Inhibition of CD38 and supplementation of nicotinamide riboside ameliorate lipopolysaccharide-induced microglial and astrocytic neuroinflammation by increasing NAD+
Neuroinflammation is initiated by activation of the brain\xe2\x80\x99s innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in various neurological conditions including multiple sclerosis and Alzheimer\xe2\x80\x99s disease. Nicotinamide adenine dinucleotide (NAD+) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that deletion of CD38, which consumes NAD+, suppressed cuprizone-induced demyelination, neuroinflammation and glial activation. However, it is still unknown whether CD38 directly affects neuroinflammation through regulating brain NAD+ level. In this study, we investigated the effect of CD38 deletion and inhibition and supplementation of NAD+ on lipopolysaccharide (LPS)-induced neuroinflammation in mice. First, intracerebroventricular injection of LPS significantly increased CD38 expression especially in the hippocampus. Deletion of CD38 decreased LPS-induced inflammatory responses and glial activation. Pre-administration of apigenin, a flavonoid with CD38 inhibitory activity, or nicotinamide riboside (NR), an NAD+ precursor, increased NAD+ level, and significantly suppressed induction of cytokines and chemokines, glial activation and subsequent neurodegeneration after LPS administration. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD+, NR or 78c, the latter a specific CD38 inhibitor. Finally, all these compounds suppressed NF-\xce\xbaB signaling pathway in microglia. These results suggest that CD38-mediated neuroinflammation is linked to NAD+ consumption and that boosting NAD+ by CD38 inhibition and NR supplementation directly suppress neuroinflammation in the brain.
Introduction<!>Chemicals<!>Animals and treatments<!>Reverse transcription quantitative polymerase chain reaction (RT-qPCR)<!>Western blot analyses<!>Differential expression analysis of published microarray studies<!>Immunohistochemistry<!>Analysis of NAD+ levels in brain tissues<!>Glial cell cultures<!>NF-\xce\xbaB nuclear translocation<!>Statistical analysis<!>CD38 expression was increased after LPS injection.<!>LPS-induced neuroinflammation and glial activation were attenuated in CD38 KO mice.<!>NR and apigenin ameliorated LPS-induced neuroinflammation and glial activation.<!>NR and apigenin attenuated LPS-induced neurodegeneration<!>NR, apigenin and 78c reduced inflammatory response in vitro.<!>NR, apigenin and 78c suppressed NF-\xce\xbaB signaling pathway.<!>Discussion<!>The role of NAD+ and CD38 in LPS-induced neuroinflammation<!>Future prospects
<p>Neuroinflammation is a biological response initiated by tissue injury or infection in the central nervous system (CNS) to eliminate pathogenic components and induce tissue remodeling. However, insufficient control of neuroinflammation leads to the progression of many neurological conditions such as multiple sclerosis, traumatic brain injury, and Alzheimer's disease (Akiyama et al. 2000; Lucas et al. 2006; Frischer et al. 2009). Glial cells, including microglia and astrocytes, are involved in the immune response in the CNS and play important roles in the development of neuroinflammation. It is well documented that sustained inflammatory responses cause the release of harmful mediators such as cytokines and chemokines from activated glial cells, and further affect neuronal cells by triggering neurodegeneration (Liu et al. 2011; Norden et al. 2016; Lian et al. 2016; Jo et al. 2017). Therefore, suppression of neuroinflammation may be an important therapeutic target in neurological diseases.</p><p>CD38 is a type II and type III transmembrane protein (Zhao et al. 2012) that catalyzes the formation of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP+) from nicotinamide adenine dinucleotide (NAD+) and related metabolites (Hogan et al. 2019) to mobilize calcium from intracellular stores (Takasawa et al. 1993b; Takasawa et al. 1993c; Lee 2004; Guse 2005; Malavasi et al. 2008; Nam et al. 2020; Hogan et al. 2019). CD38 has diverse functions. For example, it promotes the secretion of insulin from pancreatic beta cells and oxytocin from hypothalamic neurons, thereby promoting social behaviors that are deficient in mouse models of