Rat model
Transgenic (Tg) McGill-R-Thy1-APP rats, harboring the human APP751 transgene with the Swedish and Indiana mutation under the control of the murine Thy1.2 promoter, were generated using the HsdBrI: WH Wistar strain [4]. Animals were provided to Fundación Instituto Leloir (FIL) by The Royal Institution for the Advancement of Learning/McGill University, Montreal, Canada, and an in-house colony was established at FIL. Rats’ genotypes were determined by real time qPCR as previously described [5]. To avoid the litter effect, groups were made up of pups from 3 to 4 different litters. Homozygous (Tg+/+), hemizygous (Tg+/-), and littermates’ wild type (WT) control animals were maintained in polycarbonate cages in a temperature-controlled animal facility with a 12-h dark/light cycle and allowed to consume standard diet and water ad libitum. Only 9-month-old male rats were used for experiments to avoid any potential effects of female estrus cycle. All experimental procedures were performed in accordance with the guidelines of ARRIVE and OLAW–NIH. The protocol was approved by the local animal care committee (CICUAL # A5168-01).
Hippocampal tissue and plasma collection
Rats were anesthetized with ketamine (50mg/kg) and xylacine (10mg/kg), placed under a guillotine blade, decapitated and brains quickly removed. Hippocampi were dissected on an ice-cold plate, and divided into left and right hemispheres. Each hippocampus was immediately and independently frozen in liquid nitrogen, and stored at -80 °C until used. Blood samples were collected immediately after animals’ decapitation in 15 mL heparinized tubes, centrifuged at 3500 xg for 10 min. Plasma was collected, aliquoted and stored at -80 °C until use.
Human plasma samples
Plasma samples were selected from the German study on Aging, Cognition and Dementia (AgeCoDe) biobank [21]. This is a longitudinal study, where participants were recruited in primary care centers in six German cities. Inclusion criteria was to be at least 75 years old and cognitively healthy according to the general practitioner’s judgment. Every ~18 months interval participants are followed-up with personal interviews and neuropsychological assessments. To date, nine follow-ups (FUs) were completed, but results from the last one are still in process. Blood samples were obtained at the 3rd visit, processed and store at -80 0C. For this study the 3rd visit is considered the baseline. Controls (n=189) remained cognitively normal until the last FU analyzed, were 83.6 ± 3.1 years old, 64.0% were female and 20.6% were APOE4 carriers. Cases were participants with incident AD (n=68), 86.0 ± 3.6 years old, 64.7% female and 33.8% APOE4 carriers; and prevalent AD (n=29), 84.2 ± 3.1 years old, 75.8% female and 37.9% APOE4 carriers. Participants converting to AD in the next three visits following baseline (FU1, FU2 and FU3) were included in the analysis. At FU1 were 25 participants with age of 84.8 ± 3.5 years old, 80% women, and 28% APOE4 carriers; at FU2 were 37 participants with age of 83.6 ± 2.6 years old, 67.6% women, and 32.4% APOE4 carriers; and at FU3 were 23 participants with age of 82.7 ± 2.6 years old, 60.9% women, and 21.7% APOE4 carriers.
Expression of Aβ isoforms in rat hippocampus
To quantify human Aβ 38/40/42 MSD® V-PLEX PLUS Aβ Peptide Panel 1 kit was used following the manufacturer’s instructions. Briefly, hippocampus of Tg rats were processed with illustra triplePrep kit (GE) and Individual protein samples were loaded onto MULTI-SPOT® microplates pre-coated with antibodies specific to the C-termini of Aβ38, Aβ40 and Aβ42 and were detected with SULFO-TAG™-labeled 6E10 antibody. Light emitted upon electrochemical stimulation was read using the MSD QuickPlex SQ120 instrument. Data were analyzed using MSD Workbench 4.0 software. Values of concentration in pg/mg of total protein were expressed as median and interquartil range (IQR). Mann Whitney test was applied to assess significant differences between groups.
