Chemicals
All chemicals were purchased from Sigma-Aldrich (St Louis, MO) unless otherwise stated.
Animals
Alzheimer’s disease-relevant mice
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). AD animals positive for the transgenes were identified by polymerase chain reaction (PCR) using genomic DNA, isolated from the tails (Qiagen, Valencia, CA) then processed as described previously [14].
CaMKIIα-tTA and pTRE-mito/eYFP mice
Transgenic mice expressing the tetracycline-controlled transactivator protein (tTA) under regulatory control of the calcium/calmodulin-dependent kinase II (CaMKII) promoter [20] and animals expressing pTRE-mito/eYFP [21] were purchased from the Jackson Laboratory. Bigenic mice positive for both CaMKIIα-tTA and pTRE-mito/eYFP (CaMK2a-mito/eYFP) were generated by crossing these two strains. Male and female bigenic mice (2 months) were used in this study. CaMK2a-mito/eYFP bigenic mice were identified by PCR as described previously [21]. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia and tissue collection.
Nicotinamide mononucleotide (NMN) administration
APP(swe)/PS1(ΔE9) and CaMK2a-mito/eYFP male and female mice (2 months) were administered NMN (100 mg/kg, Sigma N3501) in sterile PBS (200 μl) subcutaneously (in the loose skin around the neck and shoulder area) every other day for 28 days. Non-transgenic (NTG) and vehicle control animals were administered 200 μl sterile PBS subcutaneously every other day for 28 days. Subcutaneous administration was utilized based on pilot studies. No significant differences in weight or external characteristics (fur condition, energy, size etc.) were observed between NMN and vehicle treated mice (data not shown).
N2A neuroblastoma cell culture conditions
Low passage mouse N2A hippocampal neuroblastomas (ATCC, Manassas, VA; 5,000/well) were seeded on V7 microplates (Seahorse Bioscience, North Billerica, MA) in proliferation media ((MEM, (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 h, cultures were transiently transfected (see below). After a further 24 hours, the proliferation media was replaced with differentiation media (DM) consisting of MEM, 2% horse serum (Gibco) and 1% Pen-Strep. The cultures had media changes using DM 48 hours later and oxygen consumption rates were measured 24 hours later. All wells were critically examined under the microscope to ensure cell viability prior to performing experiments.
Plasmid vector generation and transfection
The plasmid vector containing cDNA for a mitochondrially-targeted enhanced yellow fluorescent protein (eYFP), mutant APP(swe) and mutant PS1(ΔE9) described in [14] possesses a tetracycline response element thus requiring co-transfection with a tetracycline transactivator (TTA, Clontech). Co-transfection gives rise to cells possessing eYFP targeted to mitochondria and transgene-derived APP and PS1. N2A neuroblastomas were co-transfected with both constructs or TTA alone (control transfection) utilizing 1 μg of DNA/construct/well using the Magnetofection system (Oz Biosciences, San Diego, CA) with CombiMag plus lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Isolation of non-synaptic brain mitochondria
Twenty-four hours after the final NMN or vehicle injections, male and female APP(swe)/PS1(ΔE9) or non-transgenic mice (3 months) were decapitated, forebrains rapidly removed 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 [22] and as used previously [14,23]. This method has been demonstrated to show a high level of mitochondrial purity by electron microscopy [23]. Protein concentrations were determined by the method described by [24] using BSA as standards. Aliquots of brain mitochondria and homogenate had protease inhibitors (Calbiochem, San Diego, CA) added prior to storage at −20°C for later Western blot analyses.
N2A neuroblastoma cell respirometry
Prior to measurements, cultures were gently rinsed in 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. The cells were then placed in an unbuffered, humidified incubator at 37°C for 2 hours to allow temperature and pH equilibration. Cells were visually inspected prior to and after MB addition then loaded onto the Seahorse XF24-3 flux analyzer (Seahorse Bioscience). 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 carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to induce maximal oxygen consumption. Following recording of 3 more measurement loops, pyruvate was injected to determine if maximal oxygen consumption following FCCP addition was substrate limited. Antimycin A (inhibitor of mitochondrial respiration) was injected after 3 measurement loops 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, 1 μM FCCP, 10 mM pyruvate, and 1 μM antimycin A. Each plate had a subset of cells incubated with 10 mM nicotinamide adenine dinucleotide (NAD+, during the DM pre-incubation) prior to measurements.