autism spectrum disorder (ASD) (Takasawa et al. 1993a; Jin et al. 2007; Higashida et al. 2012; Kim et al. 2016; Okamoto et al. 2017; Gerasimenko et al. 2020). CD38 is also associated with glial cell functions. We previously demonstrated that under physiological conditions, CD38 regulates the maturation of astrocytes and differentiation of oligodendrocyte precursor cells using NAD+ in the brain (Hattori et al. 2017). We also recently discovered that CD38 is crucially involved in the cuprizone-induced demyelination model in mice. CD38 expression was increased in the brain after cuprizone administration in a manner associated with the production of pro-inflammatory molecules, glial activation, and subsequent neurodegeneration. Deletion of CD38 suppresses these phenotypes by increasing NAD+ levels in the brain (Roboon et al. 2019). CD38 has also been reported to play a critical role in the pathology of experimental autoimmune encephalomyelitis, another model of demyelination in mice (Herrmann et al. 2016).</p><p>NAD+ is synthesized in four gene-encoded biosynthetic pathways from tryptophan, nicotinic acid (NA), nicotinamide (NAM), and nicotinamide riboside (NR) (Bogan & Brenner 2008; Bieganowski & Brenner 2004). The NR biosynthetic pathway is both unique and highly efficient in rodents and humans (Trammell et al. 2016) and corresponds to a biosynthetic pathway that is transcriptionally induced in heart failure (Diguet et al. 2018) and central brain injury (Vaur et al. 2017). Moreover, in an Alzheimer's disease model, NR improved both cognitive function and protected against neurodegeneration (Gong et al. 2013; Hou et al. 2018). These studies suggest that NAD+ has suppressive effect on neuroinflammation, but it has not been tested directly by using neuroinflammation mouse model.</p><p>The NAD+ level in the brain can also be increased by compounds that depress NAD+ consumption. Thiazoloquin(az)olin(on)e, 78c, is a chemical that has a specific inhibitory effect on CD38 activity (CD38i) and has been reported to arrest age-related NAD+ decline (Tarrago et al. 2018; Chini et al. 2020). Similarly, a natural flavonoid apigenin (4'5,7-trihydroxyflavone) (Nijveldt et al. 2001; Shukla & Gupta 2010; Venigalla et al. 2015), was reported to inhibit CD38 enzymatic activity and increase intracellular NAD+ levels (Escande et al. 2013). In this study, to clarify roles of CD38 and NAD+ for neuroinflammation in the brain, we investigated the anti-inflammatory effects of NAD+ on lipopolysaccharide (LPS)-induced neuroinflammation by inhibition of CD38 and supplementation of NR.</p><!><p>The chemicals used in this study were as follows: LPS (Cat. No. 20389-04, Nacalai Tesque), β-NAD+ (Cat. No. 24334-97, Nacalai Tesque), nicotinamide riboside (Cat. No. ASB-00014315, ChromaDex), apigenin (Cat. No. 016-18913, Wako), and compound 78c (Cat. No. 538763, Calbiochem).</p><!><p>Wild-type (WT) and CD38 knockout (KO) male ICR mice (10–11 week old) were used for the experiments (body weight; 30-36g) (n = 260). CD38 KO mice were generated as described previously and backcrossed for more than eight times (Kato et al. 1999). This study was not pre-registered and no randomization/blinding was performed. No exclusion criteria were pre-determined, and no animals were excluded. Sample size for each experiment was determined based on our previous studies (Roboon et al. 2019). 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. All the animal experiments were performed between10:00 a.m. and 5:00 p.m. LPS injection was performed as previously described (Zhao et al. 2019). Mice were anesthetized with a mixture of anesthetic, muscle relaxant, analgesic and sedative such as medetomidine (0.3 mg/kg; Domitor®; Nippon Zenyaku Kogyo), butorphanol (5.0 mg/kg; Vetorphale®; Meiji Seika Pharma), and midazolam (4.0 mg/kg; Midazolam Sandoz®; Sandoz) to minimize the pain. 10 μg LPS dissolved in 5 μ 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. 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. The mice were deeply anesthetized with isoflurane (1ml/ml, Cat. No. 26675-46-7, Fujifilm Wako Pure Chemical Industries. Ltd.) 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).</p><!