Inhibition of CD38 and supplementation of nicotinamide riboside ameliorate lipopolysaccharide-induced neuroinflammation in the hippocampus
Authors: Roboon Jureepon , Hattori Tsuyoshi , Ishii Hiroshi , Takarada-Iemata Mika , Nguyen Dinh Thi , Heer Collin D. , O'Mealley Denis , Brenner Charles , Yamamoto Yasuhiko , Okamoto Hiroshi , Higashida Haruhiro , Hori Osamu 
Published: December 2020

Abstract
Background

Neuroinflammation is initiated by the activation of the brain’s innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in neurological conditions, including multiple sclerosis, traumatic brain injury, and Alzheimer’s disease. Nicotinamide adenine dinucleotide (NAD+) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that the deletion of CD38, an enzyme that converts NAD+ to calcium-mobilizing second messengers, increased NAD+ levels in the brain and suppressed neuroinflammation, glial activation, and demyelination in a cuprizone-induced demyelination model mouse. However, the direct effects of CD38 and NAD+ on neuroinflammation have not been clarified. Here, we investigated the effect of CD38 inhibition and NAD+ replacement in lipopolysaccharide (LPS)-induced neuroinflammation in mice.
Methods

To induce neuroinflammation, LPS (10 µg) was injected into the lateral cerebral ventricle of wild-type (WT) and CD38 knockout (KO) male ICR mice. Apigenin, a flavonoid with CD38 inhibitory activity, (40 mg/kg) or nicotinamide riboside (NR), an NAD+ precursor, (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. NAD+ levels in the hippocampus were measured, and neuroinflammation and neuronal damage in the hippocampus were assessed by qPCR, western blotting, and immunohistochemical analysis. In cell culture, mouse primary astrocytes and microglia were treated with apigenin (50 µM), NAD+ (200 µM), NR (200 µM), or 78c (0.5 µM; a specific CD38 inhibitor), 4 h before LPS (100 ng/mL) stimulation. Proinflammatory cytokine expression and NF-kB nuclear translocation were assessed by qPCR and immunocytochemical analysis, respectively.
Results

CD38 expression in the cortex and hippocampus increased after LPS administration. Inflammatory responses and glial activation after LPS injection were significantly lower in CD38 KO mice than in WT mice. Pre-administration of apigenin or NR for 7 d increased NAD+ levels in the brain and significantly suppressed the induction of cytokines and chemokines after LPS administration in mice. Moreover, LPS-induced glial activation and neurodegeneration were significantly suppressed under the same conditions. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD+, NR, or 78c. Finally, all these compounds suppressed the translocation of p65 to the nucleus by LPS in cultured microglia.
Conclusions

CD38-mediated neuroinflammation is linked to NAD+ consumption, and CD38 inhibition and NR supplementation may be beneficial for preventing neuroinflammation in pathological conditions.