All experiments were carried out in accordance with the guidelines established by the European Communities Council (Directive 114 2010/63/EU of September 22nd, 2010) and with Italian DL n.26/2014 and were approved by the local Ethical Committee and by the Italian Ministry of Health (Authorization No.97/2017-PR). The study included thirty 118/127-days-old mice (Jackson Laboratories, Bar Harbor, ME, USA): 15 B6SJL-Tg (SOD1*G93A)1Gur mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1G93A) and 15 background-matched B6SJL wildtype mice, considered as “control group” . All transgenic mice were bred in the animal facility of the Pharmacology and Toxicology Unit, Department of Pharmacy, University of Genoa (Italy) and identified by analyzing tissue extracts from tail tips, as previously described . SOD1G93A mice were studied at a late phase of disease and synchronized by motor impairment determination, defined by our validated method . Age matched wildtype mice were used as controls. Both female and male mice were used in the experiments; sexes were balanced in each experimental group to avoid bias due to sex-related intrinsic differences. Animals were housed at constant temperature (22±1° C) and relative humidity (50%) with a regular 12 h light cycle (light 7 AM–7 PM). Food (type 4RF21 standard diet; Mucedola, Settimo Milanese, Italy) and water were freely available. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable results. All mice were euthanized by cervical dislocation.
Experimental micro-PET imaging
Sample size of mice submitted to micro-PET imaging was defined based on the hypothesis of a 40% increase of skeletal muscle FDG uptake induced by ALS, coupled with an expected variation coefficient of tracer retention in this same tissue approaching 16%. These features were entered into a freely available calculator (http://www.rad.jhmi.edu/jeng/javarad/samplesize/#references) that estimated sample size to five mice per group as proposed by Eng et al. . All animals were fasted for six hours, weighted and anesthetized with intra-peritoneal ketamine (100 mg/Kg) and xylazine (10 mg/kg). Serum glucose level was tested and FDG (3-4 MBq) was injected through a tail vein. Forty minutes later, mice underwent a 10-minutes static acquisition in a dedicated micro-PET system (Albira, Bruker, USA) whose dual ring configuration allows the acquisition of the whole mouse body. After its completion mice were immediately euthanized by cervical dislocation.
Ex vivo extraction fraction of FDG and glucose
Three mice per group were sacrificed, quadriceps and hearts were harvested to analyze ex vivo FDG uptake and glucose consumption. Quadriceps muscle side was randomly selected. Ex vivo FDG uptake of quadriceps and hearts was evaluated using the Ligand-Tracer White® instrument (Ridgeview, Uppsala, SE) according to our previously validated procedure [12, 14, 19]. Briefly, the device consists of a beta-emission detector and a rotating platform harboring a standard Petri dish. The rotation axis is inclined at 30° from the vertical, so that the organ alternates its position from the nadir (for incubation) to the zenith (for counting) every minute. Slices (300 µm thick) of quadriceps or hearts were stuck in the outer ring of a Petri dish with octyl-cyanoacrylate (Dermabond, Ethicon, US) and covered with 3 mL solution collected from an input vial containing 4 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) containing glucose and FDG at the concentration of 5.5 mM (1 g/L) and 2 MBq/mL, respectively. Time-activity curves were thus obtained by subtracting decay-corrected background counting rate from the corresponding target value, as previously described [12, 14, 19-20]. After 40 min, an aliquot of 0.5 mL was sampled both from the input vial and from the Petri dish (output) to measure the radioactivity concentration using a dose calibrator with an activity resolution <10 KBq (Capintec CRC55).
Fractional FDG uptake was thus calculated as:
[Please see the supplementary files section to view the equation.]
where Ainput and Aoutput represent activity (MBq) in the surnatant before and after exposure to the muscle specimen, respectively.
After the measurement of FDG radioactivity concentration, the medium aliquots were appropriately stored for glucose assay. Glucose consumption was measured as:
[Please see the supplementary files section to view the equation.]
where Cinput and Coutput represent the corresponding mM concentrations in the surnatant before and after exposure to the muscle specimen, respectively. Medium glucose concentration was assayed following the reduction of NADP, at 340 nm, using the following solutions: 100 mM Tris-HCl pH 7.4, 2 mM MgCl2, 2 mM ATP, 4 mg HK/G6PD (Sigma-Aldrich).
Sample Preparation for Biochemical Analyses
Three dedicated quadriceps and three hearts of each group were homogenized with a Potter-Elvehjem in 1 mL of homogenization buffer (0.25 M sucrose, 0.15 M KCl, 1 mM EDTA, 10 Mm Tris-HCl pH 7.4). An aliquot of obtained homogenate was immediately frozen at -80°C whilehe remaining sample was immediately used for mitochondria isolation. Briefly, the homogenate was centrifuged at 800 x g for 10 min to precipitate nuclei and cellular debris. The supernatant collected was centrifuged for 15 min at 12,000 x g. The pellet obtained, containing mitochondria, was resuspended in a solution buffer (0.225 M sucrose, 0.075 M mannitol, 10 mM Tris-HCl pH 7.4, 1 mM EDTA), centrifuged for 10 min at 1,000 x g and washed by centrifugation at 12,000 g for 15 min . Protein concentration was tested by Bradford analysis .
