intervals using a Varioskan Flash (Thermo Fisher, Vantaa, Finland) spectrofluorometer (excitation 538 nm; emission 604 nm). Slopes were used for calculations of the rates of H 2 O 2 formation.

Flow cytometric analysis of apoptotic cell death

APC-conjugated annexin-V and 7-amino-actinomycin D (7-AAD) (Becton Dickinson Biosciences, BDB, San Jose, CA, USA) were used to determine quantitatively the percentage of apoptotic neurons by flow cytometry. Cells were stained with annexin V-APC and 7-AAD, following the manufacturer’s instructions, and were analysed on a FACScalibur flow cytometer (15 mW argon ion laser tuned at 488 nm; CellQuest software, Becton Dickinson Biosciences) using the CellQuest software (BDB). Both GFP+ and GFP− cells were analyzed separately, and the annexin V-APC-stained cells that were 7-AAD-negative were considered to be apoptotic (see, also, Supplementary Fig. S3 for the flow cytometry workflow for apoptosis).

Active caspase-3 determination

A fluorimetric caspase 3 assay kit (Sigma-Aldrich) was used following the manufacture’s protocol. This assay is based on the hydrolysis of the peptide substrate Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) by caspase-3, which results in the release of fluorescent 7-amino-4-methylcoumarin (AMC). In brief, cells were lysed with 50 mM HEPES, 5 mM CHAPS, 5 mM DTT, pH 7.4 for 20 min on ice, and the assay buffer containing the Ac-DEVD-AMC substrate (20 mM HEPES, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 16 µM Ac-DEVD-AMC, pH 7.4) was added. Aliquots of 200 µl were transferred to a 96-wells plate and the fluorescence recorded for 30 mins at 5 mins intervals at 37 °C (λ exc = 360 nm, λ em = 460 nm). CSP-3 activity was determined as AMC release rate extrapolating the slopes to those obtained from an AMC standard curve. Results are expressed as fold change, arbitrarily assigning the value of 1 to control cells.

NADPH/NADP+ ratio determination

This was performed using the colorimetric NADPH/NADP assay kit (Abcam). Cells were re-suspended in 500 µl of NADPH/NADP extraction buffer, vortexed and centrifuged at 14,000 rpm for 5 minutes to remove insoluble material. The supernatant was used for NADPH plus NADP measurement. NADPH was determined in 200 µl of the supernatant, after heated at 60 °C for 30 minutes to decompose NADP. Actual NADP and NADPH concentrations were calculated by extrapolating values to a NADPH standard curve (0–100 pmol/well).

Determination of the pentose-phosphate pathway (PPP) flux

PPP flux was measured in 8 cm2 flasks of adherent cells at 60–70% confluence containing a central microcentrifuge tube with 0.8 ml benzethonium hydroxide (Sigma) for 14CO 2 equilibration. Incubations were carried out in KRPG containing 5.5 mM D-glucose at 37 °C in the air-thermostatized chamber of an orbital shaker (Forma Benchtop Orbital Shaker, Model 420, Thermo Fischer). To ensure adequate oxygen supply for oxidative metabolism throughout the incubation period, flasks were filled with oxygen before being sealed. To measure the carbon flux from glucose through the PPP, cells were incubated in KRPG (5 mM D-glucose) buffer supplemented with 0.5 μCi D-[1-14C]glucose or D-[6-14C]glucose for 90 min, as previously described7,32. Incubations were then terminated by the addition of 0.2 ml 20% perchloric acid (Merck Millipore) for 30 min before the benzethonium hydroxide (containing 14CO 2 ) was removed, and the radioactivity was measured with a liquid scintillation analyzer (Tri-Carb 4810 TR, PerkinElmer). PPP flux was calculated as the difference between 14CO 2 production from [1-14C]glucose (which decarboxylates through the 6-phosphogluconate dehydrogenase–catalyzed reaction) and that of [6-14C]glucose (which decarboxylates through the TCA cycle).

Determination of the glycolytic flux

Glycolytic flux was measured in 8 cm2 flasks of adherent cells at 60–70% confluence containing a central microcentrifuge tube with 1 ml H 2 O for 3H 2 O equilibration. Incubations were carried out in KRPG containing 5.5 mM D-glucose at 37 °C in the air-thermostatized chamber of an orbital shaker (Forma Benchtop Orbital Shaker, Model 420, Thermo Fischer). To ensure adequate oxygen supply for oxidative metabolism throughout the incubation period, flasks were filled with oxygen before being sealed. Glycolytic flux was measured by assaying the rate of 3H 2 O production from [3-3H]glucose by incubating cells with 5 μ Ci D-[3-3H]glucose in KRPG buffer per flask for 120 min, as previously described32. Incubations were then terminated with 0.2 ml 20% perchloric acid, and the cells were further incubated for 96 h with a microcentrifuge tube containing H 2 O, suspended above the cells to allow 3H 2 O equilibration. The 3H 2 O was then measured by liquid scintillation counting (Tri-Carb 4810 TR, PerkinElmer). Under these experimental conditions, 28% of the produced 3H 2 O was recovered and used for the calculations as previously established7,32.

Lactate determination

Lactate released to the culture medium was determined as an estimation of glycolysis. To do so, the increments in absorbance of the culture medium samples were measured at 340 nm in a mixture containing 1 mM NAD+ and 22.5 units·ml−1 of lactate dehydrogenase in 0.25 M glycine/0.5 M hydrazine/1 mM EDTA at pH 9.5.

