All chemicals were purchased from Sigma-Aldrich (St Louis, MO) unless otherwise stated.
Double transgenic mice expressing a chimeric mouse/human amyloid precursor protein (APP) with the Swedish mutation (APPswe) and a mutant human presenilin 1 (PS1) with the delta E9 (PS1ΔE9) (strain # 005864) and wildtype C57BL/6 mice were purchased from the Jackson Laboratory, (Bar Harbor, ME). Male mice (3–18 months) were used in this study to avoid estrogen-related confounders. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia and tissue collection.
Animals positive for the transgenes were identified by PCR using genomic DNA, isolated from the tails (Qiagen, Valencia, CA). The primer sequences for genotyping the mice were (APP) forward 5′-GACTGACCACTCGACCAGGTTCTG-3′, and reverse 5′-CTTGTAAGTTGGATTCTCATATCCG-3′ to amplify a 350 bp fragment; (PS1) forward 5′-AATAGAGAACGGCAGGAGCA-3′ and reverse 5′-GCCATGAGGGCACTAATCAT-3′ to amplify a 608 bp fragment. One hundred nanograms of genomic DNA were used in the PCRs, with a program of one cycle of 95°C for 3 min, 33 cycles of 95°C for 45 s, 62°C for 45 s and 72°C for 45 s, and one cycle of 72°C for 5 min. The PCR products were separated on a 1% agarose gel, stained with ethidium bromide and imaged using a Gel Doc EZ Imager (Bio-Rad, Hercules, CA).
Isolation of non-synaptic brain mitochondria
After decapitation, forebrain was rapidly removed from APP(swe)/PS1(ΔE9) or non-transgenic male mice (3 months) and placed in ice-cold mannitol-sucrose (MS) buffer pH 7.4 (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, 1 mg/ml fatty acid free BSA (Roche Diagnostics, Indianapolis, IN), 1 mM EGTA). Forebrains were homogenized with 10 strokes using a Potter-Elvehjem tissue grinder (Wheaton Science Products, Millville, NJ). The brain homogenates were further processed using the Percoll isolation method described by  and as used previously  with slight modification. Briefly, the brain homogenate was centrifuged twice at 1,317 × g for 3 min. The collected supernatant was further centrifuged for 10 min at 21,074 × g and the resulting pellet resuspended in 15% Percoll (Amersham Biosciences, Piscataway, NJ) then layered on a discontinuous 40% and 24% Percoll gradient and spun at 29,718 × g for 8 min. The non-synaptic mitochondrial fraction was resuspended in MS buffer then centrifuged at 16,599 × g for 10 min. The mitochondrial pellet was resuspended in MS buffer containing 1 mg/ml fatty acid free BSA (Roche) then spun at 6,668 × g for 10 min. The mitochondrial pellet was resuspended in a small volume of MS buffer (minus EGTA) after removal of the supernatant following the final spin. Protein concentrations were determined by the method described by  using BSA as standards. Aliquots of brain homogenate had protease inhibitors (Calbiochem, San Diego, CA) added prior to storage at −80°C for later Western blot analyses.
Single fiber isolation
Flexor digitorum brevis (FDB) muscles were harvested bilaterally from APP(swe)/PS1(ΔE9) or non-transgenic male mice (3 months). The isolation procedure was then performed as previously described . Additionally, soleus, plantaris, gastrocnemius and tibialis anterior muscles were harvested from these same mice as well as from APP(swe)/PS1(ΔE9) or non-transgenic male mice (6 and 18 months). These muscles were snap frozen in liquid nitrogen and stored at −80°C for later Western blot analyses. The individual muscles for Western blotting were homogenized utilizing a BulletBlender (Next Advance, Averil Park, NY) according to the manufacturer’s protocol.