Oxygen consumption and ATP synthesis assay in isolated mitochondria
Oxygen consumption was measured in fresh mitochondria by means of an amperometric oxygen electrode (Microrespiration, Unisense A/S, Århus, Denmark) in a closed chamber (volume 1.7 ml) magnetically stirred, at room temperature. For each fresh sample, 25 µg of proteins were incubated in the respiration buffer composed of 120 mM KCl, 2 mM MgCl2, 1 mM KH2PO4, 50 mM Tris HCl, pH 7.4, and 25 μg/ml ampicillin. The following substrates were used: 5 mM pyruvate + 2.5 mM malate to stimulate complexes I – III – IV .
A luciferin/luciferase chemiluminescence method was used to measure ATP synthase in fresh mitochondria. For each sample, 25 µg of proteins were incubated for 10 min at 37° C in a medium that contained: 10 mM Tris/HCl (pH 7.4), 100 mM KCl, 1 mM EGTA, 2.5 mM EDTA, 5 mM MgCl2, 5 mM KH2PO4, 0.6 mM Ouabain, and Ampicillin (25 μg/ml). The same substrates described for the oxymetric analysis were used. After 10 min of incubation, 0.1 mM ADP was added, to induce ATP synthesis, which was measured by means of a luminometer (Glomax 20/20 Luminometer, Promega Italia, Milano, Italy). ATP standard solutions in the concentration range 10-9–10-5 M were used for calibration .
Enzymatic assays were performed using frozen homogenates of three quadriceps and three hearts of each group. Enzymatic assays were performed in a double beam spectrophotometer (UNICAM UV2, Analytical S.n.c., Italy) [10-14].
Hexokinase (HK), hexose-6-phostate dehydrogenase (H6PD) and glucose-6-phosphate dehydrogenase (G6PD) activities were assayed following reduction of NADP, at 340 nm. Phosphofructokinase (PFK) activity was assayed following oxidation of NADH, at 340 nm.
The following assays solutions were used: HK: Tris-HCl-pH 7.4-100 mM (TRIS7.4), MgCl2 2mM, glucose 200mM, ATP 1mM, NADP 0.5mM, 2 UI G6PD (Sigma-Aldrich); H6PD: TRIS7.4, 2DG6P 10 mM, NADP 0.5 mM; G6PD: TRIS7.4, G6P 10 mM, NADP 0.5 mM; PFK: Tris-HCl pH 8 100 mM, MgCl2 2 mM, KCl 5 mM, fruttose-6-phosphate 2 mM, ATP 1 mM, phosphoenolpyruvate (PEP) 0.5 mM, NADH 0.2 mM, 2 IU pyruvate kinase (PK)/ 4IU lactate dehydrogenase (LDH) (Sigma-Aldrich).
Glutathione Reductase (GR) activity and NADPH/NADP ratio were evaluated spectrophotometrically, at 405 and 450 nm respectively, using GR Assay Kit (Abcam: ab83461) and NADP-NADPH Assay Kit (Abcam: ab65349), following the manufacturer’s instructions.
To assess lipid peroxidation, malondialdehyde (MDA) levels were evaluated, by the thiobarbituric acid reactive substances (TBARS) assay, with minor modifications [13, 25]. The activity assay of the Complex I was measured on 50μg of total protein as previously described [10, 12, 26].
Western blot analysis
Western blot (WB) experiments were performed according to the standard procedure. Frozen homogenates were prepared from the quadriceps of three mice per group and were sonicated twice for 10 sec in ice, with a break of 30 sec. Denaturing electrophoresis (SDS-PAGE) was performed using Mini PROTEAN TGX Precast gels (Bio Rad). For each sample, 25 µg of total protein were loaded. Run was performed at 4 °C, at 70 mA for each gel, for 30-40 min. Running buffer contained 0.05 M Tris (pH: 8.0), 0.4 M glycine, 1.8 mM EDTA, and 0.1% SDS. Electrophoretically separated samples were transferred onto nitrocellulose (NC) membranes by electroblotting, at 400 mA for 1 h in Tris– glycine buffer (50 mM Tris and 380 mM glycine) plus 20% (v/v) methanol, at 4 °C. NC membranes were incubated with the specific antibodies diluted in Tris Buffer Saline + 0.15% tween (TBST). We tested the following antibodies: anti-Mitofusin 2 (Mfn2, ThermoFisher: PA5 72811, 1:1000), anti- Dynamin-1-like protein (Drp1, ThermoFisher: PA5 34768, 1:2000), anti-Calnexin (ThermoFisher: MA3-027, 1:500) and anti-GAPDH (Cell Signaling: #5174, 1:1000). After extensive washing with 0.15% TBST, 1 h of incubation with secondary horseradish peroxidase-conjugated Abs were diluted in TBST (1:10,000 for anti-mouse and anti-rabbit antibodies) was performed.