NMR Spectroscopy
Frozen hemi-hippocampus were homogenized with a teflon-glass grinder in 2 ml ice-cold 80% methanol and centrifuged at 4 °C for 10 min at 15000xg. Supernatants were collected and dried in a Savant SpeedVac (Thermo Scientific). Plasma proteins were precipitated with 80% methanol (1:2) as described in G. A. Nagana Gowda et al [22]. Supernatants were collected and dried in a Savant SpeedVac (Thermo Scientific). Dried hippocampus or plasma samples were solubilized in 0.5 ml sodium phosphate buffer (100 mM dissolved in D2O, pH = 7.4), supplemented with 3-trimethylsilyl-[2,2,3,3,-2H4]-propionate (TSP, final concentration 0.33 mM) as chemical shift reference. All NMR experiments were performed at 298 K on a Bruker Avance III spectrometer operating at a proton frequency of 600.3 MHz. 1H-NMR 1D spectra were acquired using a standard Bruker 1D NOESY pulse program with pre-saturation during relaxation delay and mixing time, and spoil gradients (noesygppr1d). The following experimental parameters were used in all measurements: 256 scans, 1.85 s relaxation delay, 1.36 s acquisition time, 20 ppm spectral width, 10 ms mixing time, and 32 K acquisition points. The NMR data were zero-filled, Fourier transformed, phase corrected using NMRPipe and converted to a Matlab-compatible format for further processing and analysis. All spectra were referenced to TSP (1H δ = 0 ppm) and submitted to water peak elimination, baseline correction, normalization, and scaling. The assignment was achieved using the freely available electronic databases HMDB and BMRB, and subsequently confirmed by 2D spectra including heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY). 2D 1H-1H TOCSY spectra were collected with N1=512 and N2=2048 complex data points. The spectral widths for the indirect and the direct dimensions were 9615.4 and 9604.9 Hz, respectively. The number of scans per t1 increment was set to 36. The transmitter frequency offset was 4.7 ppm in both 1H dimensions. 2D 13C-1H HSQC spectra were collected with N1=512 and N2=2048 complex data points. The spectral widths for the indirect and direct dimensions were 24,906.9 and 12,019.2 Hz, respectively. The number of scans per t1 increment was set to 256. The transmitter frequency offset was 70 ppm in the 13C dimension and 4.7 ppm in the 1H dimension. The 1H resonances assigned to Trigonelline were based on HMDB database (https://hmdb.ca/metabolites/HMDB0000875#spectra) and confirmed by 2D 1H-1H TOCSY spectra.
NAD+/NADH quantification and NAD salvage pathway enzymes levels determination
NAD levels were measured using NAD/NADH assay kit from Abcam (ab65348). Briefly, hippocampi from WT, Tg(+/-) and Tg(+/+) rats were snap frozen in liquid nitrogen, homogenized in NADH/NAD extraction buffer and filtered through a 10kD spin column (Abcam, ab93349) to remove enzymes. Assay procedure was followed per kit instructions and levels of NADH and NAD+ were determined normalized to tissue weight. NAD salvage-pathway enzymes transcript levels were estimated by reverse-transcription quantitative PCR (RT-qPCR). Protein, DNA and RNA were isolated from frozen hemi-hippocampus using Illustra™ TriplePrep Kit (GE Healthcare) following manufacturer’s instructions. Briefly, 1-3 ug of total RNA was reverse transcribed using oligo(dT) primer and SuperScript II reverse transcriptase (Invitrogen). Sequences of oligonucleotides (F, forward; R, reverse; 5′→3′) to assess transcript levels of NAD+ consumption enzymes, NAD+ synthesis enzymes and a constitutive gene were as follows: CD38 (F 5’-GGTCCCTCAGTGAGCCATTT-3’; R 5’-ATGTCATGAATTACCCAGGC-3’), PARP1 (F 5’-AGGACCCCATCGATGTCAAC-3’; R 5’-GGTCGCGTGAGTGTTCTTCAC-3’), PARP2 (F 5’-ATGACGTCGTTCAAGCG-3’; R 5’-gtcatctgttgctctgttgcc-3’) SIRT3 (F 5’-TGTGGGGTCCGGGAGTATTA-3’; R-5’GTCATCTGTTGCTCTGTTGCC-3’); NMNAT2 (F 5’-TCCCAATATGACCGAGACCAC-3’; R 5’-TTGTGCAGATAATCCCTGGCT-3’) and Eukaryotic Translation Elongation Factor 1 Alpha 1 (EEF1A1) (F 5’-AACTGACAAGCCTCTGCGAC-3’; R 5’-GCTTCATGGTGCATTTCCACA-3’). RT-PCR reactions were prepared using PowerUp™SYBR™ Green Master Mix (ThermoFisher Scientific) and run following the standard cycling mode (primer Tm > 60°C) instructions in a Light Cycler 480 Instrument II (Roche). Melt curve analysis and agarose gels verified the presence of a single desired PCR product. Absolut quantification was performed using dilutions (1, 1:10, 1:50, 1:100, 1:500 and 1:1000) of a standard sample, which was a pool of 5 ul cDNA of each analyzed sample. The relative amount of transcripts to EEF1A1 was quantified by the 2−ΔΔCt method using MxPro software. The mean ± SEM relative to WT (=1) were analyzed for each genotype and values above a fold-change of +1.5 were considered different from WT (=1).
