NAD+ supplementation reduces neuroinflammation and cell senescence in a transgenic mouse model of Alzheimer's disease via cGAS-STING

Abstract

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder. Impaired neuronal bioenergetics and neuroinflammation are thought to play key roles in the progression of AD, but their interplay is not clear. Nicotinamide adenine dinucleotide (NAD⁺) is an important metabolite in all human cells in which it is pivotal for multiple processes including DNA repair and mitophagy, both of which are impaired in AD neurons. Here, we report that levels of NAD⁺ are reduced and markers of inflammation increased in the brains of APP/PS1 mutant transgenic mice with beta-amyloid pathology. Treatment of APP/PS1 mutant mice with the NAD⁺ precursor nicotinamide riboside (NR) for 5 mo increased brain NAD⁺ levels, reduced expression of proinflammatory cytokines, and decreased activation of microglia and astrocytes. NR treatment also reduced NLRP3 inflammasome expression, DNA damage, apoptosis, and cellular senescence in the AD mouse brains. Activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are associated with DNA damage and senescence. cGAS-STING elevation was observed in the AD mice and normalized by NR treatment. Cell culture experiments using microglia suggested that the beneficial effects of NR are, in part, through a cGAS-STING-dependent pathway. Levels of ectopic (cytoplasmic) DNA were increased in APP/PS1 mutant mice and human AD fibroblasts and down-regulated by NR. NR treatment induced mitophagy and improved cognitive and synaptic functions in APP/PS1 mutant mice. Our findings suggest a role for NAD⁺ depletion-mediated activation of cGAS-STING in neuroinflammation and cellular senescence in AD.

Keywords: Alzheimer’s disease; DNA repair; NAD supplementation; inflammation; neurodegeneration.

Fig. 1.

Neuroinflammation is associated with reduced NAD⁺ levels during aging in AD mice. (A) GO term analysis of gene expression microarray from hippocampi. The most changed terms in AD compared to WT hippocampus are shown. P values for each significant changed pathway are shown. (B) The most changed genes from hippocampi microarray of AD compared to WT. P values for each significant changed gene are shown. (C) Western blots of specific inflammation-related proteins at different ages from cortical tissue from WT and APP/PS1 mice. n = 3 mice per group. (D) Quantification of NLRP3 protein level in C. (E) NAD⁺/NADH ratio in different ages of AD and WT mouse cortex. n = 3 to 8 mice per group. (F) Experimental design. The 7-mo-old AD and WT mice were treated with NR (12 mM) in their drinking water or vehicle for 5 mo. Behavioral tests were performed after 2 mo NR treatment. Mice were culled after 5 mo on NR or vehicle treatment for downstream assays. (G) NAD⁺/NADH ratio in 12-mo-old AD and WT mouse cortex with or without NR treatment. n = 5 to 9 mice per group. (H) Microarray heatmap showing significantly changed inflammation-related pathways in NR- or vehicle-treated AD or WT mice hippocampus. Data: mean ± SEM. Statistical significance was performed with two-way ANOVA followed by Tukey’s multiple comparisons test compared with control. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

NR decreases neuroinflammation, the NLRP3 inflammasome, NF-κB, and DNA damage. (A) Representative immunostaining images of IBA1 (green) and GFAP (red) with DAPI (blue) in mouse hippocampi and cortex. (B) Quantification of IBA1 intensity in A. n = 3 mice per group. (C–E) Proinflammatory cytokines or chemokines IP-10 (C), RANTES (D), and MIP-1β (E) levels detected by cytokine array in AD or WT mice cortex with or without NR treatment. n = 5 to 9 mice per group. (F and G) Proinflammatory cytokines or chemokines TNF-α (F) and IL-1β (G) levels detected by cytokine array in AD or WT mice plasma with or without NR treatment. n = 10 to 14 mice per group. (H) Western blots of specified proteins in WT and AD mice cortex with or without NR treatment. n = 3 mice per group. (I) Representative immunostaining images of NLRP3 (green) and caspase-1 (red) with DAPI (blue) in mice hippocampi and cortex. (J) Quantification of NLRP3 intensity in I. n = 3 mice per group. (K) Representative immunostaining images of γ-H2AX (green) and Cleaved-caspase-3 (red) with DAPI (blue) in mice hippocampi and cortex. (L) Quantification of γ-H2AX intensity in K. n = 3 mice per group. Data: mean ± SEM. Statistical significance was performed with two-way ANOVA followed by Tukey’s or Bonferroni multiple comparisons test compared with control. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

NR attenuates neuroinflammation through the cGAS–STING pathway. (A) Western blots of specific proteins in different ages of AD and WT mice. n = 3 mice per group. (B) Quantification of cGAS protein level in A. (C) Quantification of STING protein level in A. (D) Western blots of specific proteins from 12-mo-old AD mice with/without NR treatment (12 mM, 5 mo). n = 3 mice per group. (E) Quantification of cGAS protein level in D. (F) Quantification of STING protein level in D. (G) ELISA of IL-6 level in different conditions of HMC3 cell supernatants. Cells were treated by combining Aβ₄₂ (5 µM) and/or NR (1 mM) and/or STING inhibitor or cGAS-KD and/or STING-KD for 48 h. n = 3 biological repeats. (H) ELISA of IL-6 level in different conditions of mouse primary microglia cell supernatants. Cells were treated by combining Aβ₄₂ (5 µM) and/or NR (1 mM) and/or STING inhibitor H151 (1 µM) or cGAS-KD and/or STING-KD for 72 h. n = 3 biological repeats. (I) ELISA of IL-1β level, same cell setting as H. (J) qPCR of M2 maker Arg1 in different conditions of mouse primary microglia cell, same cell setting as H. Data: mean ± SEM. Statistical significance was performed with Student’s t test or one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Ns, not significant.