><p>RT-qPCR was performed as previously described (Hattori et al. 2010). In brief, total RNA was extracted from the cerebral cortex, hippocampus, or cultured cells using the FASTGene™ RNA Basic Kit. (Cat. No. FG-80250, Nippon Genetics Co., Ltd), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814, Applied Biosystems). Individual cDNA sequences were amplified using the Thunderbird™ SYBR qPCR® Mix (Cat. No. 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 Table S1.</p><!><p>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 (RRID: AB_1241945, R and D systems, 1:500), anti-glial fibrillary acidic protein (GFAP) (RRID: AB_477035, Sigma-Aldrich, 1:2000), anti-ionized calcium binding adaptor molecule 1 (Iba1) (RRID: AB_839504, Fujifilm Wako Pure Chemical Industries. Ltd, 1:1000), p-Stat3 (Tyr705) (RRID: AB_2491009, Cell Signaling Technology) and Stat3 (RRID: AB_331588, Cell Signaling Technology) 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 (RRID: AB_631746, Santa Cruz Biotechnology, In, 1:5000), anti-mouse (RRID: AB_2687626, Santa Cruz Biotechnology, In, 1:5000) or anti-goat (RRID: AB_628490, Santa Cruz Biotechnology, In, 1:2000) horseradish peroxidase-linked immunoglobulin G for 2 h at room temperature, and the membranes were washed several times with TBST. Immunoreactivity was detected using an enhanced chemiluminescence system (Cat. No. ELLUF0100, Merck). The intensity of each band was quantified using ImageJ software (https://imagej.nih.gov/ij/, RRID:SCR_003070).</p><!><p>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 (Davis & Meltzer 2007). 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" (Ritchie et al. 2015). 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.</p><!><p>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 (RRID: SCR_018061, Leica). 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) (RRID: AB_2564642, BioLegend, 1:500), MAP2 (RRID: AB_10693782, Cell Signaling Technology, 1:500). Subsequently, alexa488- (RRID: AB_2536161, Thermo Fisher Scientific Inc.,1:200) or Cy3-conjugated secondary antibodies (RRID: AB_2307443, Jackson ImmunoResearch Laboratories, Inc., 1:200) were used to visualize immunolabeling. Cell nuclei were visualized with DAPI (RRID: AB_2336790, Vector Laboratories, 1:5000). 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). The fluorescence intensity of each region of the hippocampus/total area was analyzed using ImageJ software.</p><!><p>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 (Cat. No. E2ND-100, BioAssay System) 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).</p><!><p>Astrocyte and microglial cultures were prepared from mixed glial cultures as previously described (Roboon et al. 2019). 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 (Cat. No. 130-093-634, microbeads conjugated to monoclonal anti-human/mouse CD11b antibody, Miltenyi Biotec, Bergisch Gladbach) and applied to a magnetic column fitted into a MidiMACS™ cell separator (Cat. No. 130-042-302, 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.</p><!><p>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 (RRID: AB_10859369, Cell Signaling Technology, 1:200). Cell nuclei were visualized with DAPI (Vector Laboratories). Immunohistochemical labeling was visualized with Alexa Fluor™ 488- (RRID: AB_143165, Thermo Fisher Scientific Inc., 1:200) or Cy3- (RRID: AB_2307351, Jackson ImmunoResearch Laboratories, Inc., 1:200) 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.</p><!><p>All statistical analyses were performed using Statcel Ver.3 (OMS Publishing Inc.). 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. Since a Shapiro-Wilk test showed normal distribution of all data sets in this study, a parametric analysis of variance (ANOVA) was applied to test for statistical differences of the means. 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. No statistical test for outliners was conducted and no data point was excluded.</p><!