The chemicals used in this study were as follows: LPS (20389-04, Nacalai Tesque, Kyoto, Japan), β-NAD+ (24334-97, Nacalai Tesque), nicotinamide riboside (NR, NIAGEN, ChromaDex, CA, USA), apigenin (016-18913, Wako, Osaka, Japan), and compound 78c (538763, Calbiochem, CA, USA).
Animals and treatments
Wild-type (WT) and CD38 knockout (KO) male ICR mice (10–11 week old) were used for the experiments (body weight; 30-36g). CD38 KO mice were generated as described previously and backcrossed for more than eight times . All mice were housed in 345 × 168 × 140 mm cages in a temperature-controlled room (24-25 °C) with 12-h light-dark cycles. Food and water were available ad libitum. LPS injection was performed as previously described . In brief, 10 µg LPS dissolved in 5 m L of sterile PBS or control sterile PBS was injected into the right lateral cerebral ventricle (0.3 mm caudal to bregma and -1.0 mm from lateral to midline at a depth of 3.0 mm) using a microsyringe and stereotaxic coordinates. The animals were sacrificed at various time points as described in the Results section. Apigenin (40 mg/kg) or NR (400 mg/kg) was administered intraperitoneally once per day for 7 consecutive days, followed by LPS injection 6 h after the final administration of apigenin or NR. Mice were anesthetized and sacrificed at the indicated times after LPS injection. To avoid the effect of injection-mediated brain damage, the contralateral (left) side of the cerebral cortex or hippocampus was analyzed in all experiments. All animal experiments were performed in accordance with the guidelines and approved by the Animal Care and Use Committee of Kanazawa University (AP-194042).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed as previously described . In brief, total RNA was extracted from the cerebral cortex, hippocampus, or cultured cells using the FASTGeneTM RNA Basic Kit. (FG-80250, Nippon Genetics Co., Ltd), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems, Warrington, UK). Individual cDNA sequences were amplified using the ThunderbirdTM SYBR qPCR® Mix (QPS-201, Toyobo Co., Ltd.) with specific primers. To measure differential expression, the comparative Ct method was used for data analyses in MxPro 4.10 (Agilent Technologies Inc.). The primer sequences are listed in Additional file 4: Table S1.
Western blot analyses
The hippocampal tissues or cultured cells were homogenized in a RIPA lysis buffer containing 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.2% deoxycholate, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin), and then centrifuged at 15,000 rpm for 15 min. Denatured protein lysates were electrophoretically separated using SDS-polyacrylamide gel electrophoresis and proteins were transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% skimmed milk for 30 min and incubated with anti-CD38 (AF4947, R&D systems, MN, USA, 1:500), anti-glial fibrillary acidic protein (GFAP) (G9269, Sigma, MO, USA, 1:5,000), anti-ionized calcium binding adaptor molecule 1 (Iba1) (019-19741, Wako, Osaka, Japan, 1:500) antibodies for 16 h at 4 °C. Membranes were washed several times with Tris-buffered saline, 0.1% Tween 20 (TBST) and incubated with anti-rabbit (SC-2004, 1:5,000, Santa Cruz Biotechnology), anti-mouse (SC-516102, 1:5,000, Santa Cruz Biotechnology), anti-goat (SC-2354, 1:1,000, Santa Cruz Biotechnology), or anti-rat (NA9350, Amersham Pharmacia biotech, 1:1000), and horseradish peroxidase-linked immunoglobulin G (Cell Signaling Technology, Tokyo, Japan) for 2 h at room temperature, and the membranes were washed several times with TBST. Immunoreactivity was detected using an enhanced chemiluminescence system (GE Healthcare Bio-Sciences, PA, USA). The intensity of each band was quantified using ImageJ software (https://imagej.nih.gov/ij/).