C2C12 cell culture conditions
Low passage C2C12 myoblasts (ATCC, Manassas, VA; 25,000/well) were seeded on V7 microplates (Seahorse Bioscience, North Billerica, MA) in proliferation media ((DMEM, (ATCC), 10% fetal bovine serum, (Gibco, Grand Island, NY), 1% Pen-Strep, (Gibco)) and maintained in a humidified incubator at 37°C and 5% CO2. After 24 hours, cultures were transiently transfected (see below). After a further 24 hours, the proliferation media was replaced with differentiation media (DM) consisting of DMEM, 2% horse serum (Gibco) and 1% Pen-Strep. The cultures had media changes using DM every other day until the myotubes covered each well in the plate. All wells were critically examined under the microscope to ensure adequate myotubes formation, with plates being used when myotubes were completely covering the plate (~ 7 days after seeding).
Plasmid vector generation and transfection
cDNA for enhanced yellow fluorescent protein possessing a mitochondrial targeting sequence (mEYFP)  was inserted into a pTRE-Tight-BI plasmid vector (Clontech, Mountain View, CA). Downstream of the mEYFP a 2A DNA sequence  and mutant PS1(ΔE9) were inserted. Mutant APP(swe) was inserted into the vector in the opposite direction. (APP(swe)/PS1(ΔE9) cDNA kind gift from Dr. David Borchelt). This construct possesses a tetracycline response element thus requiring co-transfection with a tetracycline transactivator (TTA, Clontech) giving rise to cells possessing EYFP targeted to mitochondria and transgene-derived APP and PS1. C2C12 myoblasts were co-transfected with both constructs utilizing 2 μg of DNA/construct/well using lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Proteins as determined by  from mouse muscle (50 μg) and brain (20 μg) homogenates of APP(swe)/PS1(ΔE9) and their non-transgenic litter mates (3–18 months) were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% precast Mini-Protean TGX gels (Bio-Rad) and transferred to a polyvinylidene difluoride membrane using a Trans-Blot Turbo transfer system (Bio-Rad). Immunoblotting was performed according to Li-Cor Biosciences (Lincoln, NE) protocol. Briefly, nonspecific sites were blocked in non-mammalian blocking buffer (Li-Cor Biosciences). Membranes were then incubated at 4°C overnight in mouse anti-human A-beta (Aβ) monoclonal antibody (6E10, Covance) at 1:500 dilution that detects transgene-derived full-length APP at ~100 kD  and lower molecular weight amyloid peptides. After 4 × 5 min washes in tris buffered saline (TBS), the membranes were incubated with anti mouse secondary antibody conjugated to IR green at 1:5,000 dilution (Li-Cor Biosciences). The infrared signal was captured on an Odyssey infrared imaging system (Li-Cor Biosciences) and stored as a digital image. The membranes were then stripped with stripping buffer and reprobed with rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA) monoclonal antibody at 1:20,000 dilution (muscle) and anti-rabbit secondary antibody conjugated to IR red at 1:50,000 dilution (muscle); or rabbit anti β-Actin (Cell Signaling Technology) monoclonal antibody at 1:1,000 dilution (brain) and anti-rabbit secondary antibody conjugated to IR red at 1:20,000 dilution (brain) to ensure equal loading.
C2C12 myoblasts were plated on poly-L-lysine coated cover slips in 24-well culture plates (Corning, Lowell, MA) in proliferation media (see above) and maintained in a humidified incubator at 37°C and 5% CO2. After 24 hours, cultures were transiently transfected as described above. The following day, cells were incubated for 30 min with MitoTracker Red CMXRos (Molecular Probes, 200 nM) a cell-permeant probe that will accumulate in active mitochondria. Cells were fixed in 4% paraformaldehyde for 5 min, washed in PBS and mounted on slides. A separate group of transfected myoblasts plated on coverslips were fixed as above then incubated in rabbit anti-APP polyclonal antibody (Cell Signaling Technology) at 1:50 dilution and anti-rabbit conjugated to Alexa Fluor 594 secondary antibody (Invitrogen) at 1:500 dilution according to the manufacturer’s protocol. Cell fluorescence was captured utilizing an AxioCam MRM monochrome charge-coupled device (CCD) camera and detected with a Zeiss Axio Observer Z1 with ApoTome (Zeiss, Thornwood, NY) using Axiovision software (Zeiss).