><p>We first investigated the expression of CD38 in LPS-induced neuroinflammation by RT-qPCR and Western blot analysis. Tissue samples were collected at 6, 12 and 24 h after intracerebroventricular (i.c.v.) injection of LPS (Figure 1a). Consistent with our recent report in the cuprizone-induced demyelinating model (Roboon et al. 2019), RT-qPCR analysis revealed that the expression of Cd38 mRNA gradually increased in both the cerebral cortex and hippocampus, and reached significance after 6 h of LPS injection. Furthermore, the level of Cd38 mRNA expression was higher in the hippocampus than in the cerebral cortex at any time point after LPS injection (Figure 1b; for results of ANOVA analysis, see Table 1). Western blot analysis confirmed the gradual increase in CD38 protein expression after LPS injection. The expression level of CD38 protein was also higher in the hippocampus than in the cerebral cortex (Figure 1c and Table 1). Furthermore, analysis of microarray data also showed elevated expression levels of CD38 mRNA in the brain after LPS injection and in the primary microglia after LPS stimulation (Figure S1). Interestingly, we found that other NAD+-consuming enzymes, PARPs (poly (ADP-ribose) polymerases), were also significantly increased by induction of neuroinflammation (Figure S1). These results suggest that CD38 and other NAD-consuming molecules may be involved in LPS-induced neuroinflammation.</p><!><p>To evaluate the effect of CD38 deletion on LPS-induced neuroinflammation and glial activation, we analyzed the expression of pro-inflammatory genes and glia-associated genes after LPS injection by RT-qPCR, Western blot analysis and immunohistochemistry. Tissues were collected 6, 12 and 24 h after LPS injection (Figure 2a). RT-qPCR analysis revealed that the expression of genes such as Il1b, IIl6, Tnf, Nos2, Ccl2, and Ccl3 was robustly increased in WT mice 6 h after LPS injection (Figure 2 b–g). In all cases except Il1b, mRNA expression reached a peak 6 h after LPS injection and gradually decreased thereafter (Figure 2 c–g). The expression of these genes was significantly lower in CD38 KO mice than in WT mice (Figure 2 b–g and Table 1). The expression of mRNAs for Gfap and Iba1, an astrocytic and microglial marker, respectively, gradually increased and reached significantly high levels at 12 h and 24 h, respectively, after LPS injection in WT mice (Figure 3a and Table1). Expression of Gfap and Iba1 was significantly lower in CD38 KO mice. Immunohistochemical analysis further revealed that the immunoreactivity of GFAP and Iba1 was clearly increased in both the CA1 and CA3 regions of the hippocampus 24 h after LPS injection, but the level was significantly lower in CD38 KO mice (Figure 3b, c and Table 1). These data indicate that deletion of CD38 suppresses LPS-induced neuroinflammation and glial activation.</p><!><p>The phenotypes in CD38 KO mice described above and our recent study (Roboon et al. 2019) suggest that pharmacological inhibition of CD38 and/or boosted brain NAD+ level may be capable of depressing pathological inflammation in neurological diseases. Therefore, we assessed the effect of apigenin, a natural flavonoid that acts as a CD38 inhibitor (Escande et al. 2013), and NR, an NAD+ precursor (Bieganowski & Brenner 2004), on NAD+ levels in the context of LPS-injected neuroinflammation. Apigenin or NR was administered intraperitoneally once per day for 7 consecutive days, followed by LPS injection 6 h after the final administration of apigenin or NR. Tissues for NAD+ measurement and RT-qPCR were collected 6 h after the last administration and 6 h after LPS injection, respectively (Figure 4a). The NAD+ levels in the hippocampus were significantly higher in apigenin- or NR-administered mice than in control mice, and the levels were similar in both conditions (Figure 4b and Table 1). WT mice were pre-administered with apigenin or NR for 7 d, and then injected with LPS 6 h after the final administration of each compound. RT-qPCR analysis revealed that the induction of inflammatory genes such as Il1b, Il6 and Tnf was suppressed in compound-pre-administered mice than in control mice (Figure 4c–e and Table 1). Furthermore, we examined NAD+ levels and neuroinflammation in CD38 KO mice administered NR or apigenin. Tissues were collected 6 h after LPS injection (Figure 4f). Although CD38 KO mice showed significantly higher NAD+ levels than WT mice in any group, there was not further increase of NAD+ by NR or apigenin in CD38 KO mice (Figure 4g and Table 1). Consistent with CD38 as the target of apigenin, apigenin did not increase the elevated NAD+ level in CD38 KO mice. Additionally, CD38 KO mice exhibited