Pyruvate dehydrogenase (PDH) activity

PDH was measured in 8 cm2 flasks of adherent cells at 60–70% confluence containing a central microcentrifuge tube with 0.8 ml benzethonium hydroxide (Sigma) for 14CO 2 equilibration. Incubations were carried out in KRPG containing 5.5 mM D-glucose and 1 mM pyruvate at 37 °C in the air-thermostatized chamber of an orbital shaker (Forma Benchtop Orbital Shaker, Model 420, Thermo Fischer). To ensure adequate oxygen supply for oxidative metabolism throughout the incubation period, flasks were filled with oxygen before being sealed. To measure the carbon flux from pyruvate through the tricarboxylic acid cycle (TCA), cells were incubated in KRPG (5.5 mM D-glucose and 1 mM pyruvate), buffer supplemented with 0.5 μCi D-[1-14C]pyruvate for 90 min, as previously described45. Incubations were then terminated by the addition of 0.2 ml 20% perchloric acid (Merck Millipore) for 30 min before the benzethonium hydroxide (containing 14CO 2 ) was removed, and the radioactivity was measured with a liquid scintillation analyzer (Tri-Carb 4810 TR, PerkinElmer). PDH activity was determined as the rate of [1-14C]pyruvate decarboxylation to 14CO 2 through the TCA cycle.

Mitochondrial membrane potential (Δψ m )

∆ψ m was assessed using MitoProbe DiIC 1 (5) Assay Kit for flow cytometry (Molecular Probes Europe BV, Leiden, Netherlands) on a FACScalibur flow cytometer (15 mW argon ion laser tuned at 488 nm; CellQuest software, Becton Dickinson Biosciences). Δψ m values were expressed as percentages, using carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (CCCP; 10 μM) for 15 min to define the 0% Δψ m values21 (see, also, Supplementary Fig. S4 for the flow cytometry workflow for mitochondrial membrane potential).

Transient middle cerebral artery occlusion (MCAO)

Surgical endovascular insertion of a silicon-coated monofilament (602012PK10; Doccol Corporation, Sharon, MA, USA) was performed to induce transient middle cerebral artery occlusion (MCAO) for 30 minutes of ischemia, followed by filament removal to allow reperfusion31,33. Briefly, 10-weeks-old C57BL/6J mice were anesthetized with sevoflurane (4% for induction, 3% for maintenance) in a mixture of O 2 /N 2 O (30/70%). After surgical exposure of the right carotid artery tree, the filament was inserted through the external carotid artery and advanced through the internal carotid artery until it reached the middle cerebral artery. The regional cerebral blood flow was monitored during surgery with a laser Doppler probe (Moor Instruments, Devon, UK). After 30 minutes of ischemia, the filament was removed to allow reperfusion. AZ67 (60 mg/kg of body weight) or vehicle were administered in a bolus (200 µl) via the jugular vein immediately after reperfusion. Body temperature was maintained at 37 ± 0.5 °C using a heating pad connected to a rectal probe (BAT-12 thermometer; Physitemp Instruments Inc., Clifton, NJ, USA). Mice were then sutured and returned to the cages. Sham-operated mice underwent the same surgical procedure without middle cerebral artery occlusion.

Rotarod analysis

An accelerating rotarod test was used to determine motor coordination. Animals were trained during the immediate three previous days of the MCAO surgery. The first day, mice stayed on the rotating rod at a constant speed of 4 rpm, and the remaining 2nd and 3rd day they stayed at an accelerating speed (4 to 40 rpm in 5 mins). For the test, which was performed 24 hours after the MCAO surgery, mice were subjected to three consecutive trials at the accelerating speed for 5 mins (at 15 mins intervals). The latency to fall was determined and expressed in seconds.

Neurological severity score (NSS)

For the NSS test, mice were examined to assess the neurological status using a 0–5 grading scale as described34. Mice treated with the vehicle (DMSO) were scored 0, while dead mice were scored 5. The rest of the animals were examined and assigned a score for each of the following five items, following the test description34, namely (i) spontaneous activity, (ii) spontaneous rightward rotation, (iii) rightward rotation after grabbing the animal by the tail with both forelimbs placed at a platform, (iv) left forepaw extension deficit after grabbing the animal by the tail and (v) moving it closer to the platform.

Infarct volume

Immediately after the rota-rod test, mice were euthanized by cervical dislocation after CO 2 overdose, and the brain extracted and sliced in 2-mm coronal sections with a brain matrix on ice, which were used to determine the infarct volume after incubation of the slices in 2% (wt/vol) 2,3,5-triphenyltetrazolium chloride in phosphate-buffered saline (136 mM NaCl, 27 mM KCl, 7.8 mM Na 2 HPO 4 , 1.7 mM KH 2 PO 4 , pH 7.4) for 20 minutes at room temperature. Pictures of the brain sections were taken, and the images processed using the NIH image-processing package ImageJ 1.43n. Infarct volumes were determined by multiplying the selected infarcted area by the width of the slices. In order to correct the infarct volume by the edema, the ratio lesion volume of the ipsilateral (affected) versus that of the contralateral (unaffected) hemispheres was calculated. The percentage of infarct volume was calculated using the following formula: (infarcted volume corrected by edema × 100)/Infarcted hemisphere volume.

Statistical analysis

Results from cultured cells were obtained from 3 independent culture preparations using 4–6 technical replicates per sample. Data were expressed as mean ± standard error of the mean (SEM) values, using as “n” the number of independent culture preparations. Statistical analysis of the results was performed by one-way or two-way analysis of variance (ANOVA), followed by the least significant difference multiple range test. In all cases, p < 0.05 was considered significant. Statistics were performed using Microsoft Excel or the IBM SPSS Statistics software.