Brain Mitochondrial respirometry
Following calibration of the Seahorse XF24-3 flux analyzer (Seahorse Bioscience), the final non-synaptic mitochondrial pellets from individual mouse brains were resuspended in MAS1 buffer  pH 7.2 (70 mM sucrose, 220 mM D-mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM Hepes, 1 mM EGTA, 0.2% fatty acid free BSA (Roche)) and 5 μg protein as determined above  loaded into each of 20 wells of an XF24 V7 cell culture plate (Seahorse Bioscience). The plates were centrifuged at 1,600 × g at 4°C for 5 min. MAS1 buffer with 5 mM L-malate plus 5 mM sodium pyruvate (freshly made in MAS1 buffer) was gently added to the wells and the plates immediately loaded onto the instrument and oxygen consumption measurements were recorded. The experimental measurements consisted of cycle 1 (1 min mix, 1.5 min wait, 0.5 min mix, 2 min measure, 1 min mix) prior to injection of ADP (4 mM). Then cycle 2 (0.5 min mix, 2 min measure, 1 min mix) followed by injection of oligomycin (2.5 μg/ml). Following oligomycin addition, cycle 2 was repeated then carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 4 μM) was injected. A final 0.5 min mix and 2 min measure was performed prior to termination of the experiment. Reagent concentrations were chosen based on previous experiments utilizing isolated mitochondria measured with the Seahorse XF24 . All measurements were performed at 37°C.
Individual, intact muscle fiber respirometry
Bioenergetic analyses were performed utilizing an XF24-3 Seahorse Extracellular Flux Analyzer (Seahorse BioScience). The procedure was performed as previously described  with modifications. Specifically, prior to measurements, pre-warmed (37°C) assay measurement buffer (MB) consisting of 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES (pH 7.4) supplemented with 2.5 mM D-glucose and 0.5 mM L-carnitine was gently added to the fibers. The fibers were then placed in an unbuffered, humidified incubator at 37°C for 2 hours to allow temperature and pH equilibration. Fibers were visually inspected prior to and after MB addition then loaded onto the instrument. After an equilibration step, basal oxygen consumption rates (OCR, pMoles/min) were recorded using 3-min mix, 2-min wait, and 3-min measure (looped 3 times) cycles prior to injection of oligomycin to inhibit the ATP synthase. Three more measurement loops were recorded prior to injection of substrate plus FCCP to induce maximal oxygen consumption. Following recording of 3 more measurement loops, antimycin A (inhibitor of mitochondrial respiration) was injected to assess non-mitochondrial OCR. Two measurement loops were recorded after antimycin A injection then the experiment was terminated. The injectates prepared in MB (75 μl volumes) were preloaded, then sequentially injected as indicated through ports in the XF24 calibration cartridge to final concentrations of 1 μg/ml oligomycin, 400 nM FCCP + 10 mM pyruvate, and 1 μM antimycin A.
Both DNA constructs described above were injected into the right footpad of male wild type C57BL/6 mice (3 months). As a contralateral control, the left footpad received only one of the constructs. The feet were electroporated as previously described  and the FDBs harvested (as described above) one week later. Following isolation, the individual intact fibers were seeded on a V7 microplate (Seahorse Bioscience) for respirometry measurements as detailed above but without oligomycin.
C2C12 myotube respirometry
Prior to measurements, cultures were gently rinsed in pre-warmed (37°C) MB (see above) then placed in a 37°C humidified, unbuffered incubator for 2 hours to allow for temperature and pH equilibration. Myotubes were visually inspected prior to and after MB addition then loaded onto the instrument and the experimental procedure was performed as above with the FDBs.
Data are expressed as means ± SE, and the comparisons between experimental groups were made with SPSS statistical software (SPSS, Inc., Chicago, IL) using analysis of variance (ANOVA). Posthoc Holm-Sidak method was used for all pairwise comparisons after ANOVA tests. Significance was assumed at p < 0.05.