lower expression of proinflammatory genes as shown in Fig. 2, and apigenin or NR did not further decrease these gene expression programs (Figure 4h–j and Table 1). To assess whether boosting NAD+ prior to LPS-induced neuroinflammation is important or not, apigenin or NR was administered once immediately after LPS injection. Tissues are collected 6 h after LPS injection (Figure 4k). Single-dose administration of apigenin did not increased NAD+ level. NR slightly, but not significantly, increased NAD+ level. However, the level was much lower than that of CD38 KO mice (Figure 4l and Table 1). Correlated with these NAD+ levels, NR had weak suppressive effect on expression of inflammatory genes compared with that of CD38 KO mice (Figure 4m–o and Table 1).</p><p>To assess the effect of apigenin and NR on glial activation, these compounds were pre-administered intraperitoneally once per day for 7 consecutive days, followed by LPS injection. Brains were fixed 24 h after LPS injection (Figure 5a). Immunohistochemical analysis revealed enhanced levels of immunoreactivity for GFAP and Iba1 24 h after LPS injection, but levels were reduced in apigenin or NR pre-administered mice compared to the control group of mice (Figure 5b–d and Table 1). These results suggest that the levels of CD38 and NAD+ determine the state of neuroinflammation and the activation of both astrocytes and microglia in the brain.</p><!><p>As LPS-induced neuroinflammation often leads to neurodegeneration (Qin et al. 2007; Pintado et al. 2012; Deng et al. 2014; Wang et al. 2019), the effect of apigenin and NR on neuronal damage was evaluated after LPS injection. Immunohistochemistry for non-phosphorylated neurofilament H (SMI32), a marker of damaged axons, revealed that neurodegeneration occurred within 24 h after LPS injection in both the CA1 and CA3 regions of the hippocampus, and the level was significantly lower in apigenin or NR pre-administered mice (Figure 6a, b and Table 1). Consistent with these results, immunohistochemistry for MAP2 revealed that the intensity of normal axons and dendrites decreased after LPS injection, and this decrease was partially recovered by apigenin or NR pre-administration (Figure 6a, c and Table 1). These results suggest that administration of NR or apigenin attenuated not only LPS-induced neuroinflammation but also subsequent LPS-induced neurodegeneration.</p><!><p>Silencing of the cd38 gene and the addition of NAD+ suppressed LPS-induced activation of astrocytes and microglia in vitro (Hattori et al. 2017; Roboon et al. 2019), the effects of NR, apigenin, and 78c, the latter a CD38-specific inhibitor, on the inflammatory response were examined using cultured microglia and astrocytes. RT-qPCR analysis revealed that LPS strongly upregulated the expression of pro-inflammatory genes, such as Il1b, Il6, Tnf, Nos2 in microglia and/or astrocytes. In microglia, the expression of Il1b and Il6 was significantly reduced by all compounds, and that of Tnf and Nos2 was significantly decreased by NAD+ and NR, and apigenin, respectively (Figure 7a–d and Table 1). In addition, in astrocytes, all compounds showed a tendency to suppress the induction of inflammatory genes, although the expression of Il6 was significantly decreased by apigenin, and that of Tnf was significantly reduced by NAD+ after LPS injection (Figure 7e–h and Table 1). These results suggest that inhibition of CD38 and increased NAD+ status directly suppress LPS-induced inflammatory responses, especially in microglia.</p><!><p>It is known that LPS binds to toll-like receptor 4, which activates the NF-κB cascade and consequently induces pro-inflammatory genes. To determine the relevance of CD38 inhibition and supplementation of NAD+ in the NF-κB signaling pathway, the nuclear translocation of NF-κB was examined after LPS stimulation in cultured microglia. Immunocytochemical analysis revealed that the intensity of p65, a major component of NF-κB, in the nucleus was strongly increased 1 h after LPS stimulation. In contrast, it was significantly reduced by all the compounds (Figure 8a, b and Table 1). NF-κB activation in astrocytes was also reduced by these compounds 1 h after LPS stimulation (Figure S2). Furthermore, to investigate effect of NAD+ on JAK/STAT signaling pathway, we examined Stat3 and p-Stat3 expression in CD38 KO mice and NR- or apigenin-administered mice after LPS injection. Although p-Stat3 was clearly increased by LPS injection, its expression was not changed by deletion of CD38 nor administration of these compounds (Figure S3). These results suggest that intracellular NAD+ suppresses LPS-induced NF-κB activation.