Differential expression analysis of published microarray studies
Microarray expression profiles and associated platform data were downloaded from the Gene Expression Omnibus (GEO) database for three studies: GSE49329, GSE102482, and GSE122815 using the “GEOquery” Bioconductor package . Specific information regarding sample treatment and preparation can be found at the respective GEO accession numbers. Differentially expressed genes between control and LPS-treated cells or mice were identified using the GEO2R wrapper (https://www.ncbi.nlm.nih.gov/geo/geo2r/) around the Bioconductor package “limma” . P-values were adjusted to correct for false positives using the Benjamini-Hochberg method. Plots of NAD-related genes were generated using GraphPad Prism v8.
Mice were anesthetized with isoflurane and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed from mice and subjected to post-fixation in 4% PFA, followed by dehydration in 30% sucrose. Twenty micrometer-thick sections from -3.14 mm -4.30 mm bregma were obtained using a cryostat (CM1950, Leica, Nussloch, Germany). To measure glial and neuronal cell loss in the mouse hippocampus sections were processed for immunostaining with antibodies against GFAP (1:1000), Iba1 (1:500), and neurofilament H non-phosphorylated (SMI-32) (801701, Biolegend, CA, USA 1:500), MAP2 (4542, Cell Signaling Technology, Tokyo, Japan 1:500). Subsequently, alexa488- (Thermo Fisher Scientific Inc.,1:200) or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., PA, USA, 1:200) were used to visualize immunolabeling. Fluorescence images in the CA1 or CA3 of the hippocampus were obtained from 2 sections per mouse using a laser scanning confocal microscope EZ-C1 (Eclipse TE2000U, Nikon, Tokyo, Japan). The fluorescence intensity of each region of the hippocampus/total area was analyzed using ImageJ software.
Analysis of NAD+ levels in brain tissues
After administration of apigenin or NR for 7 d, hippocampal tissues were harvested from WT mice, and NAD+ levels were measured with a commercially available NAD+ /NADH assay kit (E2ND-100, bioassay System, SFC, USA) according to the manufacturer’s protocol. The absorbance at 570 nm for each sample was measured using a Multiskan GO Microplate Spectrophotometer (Thermo Fischer Scientific, MA, USA).
Glial cell cultures
Astrocyte and microglial cultures were prepared from mixed glial cultures as previously described . In brief, mixed glial cells were harvested from the cerebral cortices of WT neonatal mice (P1 to P3). After 14 d of cultivation, cells were collected and incubated with CD11b MicroBeads (130-093-634, microbeads conjugated to monoclonal anti-human/mouse CD11b antibody, Miltenyi Biotec, Bergisch Gladbach, Germany) and applied to a magnetic column fitted into a MidiMACSTM cell separator (Miltenyi Biotec). The cells were separated into CD11b-positive and CD11b-negative fractions. The CD11b-positive fraction, which contained microglia, was used for the experiments 24 h after plating. The CD11b-negative fraction, which contained astrocytes, was plated and used for experiments after reaching confluence.
NF-kB nuclear translocation
Cultured microglia were plated in eight chamber slides and treated with CD38 apigenin, NAD+, and NR for 4 h followed by LPS stimulation for 1 h. Cells were then fixed with 4% PFA containing 0.2% NP-40. Cells were processed for immunocytochemistry experiments with antibodies against Iba1 and p65 (8242, Cell Signaling Technology, Tokyo, Japan 1:200). Cell nuclei were visualized with DAPI (Sigma). Immunohistochemical labeling was visualized with alexa488- or Cy3-conjugated secondary antibodies, and images were obtained using a laser scanning confocal microscope EZ-C1. The nuclear fluorescence intensity of p65 was determined by Integrated Density – (Area of selected cell × mean fluorescence of background) using ImageJ software.
The experimental results are expressed as mean ± standard error of the mean (SEM), with the number of experiments indicated by “n.” No statistical evaluations were performed to predetermine sample size, but our sample sizes were similar to those generally used in the field. One-way ANOVA followed by the Tukey-Kramer test or two-way ANOVA followed by Scheffe’s F test was used for the statistical analysis. P values < 0.05 were considered statistically significant.