Fig. 4.

NR decreases cGAS–STING by decreasing cytoplasmic DNA. (A) Immunostaining of dsDNA and ssDNA with DAPI in mouse brain sections. n = 3 mice per group. (B) Immunostaining of dsDNA and ssDNA with DAPI in AD human fibroblasts and control fibroblasts. n = 21 to 58 cells per group were analyzed. (C) Quantification of dsDNA and ssDNA in A. n = 3 mice per group. (D) Quantification of dsDNA and ssDNA in B. (E) Total DNA was harvested from cytosolic and mitochondrial fractions of human fibroblasts and analyzed by qPCR. Cytosolic mtDNA genes were normalized to respective mitochondrial mtDNA genes (mt-ND1, D-loop, MT-CO₂, MT-ATP6) and presented as fold enrichment over vehicle-treated controls (Methods). n = 3 biological repeats. (F) Immunostaining of TOMM20 and LAMP2 with DAPI in mouse brain sections. (G) Quantification of mitophagy (colocalization of TOMM20 and LAMP2) in F. n = 3 mice per group. (H) Western blots of specific proteins in NR- or vehicle-treated AD and WT mice brain cortex. n = 3 mice per group. (I) Relative mitophagy intensity in AD human fibroblasts after some key mitophagy genes knockdown. Quantification of mitophagy in SI Appendix, Fig. S5A. n = 8 to 10 images per group were analyzed. (J) Immunostaining of dsDNA with DAPI in AD human fibroblasts after some mitophagy genes knockdown. (K) Immunostaining of ssDNA with DAPI in AD human fibroblasts after some mitophagy genes knockdown. (L and M) Quantification of dsDNA and ssDNA in J and K. n = 20 cells per group were analyzed. Data: mean ± SEM. Statistical significance was performed with two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Ns, not significant.

Fig. 5.

NR decreases cellular senescence in AD mouse brains. (A) Representative SA-β-gal staining in NR- or vehicle-treated AD or WT mice hippocampi and cortex. The panels of the hippocampus are part of the pictures after stitching the entire hippocampus with mosaic tile scan of microscope, and some of the stitching areas are still visible. (B) Quantification of SA-β-gal staining densities in A. n = 3 mice per group. (C) Representative p16^INK4a staining in NR- or vehicle-treated AD or WT mouse brains. (D) Quantification of p16^INK4a staining intensity in C. n = 3 mice per group. (E and F) Real-time qPCR of relative p16^INK4a and p21 mRNA levels. n = 5 to 7 mice per group. (G) Immunofluorescence of microglia marker IBA1 (red) or astrocyte marker GFAP (red) with cellular senescence marker p16^INK4a (green) in AD mouse brains. White arrows point to colocalization. (H) Immunofluorescence of neuron marker NeuN (red) or oligodendrocyte progenitor cell marker Olig2 (red) with cellular senescence marker p16^INK4a (green) in AD mouse brains. White arrows point to colocalization. (I) Representative SA-β-gal staining in etoposide- (Eto, 3 µM for 24 h and then washed the cells with fresh medium and incubated them an additional 4 d) or vehicle-treated HMC3 microglia cells. Cells were treated with STING inhibitor, or cGAS siRNA, or STING siRNA. (J) Quantification of SA-β-gal positive cells in I. n = 18 pictures per group. Data: mean ± SEM. Statistical significance was performed with two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Ns, not significant.

Fig. 6.

NR promotes a protective microglial phenotype and improves cognition and LTP in AD mice. (A) Representative immunostaining colocalization of Aβ (6E10) and microglia (IBA1) in NR- or vehicle-treated AD mouse brains. (B) Quantification of Aβ-positive microglia per region of interest (ROI) in NR- or vehicle-treated NR mouse brains (related to A). n = 3 mice per group. (C) Western blots of specific proteins in NR- or vehicle-treated AD and WT mice brain cortex. n = 3 mice per group. (D) Quantification of CSF2RA protein levels in C. (E) Real-time qPCR of protective microglia markers Arg1, Fizz1, and YM1 in AD or WT mice brain cortex with or without NR treatment. n = 5 to 7 mice per group. (F) Training phase of the Morris water maze test. The latency to find the platform was shown in NR- or vehicle-treated AD and WT mice. n = 15 to 17 mice per group. # comparison between AD versus WT. ^#P < 0.05. ^###P < 0.001. * comparison between NR-treated AD versus vehicle-treated AD. *P < 0.05, ***P < 0.001. (G) Time in the target quadrant of mice in the probe trial of Morris water maze. (H) Frequency to cross the location of the platform of mice in the probe trial of Morris water maze. n = 15 to 17 mice per group. (I) The LTP in mice hippocampus CA1. n = 5 mice per group. (J) Quantification of 50 to 60 min LTP magnitude in mice hippocampus CA1. Data: mean ± SEM. Statistical significance was performed with Student’s t test for two groups or two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.