</p><!><p>In the current study, we investigated the effect of CD38 inhibition and supplementation of NAD+ on LPS-induced neuroinflammation in mice. CD38 expression was increased both in the hippocampus and in the cerebral cortex after LPS injection. Deletion of CD38 decreased LPS-induced neuroinflammation and glial activation Consistently, pre-administration of apigenin and NR increased NAD+ concentration in the brain, and suppressed LPS-induced inflammatory response, glial activation, and neurodegeneration. In primary cultured glial cells, compounds such as 78c, apigenin, NAD+, and NR effectively suppressed the activation of NF-κB and the induction of cytokines and chemokines after LPS treatment. These results suggest that boosting NAD+ by CD38 inhibition and NR supplementation has suppressive effect on neuroinflammation in the brain.</p><!><p>To date, several studies, including ours, have suggested the involvement of CD38 in neuroinflammation. Deletion of CD38 in the APP.PS Alzheimer's disease model exhibited a significant reduction in Aβ plaque load and microglia/macrophage accumulation in the brain compared with APP.PS mice with CD38 intact (Blacher et al. 2015). Our previous study also revealed that CD38 KO mice ameliorated demyelination, glial activation, and neuroinflammation in cuprizone-induced demyelination (Roboon et al. 2019). To explore the direct effects of CD38 deletion or inhibition, and supplementation of NAD+ on neuroinflammation, we employed a mouse model of LPS-injection into the lateral cerebral ventricle in the current study. Our results demonstrated that CD38 deletion or inhibition, and supplementation with NAD+ suppressed neuroinflammation at earlier stages (within 6 h) after LPS administration in vivo (Figure 2 and 4) and in vitro (Figure 7 and 8), and these effects seem to be correlated with the increased level of NAD+ in the brain (Figure 4b and g) (Roboon et al. 2019). Administration of NR did not have further suppressive effect on neuroinflammation in CD38 KO mice (Figure 4h–j). This might because CD38 KO mice had much higher NAD+ levels than WT mice in any group and there was no further increase of NAD+ by NR administration in CD38 KO mice (Figure 4g). Although pre-administration of apigenin or NR for 7 days significantly reduced LPS-induced neuroinflammtion, single-dose administration of these compounds simultaneously with LPS injection had no or weak effect of increasing NAD+ and suppression of neuroinflammation (Figure 4). To boost NAD+ level enough to mitigate neuroinflammation, multiple pre-administration of apigenin and NR seems to be necessary. Furthermore, NAD+ level was not significantly decreased by LPS injection in WT mice (Figure 4l). Therefore, boosting NAD+ prior to LPS-induced neuroinflammation seems to be more important than reducing depletion of NAD+ after LPS injection.</p><p>The biology of inflammation suggests that expression of inflammatory markers, including CD38, cytokines, and chemokines, constitutes a homeostatic attempt at controlling infection or environmental damage. However, uncontrolled inflammatory responses are clearly neurotoxic. The behavioral deficits of mice with CD38 deletion suggest that the formation of CD38-dependent second messengers, including cADPR (Takasawa et al. 1993c) and NAADP (Nam et al. 2020), is required for oxytocin signaling, social intelligence, and parenting (Jin et al. 2007). In the case of neuroinflammatory responses, we consider that the formation of CD38-dependent signals might induce such activities and/or elevated NAD might depress such activities.