Nicotinamide as potential biomarker for Alzheimer's disease: A translational study based on metabolomics

Abstract

Introduction: The metabolic routes altered in Alzheimer's disease (AD) brain are poorly understood. As the metabolic pathways are evolutionarily conserved, the metabolic profiles carried out in animal models of AD could be directly translated into human studies. Methods: We performed untargeted Nuclear Magnetic Resonance metabolomics in hippocampus of McGill-R-Thy1-APP transgenic (Tg) rats, a model of AD-like cerebral amyloidosis and the translational potential of these findings was assessed by targeted Gas Chromatography-Electron Impact-Mass Spectrometry in plasma of participants in the German longitudinal cohort AgeCoDe. Results: In rat hippocampus 26 metabolites were identified. Of these 26 metabolites, nine showed differences between rat genotypes that were nominally significant. Two of them presented partial least square-discriminant analysis (PLS-DA) loadings with the larger absolute weights and the highest Variable Importance in Projection (VIP) scores and were specifically assigned to nicotinamide adenine dinucleotide (NAD) and nicotinamide (Nam). NAD levels were significantly decreased in Tg rat brains as compared to controls. In agreement with these results, plasma of AD patients showed significantly reduced levels of Nam in respect to cognitively normal participants. In addition, high plasma levels of Nam showed a 27% risk reduction of progressing to AD dementia within the following 2.5 years, this hazard ratio is lost afterwards. Discussion: To our knowledge, this is the first report showing that a decrease of Nam plasma levels is observed couple of years before conversion to AD, thereby suggesting its potential use as biomarker for AD progression.

Keywords: NAD salvage pathway; alzheimer’s disease; biomarkers, brain alterations; case-control analysis; nicotinamide (NAM); transgenic rats; vit B3.

FIGURE 1

Untargeted ¹H-NMR metabolomics of hippocampus of AD-like amyloid pathology transgenic rats (A) Typical 600 MHz ¹H-NMR spectrum of WT rats, representative of all the registered spectra. Assigned resonances of specific metabolites are indicated in red. Expanded views of the spectrum between 3.1–4.4 ppm (A) and 1.7–3.2 ppm (B) are shown (B) Overlaid of averaged ¹H-NMR spectra of WT rats (blue) (n = 10), Tg +/- (green) (n = 12) and Tg+/+ (red) (n = 10) in the 9.5–8.0 ppm zone (dashed box in A). The resonances assigned to NAD and Nam protons are indicated (C) Overlaid of representative ¹H-NMR spectra of WT rats (black, sample), NAD standard (red, upper panel) and NADP standard (red, lower panel) (D) Lower panel: correlation between NAD and Nam levels of samples analyzed. The AUC of H6 of NAD and H2 of Nam were plotted (n = 32). The linear regression (dashed line), the Pearson’s correlation coefficient and the p-value (two tailed) are shown. Upper panels: differences in the AUC of NAD (left) and Nam (right) among groups (WT, blue; Tg+/-, green and Tg+/+, red) were analyzed by one-way ANOVA. Significant differences are indicated accordingly to Fishers´s LSD test. ****p < 0.0001.

FIGURE 2

Hippocampal Aβ deposition alters the brain NAD + metabolism (A) Bars show mean ± SEM levels of NADH (left panel) and NADH/NAD + ratio (right panel) in hippocampal homogenates of control (WT; n = 3), hemizygous (Tg+/; n = 3) and homozygous (Tg+/+; n = 3) transgenic rats. One-way ANOVA tests and post hoc analyses revealed that Tg+/+ showed significantly lower levels of NADH (F_{(2, 6}) = 10.76, p = 0.01; post-hoc: **p < 0.01) and NADH/NAD + ratio (F_{(2, 6}) = 6.71, p = 0.02; post-hoc:*p < 0.05) compared with those observed in WT (B) Transcript levels of rate-limiting (NAMPT), NAD + -generation (NMNAT2) and NAD + consuming enzymes (CD38, PARP1, PARP2 and SIRT3) in hippocampal homogenates of Tg+/+ rats. Each bar represents the mean ± SEM of at least three independent experiments performed by triplicate for each sample normalized by GAPDH or Eukaryotic Translation Elongation Factor 1 Alpha 1 (EEF1A1). The mean ± SEM relative to WT (=1) is shown. Values above the dashed line (+1.5) were considered different from WT (=1).

FIGURE 3

Plasma levels of Nam in association with AD (A) Meta-analysis forest plot of Nicotinamide plasma levels in human samples. Discovery experiment includes 68 cases and 93 controls. Replication experiment includes 29 cases and 93 controls. Estimates are in Odds Ratios; CI, confidence interval; FE Model, fixed effects meta-analysis results. (B) Kaplan-Meier conversion to AD survival of 85 participants after blood test for Nicotinamide, stratified in high, medium or low levels. High levels of Nicotinamide seem to be a predictor of dementia survival for 2.5 years (HR = 0.73, p = 0.04) (C) Box plots represent the normalized GC-EI-MS spectral areas of Nicotinamide in human plasma of CN (cognitive normal) subjects (n = 189); AD (n = 85) patients and FU1 (n = 25); FU2 (n = 37) and FU3 (n = 23) participants.*p < 0.05.