Nicotinamide forestalls pathology and cognitive decline in Alzheimer mice: evidence for improved neuronal bioenergetics and autophagy procession

Abstract

Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline and the pathologic accumulations of amyloid β-peptide (Aβ) and hyperphosphorylated tau in Alzheimer's disease (AD). To determine whether improving brain energy metabolism will forestall disease progress in AD, the impact of the β-nicotinamide adenine dinucleotide precursor nicotinamide on brain cell mitochondrial function and macroautophagy, bioenergetics-related signaling, and cognitive performance were studied in cultured neurons and in a mouse model of AD. Oxidative stress resulted in decreased mitochondrial mass, mitochondrial degeneration, and autophagosome accumulation in neurons. Nicotinamide preserved mitochondrial integrity and autophagy function, and reduced neuronal vulnerability to oxidative/metabolic insults and Aβ toxicity. β-Nicotinamide adenine dinucleotide biosynthesis, autophagy, and phosphatidylinositol-3-kinase signaling were required for the neuroprotective action of nicotinamide. Treatment of 3xTgAD mice with nicotinamide for 8 months resulted in improved cognitive performance, and reduced Aβ and hyperphosphorylated tau pathologies in hippocampus and cerebral cortex. Nicotinamide treatment preserved mitochondrial integrity, and improved autophagy-lysosome procession by enhancing lysosome/autolysosome acidification to reduce autophagosome accumulation. Treatment of 3xTgAD mice with nicotinamide resulted in elevated levels of activated neuroplasticity-related kinases (protein kinase B [Akt] and extracellular signal-regulated kinases) and the transcription factor cyclic adenosine monophosphate (AMP) response element-binding protein in the hippocampus and cerebral cortex. Thus, nicotinamide suppresses AD pathology and cognitive decline in a mouse model of AD by a mechanism involving improved brain bioenergetics with preserved functionality of mitochondria and the autophagy system.

Published by Elsevier Inc.

Figures

Figure 1

NAM preserves mitochondrial mass, morphology and functionality under conditions of oxidative stress in neurons. Cortical neurons were loaded with the fluorescent probe MitoTracker Green (MitoTracG) and the mitochondrial membrane potential (MMP) indicator TMRE following exposure to vehicle (Control), H2O2 (20 μM) or rotenone (1 μM) for 6 h. (A) Representative images showing mitochondrial morphological changes with reduced mitochondrial mass (MitoTrac Green) and MMP (TMRE) in neurons exposed to oxidative stress. Images are representative of more than 40 neurons examined in each group. (B) Reduced mitochondrial size and loss of the normal elongated shape of the mitochondria occurred in neuronal processes (MitoTracker Green) after exposure of neurons to the indicated oxidative insults. (C) MMP is reduced in neurons exposed to oxidative insults. Values are the mean ± SD of measurements made on 3-4 separate cultures (10-15 neurons evaluated/culture). *p<0.05. **p<0.01. (D and E) NAM treatment preserves mitochondrial mass in neurons exposed to oxidative stress. Values are the mean ± SD of measurements made on 3-4 separate cultures (10-15 neurons evaluated/culture). (F) Levels of DLP1 (a mitochondrial fission-related protein) were elevated in cells exposed to oxidative insults, and NAM treatment attenuated the DLP1 response to oxidative stress. Levels of the mitochondrial fusion protein mfn2 were reduced in cells exposed to oxidative stress. (G) Densitometric values of protein bands (normalized to the β-actin band). Values are the mean ± SD of determinations made on samples from 3 different experiments (samples from 6-7 cultures/experiment were pooled). *p<0.05. (H) Electron microscopy showing ultrastructural alterations in neurons caused by exposure to H2O2 (20 μM) for 6 h. Panel a shows a neuron in a control culture with the inset showing an enlarged view of the boxed area in the lower magnification image. The images illustrate the ‘healthy’ appearance of the mitochondria which are elongated with cristae evident. Panel b shows a neuron in a culture that had been exposed to hydrogen peroxide for 6 h, with the inset showing an enlarged view of the boxed area in the lower magnification image. The latter images illustrate abnormal mitochondria which are round and swollen, with cristae damaged. Panels c and e show neurons that were treated with NAM and then exposed to hydrogen peroxide; mitochondria in the cell body (panel c) and neurites (panel e) appear healthy. Panel d shows the loss of mitochondria in a neurite of a neuron exposed to hydrogen peroxide. Nuc, nucleus.