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 [25] and 5 μg protein as determined above [24] 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 as previously described [14]. All measurements were performed at 37°C.
Immunoblotting
Proteins as determined by [24] from brain homogenates or non-synaptic mitochondria (50 μg) of APP(swe)/PS1(ΔE9) and their non-transgenic litter mates (3 months) were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on precast Mini-Protean TGX any KD gels (Bio-Rad, Hercules, CA) 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). After blocking, the membranes were incubated with primary antibodies to Beta amyloid 1–16 (6E10, 1:1,000; Covance); Histone deacetylase sirtuin 1 (SIRT1, 1:500; Millipore); NAD+ glycohydrolase CD38 (CD38; 1:2,000; R&D, Minneapolis, MN); Dynamin-related protein 1 (DRP1, 1:1,000; BD Biosciences, San Jose, CA); Phospho-DRP1 (P616-DRP1, 1:1,000; Cell Signaling Technology, Danvers, MA); Mitofusin 2 (MFN2, 1:1,000; Abcam, Cambridge, MA); Optic atrophy protein (OPA1, 1:1,000; BD Bioscience); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:14,000; Cell Signaling Technology); Voltage dependent anion channel (VDAC, 1:1,000; (rabbit); Cell Signaling Technology); VDAC (mouse; 1:1,000; Mitosciences (Eugene, OR); β-Actin (1:10,000; Sigma) at 4°C overnight. After 4 × 5 min washes in phosphate buffered saline (PBS) with 0.1% tween-20 (PBST), the membranes were incubated in the appropriate infrared (IR) fluorophore conjugated secondary antibody (Li-Cor Biosciences) for 30 min in the dark. Following PBST washes, the IR signal was captured on an Odyssey infrared imaging system (Li-Cor Biosciences) and stored as a digital image. VDAC was utilized as the loading control for mitochondria and GAPDH or β-Actin for brain homogenate to ensure equal loading.
Histology
Male and female CaMK2a-mito/eYFP, (3 months) were perfusion-fixed under deep anesthesia then processed as previously described [26].
Laser scanning confocal microscopy and quantitation of mitochondrial morphology
Forty μm-thick coronal brain tissue sections from CaMK2a-mito/eYFP mice (3 months) were washed with potassium phosphate buffered saline (KPBS) then mounted and coverslipped using Vectashield Hard set (Vector, Burlingame, CA) mounting media. Mitochondria in brain sections from CA1 hippocampal sub-regions were imaged utilizing a Zeiss LSM 510 laser scanning confocal microscope using a Plan-Apochromat 63x/1.4 oil lens. Single planes of 1024 × 1024 pixels were recorded at 1.0 – 1.5 Airy unit pinhole every 0.2 μm z-spacing throughout the entire tissue section as previously described [26] with modifications. Specifically, z-stack images were obtained from the striatum oriens of the CA1 sub-region. A 488 nm laser was used to visualize eYFP. Four z-stack images were taken per mouse brain. Recorded images were analyzed with Volocity software (Perkin Elmer, Waltham, MA). Quantification of mitochondrial morphology utilizing Volocity software was performed as previously described [26]. The following equation was used to determine 3D shape factor (ratio of the surface area of a sphere (with the same volume as the given object) to the surface area of the object): V0 = volume of object; A0 = area of object
$$ 3\mathrm{D}\ \mathrm{Shape}\ \mathrm{Factor}=\frac{\pi^{1/3}{\left(6{V}_0\right)}^{2/3}}{A_0} $$
Statistical analysis
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. Student t-test was used when direct comparison of two groups were analyzed (volocity data). Significance was assumed at p < 0.05.