Bioimaging by confocal microscopy
The experiments were performed on dedicated fresh skeletal muscle sections from three SOD1G93A and three control mice, promptly after excision. The sections were gently stretched to reduce thickness without affecting viability of tissue cells (verified by exclusion of the non-permeable dye DAPI in parallel samples) and immediately incubated at 37°C for 10 minutes with the fluorescent probes 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) (50 microM ) and ER-TrackerTM Red (1 µM), both from Molecular Probes (InVitrogen, Eugene, OR). Nuclei were stained with the cell-permeable dye Hoechst 33342 (10 µM). Images were obtained using the SP2-AOBS confocal microscope (Leica Microsystems, Mannheim, Germany).
Immunofluorescence and immunohistochemical analysis
Soon after sacrifice, 3 half-hearts and 3 quadriceps were harvested from controls and from SOD1G93A mice, embedded in OCT and snap-frozen in precooled isopentane. Six µm-thick serial cryostat sections were obtained.
For the immunofluorescence analysis: immediately after cutting the sections were fixed for 10 min in cold acetone, followed by incubation with MitoTracker® Red (Molecular Probes), at 10 nM for 20 min at 37°C. For reactive oxygen species (ROS) staining, serial sections from the same samples were fixed for 10 min in acetone, followed by incubation with 10 µM 2’,7’-dichlorofluorescein diacetate (H2DCFDA; Molecular Probes) for 30 min and a wash in PBS.
For immunohistochemical staining of skeletal muscles infiltrated macrophages the following antibodies were used: rat anti-mouse CD206 (AbD Serotec, 1:200, 5mg/ml) to detect macrophages M2; rat anti-mouse CD86 clone PO.3 (Millipore, 1:100, 5mg/ml) to detect macrophages M1; rat anti-mouse CD11b (Novus Biologicals, 1:200, 5mg/ml).
Three quadriceps and three hearts per group were dissected and fixed in 0.1M cacodylate buffer containing 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield, PA, USA), for 2 h at room temperature. Samples were post fixed in osmium tetroxide for 2 h and 1% uranyl acetate for 2 h. Samples were next dehydrated through a graded ethanol series and propylene oxide and embedded in epoxy resin (Poly-Bed; Polysciences, Inc., Warrington, PA, USA) overnight at 42°C and 2 days at 60°C. Ultrathin sections (50 nm) were counterstained with 5% uranyl acetate and lead citrate and observed with transmission electron microscope (TEM) HT7880, Hitachi, Japan.
Micro-PET images were reconstructed using a Maximum-Likelihood Expectation-Maximization (MLEM) algorithm and were qualitatively inspected. Images were analyzed using the routine of a commercial software (PMOD, Zurich, CH). Two nuclear medicine physicians, unaware of model nature (transgenic or wildtype), drew volumes of interest (VOIs) on the left and right hindlimb skeletal muscles and on the metabolically active left ventricular myocardium to measure the mean standardized uptake value (SUV) according to the formula:
[Please see the supplementary files section to view the equation.]
Where [FDG] indicates average FDG concentration in kBq/ml within any given VOI, body weight is expressed in kilograms and injected activity in MBq. Skeletal muscle and myocardial FDG concentration were divided by the corresponding value in the blood pool to obtain the SUV ratio (SUVr). For WB analysis, the signal was acquired with Alliance 6.7 WL 20M (UVITEC, Cambridge, UK), and UV1D software (UVITEC). Densitometry analysis was performed using the dedicated routine of ImageJ software (ImageJ Version 2.0.0-rc-65/1.51s). For colocalization analysis, six randomly selected fields were analyzed in three independent samples harvested from either SOD1G93A and control mice. Original unadjusted and uncorrected images were processed using ImageJ software for the evaluation of colocalization, which was expressed as the percentage of above-background pixels in 2-NBDG images that overlapped above-background pixels in ER images, with background threshold set by the Costes’ method .
For immunofluorescence analysis, images were acquired by the Fluoview FV500 software and the fluorescence was quantified using the same ImageJ software. Immunohistochemical images were acquired with Leica DM RX microscopy and were analyzed using Scion Image software .
Finally, for ultrastructural analysis, digital images were acquired with Megaview 3 camera (EMSIS GmbH, Germany).
Data are presented as mean ± standard deviation. Two-tailed student t test for unpaired data, was used. Levene’s test for equality of variances was used to test the homogeneity of variances of the two groups. When homogeneity wasn’t satisfied the Welch-Satterthwaite method was used to adjust the p-value for this assumption violation. Statistical significance was considered for p < 0.05. In all analyses, p value was complemented by the evaluation of standardized difference between the two means estimated by Cohen's d index. A complete description of these evaluations is reported in Supplementary Table 1. Statistical analyses were performed using SPSS software 26.0 (Chicago, IL, USA).