Figure 2

Oxidative stress induces autophagy/mitophagy in neurons. Cortical neurons were exposed to H2O2 for 6 h. (A) Electron microscopy revealed numerous autophagosomes in the cell body (b, d), neurites (e) and neurite terminals (f, arrow) in H2O2-treated neurons, compared to a relative paucity of autophagosomes in control neurons (a). Black arrows indicate autophagosomes and red arrows indicate mitochondria. Large autophagosomes contained membranous structures and dense aggregates (g), autolysosomes (h), and degenerated mitochondria (mitophagy) (i, arrows). The remaining mitochondria in H2O2-treated neurons had degenerative features including swelling, enlarged matrix and disrupted cristae (j, arrows). These electron micrographs are representative of ultrastructural features observed in more than 60 neurons examined from at least 3 separate cultures for each treatment condition. (B) Immunoblot revealed slightly increased levels of the LC3-II/LC3-I ratio in cells exposed to oxidative insults at 6 h, while at 18 h the levels of LC3-I were reduced resulting in an elevated LC3-II/LC3-I ratio. The LC3-I level and the LC3-II/LC3-I ratio were maintained in NAM-treated neurons exposed to H2O2. (C) Results of densitometric analysis of immunoblots. Values are the mean ± SD of determinations made on samples from 3 different experiments (samples from 6-7 cultures/experiment were pooled); values were normalized to the β-actin band.

Figure 3

NAM enhances the survival of neurons exposed to oxidative stress and Aβ oligomers. (A) Cortical neurons were treated for 20 h with the autophagy inhibitor Bafilomycin A1 (1 nM) or with FK866 (1 μM), an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Values are the mean and SD of measurements made in 4 separate cultures. (B) NAM protected neurons against oxidative insults, and the neuroprotective effects were significantly attenuated by co-treatment with Bafilomycin A1 (1 nM) or FK866 (1 μM). Values are the mean and SD of measurements made in 4 separate cultures. (C) Wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), also attenuated the neuroprotective effects of NAM. Values are the mean and SD of measurements made in 4 separate cultures. (D) NAM protected cortical neurons against the toxicity of Aβ oligomers (mean and SD of 4 separate cultures). (E) Cellular NAD+ levels were reduced after exposure to Aβ oligomers, but were maintained in NAM-treated neurons exposed to Aβ oligomers. Values are the mean and SD of measurements made in 3 separate cultures. (F) Immunoblot analysis revealed that levels of the NAD+-dependent deacetylase SIRT1 were preserved in NAM-treated cortical neurons exposed to oxidative stress. Values are the mean ± SD of determinations made on samples from 3 different experiments (samples from 8-10 cultures/experiment were pooled). *p<0.05; **p<0.01.

Figure 4

NAM ameliorates spatial memory deficits and an anxiety-like phenotype in 3xTgAD mice. 3xTgAD mice were treated with NAM or vehicle (control) for 8 months beginning at 4 months of age (see Methods) and were then tested in the water maze (A-F) and the open field (G-J). Mice were tested in the hidden platform version of the water maze on 6 consecutive days. Goal latencies (A), path length (B), swim speed (C) and percentage of time floating (D) were measured. Values are the mean ± SD (n = 10 mice per group). *p<0.05, **p<0.01 compared to corresponding value for NAM-treated 3xTgAD mice. Results of probe trial test after 6 consecutive training days showed that NAM-treated 3xTgAD spent significantly more time in the target quadrant compared to control 3xTgAD mice (E and F). There were no significant differences in distance traveled (G) and ambulatory counts (H) in the open field between control and NAM-treated 3xTgAD mice. However, NAM-treated 3xTgAD mice exhibited a greater number of vertical counts (I) and stereotypical behavior counts (J) compared to vehicle-treated 3xTgAD mice. *p<0.05.

Figure 5

NAM treatment ameliorates Aβ and tau pathologies in the brains of 3xTgAD mice. (A) Immunoblots showing levels of full-length APP, Aβ oligomers, Tau and p-Tau in samples of cortical tissue from NAM-treated 13 month-old 3xTgAD mice compared to vehicle-treated 3xTgAD mice. Each lane represents a sample from an individual mouse. (B and C) Results of densitometric analysis of immunoblots of full-length APP, Aβ oligomers (trimers, about 17 kDa), total Tau (T46), and hyper-phosphorylated tau (AT180) from cortical tissue samples of 13 month-old untreated and NAM-treated 3xTgAD mice (4 mice/group). (D - F) Immunoblots and densitometric analysis of APP, Aβ, total Tau and p-Tau (AT180) in protein samples from the hippocampus of untreated and NAM-treated 3xTgAD mice. Density of protein bands were normalized to β-actin bands; values are the mean ± SD (6 mice/group).

