Background
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, with aging being the most important risk factor [
1]. Mitochondrial dysfunction is a hallmark of neurodegenerative diseases with morphological and functional abnormalities limiting the electron transport chain and adenosine triphosphate (ATP) production seen in AD [
2].
Nicotinamide adenine dinucleotide (NAD) is a cofactor that is essential for many biological reactions in either its oxidized (NAD
+) or reduced (NADH) forms [
3]. NAD
+ and NADH mediate transfer of hydrogens in oxidative and reductive metabolic reactions [
4]. NAD
+ is essential to many mitochondrial enzymatic reactions and appropriate cellular bioenergetic metabolism [
5,
6]. NAD
+ levels naturally decline with aging [
4]. In normal conditions, the loss of NAD
+ inhibits cellular respiration, resulting in loss of mitochondrial ATP production and potentially cellular death [
5]. NAD
+ is used as a substrate by several NAD
+ dependent enzymes including poly(ADP-ribose) polymerase 1 (PARP1), Sirtuin 1 (SIRT1), and ADP-ribosyl cyclase (CD38) [
4,
5,
7,
8].
Preventing NAD
+ depletion and cellular energy deficits could be a therapeutic target for neurodegenerative diseases and act as a neuroprotective mechanism [
7]. Four pathways can synthesize NAD
+ in mammals. NAD
+ can be synthesized from the salvage pathway (primary route) utilizing nicotinamide, nicotinic acid, nicotinamide riboside, or the
de novo pathway using tryptophan [
9]. Nicotinamide phosphoribosyltransferase (Nampt) helps transfer a phosphoribosyl residue to nicotinamide forming nicotinamide mononucleotide (NMN) [
9]. NAD
+ consists of NMN covalently bound to adenosine monophosphate (AMP) [
4]. The enzyme NMN adenyltransferase (NMNAT) converts NMN to NAD
+ in one step [
4,
9,
10] making NMN a key precursor with possible therapeutic implications for increased NAD
+ levels [
11,
12]. Further, NMN is more soluble than NAD
+ in phosphate buffered saline (PBS) and is taken up more efficiently through the plasma membrane [
9,
13].
We have recently demonstrated in the well-studied AD chimeric APP
(swe)/PS1
(ΔE9) mouse model, deficits in mitochondrial oxygen consumption rates (OCR) in both brain and muscle [
14]. These deficits in OCR may have resulted from lack of sufficient NAD
+ due to increased catabolism. Thus, in the present study we tested the hypothesis that increasing NAD
+ availability by administering the precursor NMN would reverse mitochondrial OCR deficiencies in these AD disease-relevant animals. Mitochondrial respiration, calcium homeostasis and organelle transport have also been demonstrated to be influenced by mitochondrial morphology [
15,
16].
Fusion of two mitochondria causes an elongated morphology that can play a protective role in the nervous system, while fission allows proper distribution of mitochondria and is also used to remove damaged organelles [
17]. Mitochondrial dynamics are the balance of fission and fusion, controlling the morphology, number, and function of mitochondria [
17-
19]. Abnormal changes in these dynamics have been linked with aging and several neurodegenerative diseases (e.g. AD, Huntington’s disease (HD), Parkinson’s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS)). In these diseases mitochondrial morphology tends to shift towards increased fragmentation, indicating either an increase in fission or decreased fusion [
19]. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.
We demonstrate restoration of OCR in the NMN-treated AD double transgenic (AD-Tg) mice, indicating NAD+ levels were probably limiting. To further evaluate the basis of this effect we measured immunoreactivity of the NAD+-consuming proteins SIRT1 and CD38 and determined that they change with age as well as a function of NMN-treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN treated mice. This is the first study to directly examine amelioration of NAD+ catabolism and changes in mitochondrial morphological dynamics in AD mouse brain utilizing the immediate precursor NMN as a potential therapeutic compound.
Methods
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 (APP
swe) and a mutant human presenilin 1 (PS1) with the delta E9 (PS1
ΔE9) (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 CaMKII
α-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 (P
616-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): V
0 = volume of object; A
0 = 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.
Discussion
Alzheimer’s disease, along with other neurodegenerative diseases, has a complex multifactorial pathology with known mitochondrial deficits. This study is the first to directly examine the amelioration of NAD
+ catabolism and changes in mitochondrial morphological dynamics in AD mouse brain utilizing the immediate precursor nicotinamide mononucleotide (NMN) as a potential therapeutic compound. At three months, the well-studied AD chimeric APP
(swe)/PS1
(ΔE9) (AD-Tg) mouse model have mitochondrial oxygen consumption rate (OCR) deficits [
14] that precede amyloid deposition and plaque formation in brain [
33]. In the present study, deficiencies in OCR were successfully reversed using the same murine AD model administered NMN. In addition, NMN reduced levels of full length mutant APP in the AD-Tg mice. Further, the effects of NMN on normal mitochondrial morphology were examined using mice possessing fluorescent proteins targeted to neuronal mitochondria (CaMK2a-mito/eYFP) and demonstrate mitochondrial elongation and decreased fragmentation in NMN treated animals.
Nicotinamide (NAM) administration has been demonstrated to cross the blood–brain barrier and be converted to NAD
+, thus increasing cellular NAD
+ levels in the brain [
34]. Further, NMN has been shown to be highly enriched in mitochondria by sub-cellular fractionation studies suggesting intramitochondrial NAD
+ synthesis [
35] and may inhibit CD38 NAD
+ glycohydrolase activity [
27,
36]. Several disease/injury model studies have previously utilized NMN as a therapeutic to ameliorate NAD
+ deficiencies.