Gas chromatography Electron Impact Mass Spectrometry (GC-EI-MS)
Plasma samples were thawed on ice, and 100 ul were extracted with 900 ul of cold extraction buffer containing 40:40:20 methanol:acetonitrile:water [v:v:v]. After 30 min in an orbital mixer at 4ºC., samples were sonicated for 10 min in an ice-cooled bath-type sonicator and centrifuged for 10 min at 16000xg at 4ºC. Supernatants were collected and dried in a SpeedVac until complete dryness. Standard curves were prepared with concentrations ranging from 0.005 to 50 ug/ml of nicotinamide and trigonelline, processed in the same way as samples. Dried down samples and standards were derivatized using methoxyamine and MSTFA/FAMEs solution (N-methyl-N-trimethylsilyl-trifluoracetamid/Fatty acid methyl esters) following standard procedures [23, 24]. After that, samples were analyzed in a GC-EI-MS (Q Exactive GC Orbitrap system, ThermoFisher) using a 30-m DB-35MS capillary column. Representative fragments from the GC-EI-MS analysis of Nam were extracted using TraceFinder (Version 4.1, ThermoFischer) and quantified using the linear range of the obtained standard curve. All analysis were performed using peak areas, transformed into Z-scores, for easier comparation among experiments.
Statistical analysis
Differences among groups in the AUC of peaks from 1H-RMN spectroscopy experiments were analyzed by one-way ANOVA, taking p<0.05 and p<0.0001 as significant according to Dunnett’s multiple range test. Data of protein levels of NADt/NADH and NAD+ were analyzed by one-way ANOVA tests followed by post-hoc Tukey's multiple comparisons tests. Two-tailed unpaired t-tests were used to compare transcripts expression of those genes that showed more than 1.5-fold change in their expression levels compared to WT (=1). For easier comparation among experiments of rat plasma metabolome a Z-score standardization was applied and subsequently data were analyzed by one-way ANOVA tests followed by post-hoc Tukey's multiple comparisons tests. In all cases, assumption of normality was examined using Kolmogorov-Smirnov or Shapiro–Wilk tests. A probability equal or less that 5 % was considered as significant. All analyses were carried out using GraphPad Prism for Windows (version 7.0). Statistical analysis and plots of data from GC-EI-MS experiments performed with human plasma were done using R-project v. 4.0.0 (https://www.R-project.org) and R-studio v.1.2.5042 (http://www.rstudio.com/). Normal distribution was visualized using qqnorm plots, and outliers (defined as mean +/-3 standard deviation) were eliminated from the analysis (n=2). Linear regression models adjusted for sex, age and apoe4 were used to estimate the association of Nam levels vs CN in the discovery and replication experiments, as well as in FUs groups. Meta-analysis was performed using the R-package “metafor” [25] and visualized with the general function forest. The cox proportional hazards regression model, which relates time dependent variables, time dependent strata, and multiple events per subject, were performed with the R-package “survival” [26] and “survminer” [27]. Only samples with incident AD at FU1-3 were included, time variable was time to conversion to AD in years, and the event per subject was conversion (no=0, yes=1). Proportional hazard assumption was tested by Schoenfeld’s test, and consequently two cox regressions were performed with a split-time=2.5 years.