Figure 6

NAM treatment reduces Aβ accumulation and neuronal p-Tau immunoreactivity in 3xTgAD mice. Representative brain sections showing the indicated brain regions from control 3xTgAD mice and NAM-treated 3xTgAD mice immunostained with either Aβ or p-Tau (AT180 ) antibodies as indicated. Note that levels of extracellular and cell-associated Aβ immunoreactivity and intracellular p-Tau (Thr 231) immunoreactivity are reduced in the NAM-treated mice. These images are representative of results observed in 6 vehicle-treated and 6 NAM-treated 3xTgAD mice. Scale bars = 50 μm.

Figure 7

NAM normalizes mitochondrial dynamics and improves autolysosomal processing in 3xTgAD mouse brains. (A- C) Immunoblots and densitometric analysis showing relative levels of DLP1 and OPA1in cortex samples from age-matched wild type mice (WT), vehicle-treated 3xTgAD mice and NAM-treated 3xTgAD mice. (C- E) Immunoblot and densitometric analysis of LC3-I, LC3-II, beclin, pmTOR and p70p-S6 protein levels in cerebral cortex samples from vehicle- and NAM-treated 3xTgAD mice (4 mice/group). (F and G) Immunoblot and densitometric analysis of LC3-I, LC3-II levels and LC3-II/LC3-I ratio in hippocampal tissue samples of NAM-treated and control 3xTgAD mice. Values are the mean ±SD (6 mice/group).

Figure 8

NAM enhances organellar acidification and autolysosomal processing in neurons. (A) Rat primary cortical neuronal cultures were loaded with the acidic organelle pH indicator LysoSensor™ Green, which selectively accumulates in acidic organelles including lysosomes and autolysosomes. The fluorescence intensity was monitored by time-lapse confocal imaging images (images were acquired every 2 seconds). Adding NAM to cells induced a transient increase of LysoSensor G fluorescence intensity, indicating enhanced acidification of the organelles. Values are the mean and SD of measurements made in 12 neurons from 3-4 separate cultures. (B) Representative image of cortical neurons loaded with LysoSensor™ Green. Flourescence intensity increases as the pH decreases. (C) Cells were loaded with mtΔψ indicator TMRE (red) and LysoSensor™ G. Under basal culture conditions there was little or no co-localization of the TMRE and LysoSensor signals (Ca-c). Acidic organelle clustering with increased intensity (green) and reduced mitochondrial Δψm (red) were detected in neurons exposed to rotenone for 6 h (Ce). In NAM-treated cells, MMP was partially preserved with co-localization of TMRE and LysoSensor G labeling (yellow), suggesting occurrence of mitophagy (Cf).

Figure 9

Evidence that NAM enhances Akt, ERK, and CREB signaling pathways. (A and C) Immunoblots of hippocampal and cerebral cortex tissue samples from NAM-treated and age-matched (13 month-old) control 3xTgAD mice showing relative levels of Akt, p-Akt, CREB, p-CREB, p-ERK1, p-ERK2 and β-actin. (B and D) Results of densitometric analysis of relative levels of the indicated proteins (normalized to β-actin levels in the same samples). Values are the mean ± SD (n = 4-6 mice/group). (E) Immunoblots performed on tissue samples from the cerebral cortex and hippocampus of NAM-treated and age-matched control 3xTgAD mice showing relative levels of SIRT1, SIRT3, NAMPT and β-actin. (F) Results of densitometric analysis of relative levels of the indicated proteins (normalized to β-actin levels in the same samples). Values are the mean and SD (n = 5 mice). *p<0.05. (G) NAD+ levels in cerebral cortex tissue samples from vehicle-treated and NAM-treated (250 mg/kg) mice 6 h after i.p. injection. Values are the mean ± SD (n = 5 mice). *p<0.05.

Figure 10

Model for the mechanisms by which NAM counteracts AD-like disease processes and maintains cognitive function in 3xTgAD mice. Compromised brain energy metabolism, and age-related accumulation of oxidative stress may contribute to the adverse effects of APP, PS1 and tau mutations on neuronal plasticity and cognitive function. Important aspect of the pathogenic process are perturbed mitochondrial bioenergetics and aberrant mitochondrial dynamics, oxidative stress and impaired autophagy. NAM, the precursor in NAD+ salvage biosynthesis increases mitochondrial resistance to oxidative stress by modulating the NAD+: NADH redox state to reduce ROS and enhances autophagylysosomal processing of damaged organelles. NAM may also preserve mitochondrial function by acting as an antioxidant to suppress ROS production. NAM increases activation of PI3K-Akt, MAPK/ERK1/2 and SIRT1 cell survival and stress response signaling pathways, as well as transcription factor CREB, a protein critical for synaptic plasticity and long-term memory, thereby ameliorating cognitive decline in AD mice.