Yoshino et al. [
37] reversed NAD
+ deficits in a diabetic mouse model with intraperitoneal (IP) injections of NMN (500 mg/kg body weight/day for 7 days) a higher total dosage compared to the present study. In a separate study, NAD
+ levels were increased within 30 minutes after NMN administration (IP, 500 mg/kg body weight) directly following ischemia and reperfusion in mouse heart [
9]. Further, NMN prevented decreases in NAD
+ if injected 30 minutes prior to the insult [
9]. In a cell culture model of Parkinson’s disease consisting of rotenone treated PC12 cells, NMN intervention reduced apoptosis and restored intracellular levels of NAD
+ and ATP [
11]. In the present study, we used early low dose NMN administration beginning at two months of age to prevent the OCR deficits seen previously in three months old AD-Tg mice [
14].
Studies of NAD
+ deficiencies in diabetes, hepatic steatosis, and aging have historically used the NAD
+ salvage pathway supplements: NAM or nicotinic acid (NA) [
38,
39]. Nicotinic acid can bind to the GPR109A receptor, a G protein-coupled receptor that binds NA resulting in severe flushing as a side effect, making it unfavorable to most patients [
39]. Alternatively, nicotinamide riboside (NR) and NMN do not bind to the GPR109A receptor and are considered to have fewer unfavorable side effects [
39]. Several studies have looked at NAM and NR as possible therapeutics for Alzheimer's disease, although none have used NMN. Liu et al. [
40] administered NAM for 8 months in triple transgenic AD mice, finding NAM reduced beta amyloid (Aβ) and tau pathologies, elevated brain NAD
+ levels, improved brain bioenergetics, and preserved mitochondrial functionality. In a separate study, Green et al. [
41] treated four months old triple transgenic AD mice for 4 months, finding NAM treatment decreased tau levels and improved cognition. Similarly, when adult neurons isolated from triple transgenic AD mice aged 2 or 21 months were treated with NAM for 15 hours, NADH regenerating capacity was completely restored [
42]. In a different AD disease mouse model, Gong et al. [
43] treated Tg2576 animals with NR from 5–6 month of age until 10–11 months of age. NR treated mice had increased brain NAD
+ levels, elevated Peroxisome proliferator-activated receptor gamma coactivator 1
-alpha (PGC-1α), reduced Aβ, and reduced Beta site APP cleaving enzyme 1 (BACE1) [
43]. Our present study is the first report using NMN in any AD mouse model, treating them temporally prior to amyloid deposition to focus on mitochondrial bioenergetics.
Sirtuins (SIRT 1–7) are class III histone deacetylases and NAD+ dependent enzymes [
44]. Activation of SIRT1 mainly exerts neuroprotective actions [
1] for example by deacetylating target proteins including PGC-1α, demonstrated to be deficient in human AD brain [
45]. PGC-1α activation gives rise to mitochondrial biogenesis. In another study in APP/PS1 AD mice, overexpression of SIRT1 improved behavior and reduced Aβ [
46]. In the present study, AD-Tg mice had significantly increased SIRT1 immunoreactivity compared to non-transgenic (NTG) mice that NMN treatment reduced (Figure
4) potentially preserving overall NAD
+ pools for functional mitochondrial energetics.
Mitochondrial energetics in NMN treated animals could also be influenced by dynamic efficiency and the balance between fission and fusion. Mitochondrial fusion is regulated by the short and long isoforms of the protein Optic atrophy 1 (Opa1), as well as Mitofusin proteins (Mfn1 and Mfn2). Long and short OPA1 isoforms are both required for fusion. There is a reduction in fusion and an increase in fragmentation when the long OPA1 isoforms are converted to the short soluble OPA1 isoforms [
47]. This can be the result of a reduction in mitochondrial membrane potential and can lead to mitophagy and cellular death [
47]. Mitochondrial fission is regulated by Dynamin related protein Drp1, specifically by post-translational modifications of Drp1 [
18]. Short round mitochondria occur more commonly with dysfunctional mitochondria resulting from treatment with a toxin or mtDNA depletion [
48]. In this study we found a significant increase in P
616-DRP1 immunoreactivity in AD-Tg mouse mitochondria compared to NTG and AD-Tg NMN treated animals. This would indicate AD-Tg mice might have more fission and fragmentation of mitochondria than the NTG mice. Furthermore, NMN reduced fission in the treated AD-Tg mice reverting P
616-DRP1 levels to those of NTG animals.
To more clearly investigate mitochondrial morphology, CA1 hippocampal sections from CaMK2a-mito/eYFP mice were examined. These bigenic mice were previously determined to have functional mitochondrial bioenergetics similar to non-transgenic littermates [
21]. NMN treated CaMK2a-mito/eYFP mice had longer mitochondria and reduced fragmentation, while vehicle treated mice had a greater proportion of spherical shaped mitochondria, possibly indicating more fission. There were no differences in total mitochondrial volume and the NMN treated samples had slightly fewer mitochondria, suggesting, NMN reduces fission or increases fusion. Taken together, NMN treatment decreased fission (P
616-DRP1, AD mice) and reduced fragmentation (CaMK2a-mito/eYFP mice) suggesting a shift in dynamics from fission to fusion. This shift in dynamics could explain amelioration of mitochondrial bioenergetic deficits in the AD-Tg mice given NMN.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ANL performed the confocal microscopy, analyzed the morphology data, Western blots and drafted the manuscript. KO assisted with confocal microscopy and morphology analysis. AES performed the N2A cell culture experiments. TK assisted with morphology data analysis, study design and data interpretation. PSF assisted with drafting the manuscript, study design and data interpretation. RAS designed the study, performed the mitochondrial oxygen consumption and Western blotting experiments, analyzed the data and co-drafted the manuscript. All authors read and approved the final manuscript.