</p><p>Expression of glial markers such as GFAP and Iba1 (Figure 3 and 5), and those of neurodegeneration such as SMI32 (Figure 6) were increased after LPS injection, and the levels were reduced in the conditions where CD38 was deleted or inhibited, and NAD+ synthesis was supported by precursor supplementation. Since proinflammatory genes were elevated at earlier stages than glial markers after LPS administration, it is likely that the suppressive effects on glial activation are secondary to the anti-inflammatory effect of CD38 deletion or inhibition, and support of NAD+ synthesis. However, we cannot rule out the possibility that CD38 directly regulates glial activation, since CD38 enhances GFAP expression during the postnatal development stages in vivo (Hattori et al. 2017).</p><p>In contrast to the potential mechanisms by which CD38 and calcium release promote inflammatory gene expression, CD38 inhibition and NR supplementation both converge on elevated NAD+- mediated suppression of inflammatory responses. We recently showed that coronavirus infection induces transcriptional induction of a set of PARP-related genes that depleted cellular NAD+ and that supplementation with NAD+ precursors or pharmacological activation of NAD+ synthesis can boost the activity of the highly transcribed PARP genes while providing partial protection against viral replication (Heer et al. 2020). Here, we show that many of the same PARP genes are upregulated after LPS stimulation in vitro and in vivo (Figure S1). We therefore suggest that depressed activities of NAD-dependent enzymes (other than CD38) mediate inflammatory gene transcription. Three potential mediators are SIRT2, which reportedly prevents microglial activation by promoting NF-κB deacetylation (Pais et al. 2013), SIRT1, which potentially depresses NF-κB activity via deacetylation of p65/RelaA (Caon et al. 2020) and PARP10, which inhibit activation of NF-κB and downstream target genes in response to IL-1β and TNF-α in a manner that depends on its catalytic activity and poly-ubiquitin binding activities (Verheugd et al. 2013). As glia-derived cytokines such as IFN-γ and TNF-α synergistically promote neuronal degeneration with other toxic factors (Suzumura et al. 2006), the reduced levels of neuronal damage by NR or apigenin administration are likely attributed to decreased induction of pro-inflammatory molecules. Consistent with this proposed mechanism, three weeks of oral high-dose NR has been shown to depress the circulation of inflammatory cytokines in a small placebo-controlled trial of older men (Elhassan et al. 2019). NAD+ may also protect neurons directly because NAD+ degradation has been directly linked to axonal degeneration. Sterile alpha and the TIR motif containing 1 (SARM1) initiates a local destruction program of axons through a process that involves the catastrophic depletion of axonal NAD+ (Essuman et al. 2017).</p><p>These results are not without caveats. For example, apigenin showed stronger effects than other compounds such as NR and 78c to suppress Il6 and Nos2 in cultured microglia and Il6 in cultured astrocytes (Figure 7). Moreover, apigenin decreased cytokine expression even in LPS-injected CD38 KO mice (Figure 4h–j), suggesting that it has targets beyond CD38, which could include MAPK, Akt, JNK (Ginwala et al. 2019) or the GSK3b/Nrf2 signaling pathway (Chen et al. 2019).</p><!><p>Neuroinflammation is a contributing factor in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease (Glass et al. 2010; Ginwala et al. 2019). Importantly, the vulnerability of hippocampal neurons that leads to cognitive impairment is strongly associated with neuroinflammation (Semmler et al. 2013; Tan et al. 2014). In fact, apigenin and NR have been reported to have beneficial effects on neurodegenerative disease models (Zhao et al. 2013; Hou et al. 2018). Apigenin has been reported to cross the blood-brain barrier (BBB) and increase NAD+ concentration in the brain (Balez et al. 2016). NR can also increase NAD+ levels in the brains of Alzheimer's disease model mice when orally administered (Gong et al. 2013). In ongoing work, we aim to clarify the neuroprotective mechanisms in disease models such as multiple sclerosis and Parkinson's disease models in order to better identify biomarkers that will enable successful clinical translation.</p><p>We identified novel effects of NR and apigenin on LPS-induced neuroinflammation. Testing these compounds in neurodegenerative diseases, aging, and neurodevelopmental disease models may be important for the development of new therapies for these diseases.</p>