Targeting sirtuin activity with nicotinamide riboside reduces neuroinflammation in a GWI mouse model

Highlights

• Blood NAD and Sirt1 are low in GW veterans with GWI.

• Treatment with NR increased NAD and alleviated fatigue-like behavior in a mouse model of GWI.

• Consistent with human GWI, blood NAD and Sirt1 are low in our mouse model of GWI.

• Brain NAD levels were normal in GWI mice but are increased in both GWI and control mice after NR treatment.

• Treatment with NR increased Sirt1 activity and reduced neuroinflammation in the brains of GWI mice.

• Targeting sirtuin activity with NR may represent a new avenue for treating GWI.

Abstract

Gulf War Illness (GWI) affects 30% of veterans from the 1991 Gulf War (GW), who suffer from symptoms that reflect ongoing mitochondria dysfunction. Brain mitochondria bioenergetics dysfunction in GWI animal models corresponds with astroglia activation and neuroinflammation. In a pilot study of GW veterans (n = 43), we observed that blood nicotinamide adenine dinucleotide (NAD) and sirtuin 1 (Sirt1) protein levels were decreased in the blood of veterans with GWI compared to healthy GW veterans. Since nicotinamide riboside (NR)-mediated targeting of Sirt1 is shown to improve mitochondria function, we tested whether NR can restore brain bioenergetics and reduce neuroinflammation in a GWI mouse model. We administered a mouse diet supplemented with NR at 100μg/kg daily for 2-months to GWI and control mice (n = 27). During treatment, mice were assessed for fatigue-type behavior using the Forced Swim Test (FST), followed by euthanasia for biochemistry and immunohistochemistry analyses. Fatigue-type behavior was elevated in GWI mice compared to control mice and lower in GWI mice treated with NR compared to untreated GWI mice. Levels of plasma NAD and brain Sirt1 were low in untreated GWI mice, while GWI mice treated with NR had higher levels, similar to those of control mice. Deacetylation of the nuclear-factor κB (NFκB) p65 subunit and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) was an increase in the brains of NR-treated GWI mice. This corresponded with a decrease in pro-inflammatory cytokines and lipid peroxidation and an increase in markers of mitochondrial bioenergetics in the brains of GWI mice. These findings suggest that targeting NR mediated Sirt1 activation restores brain bioenergetics and reduces inflammation in GWI mice. Further evaluation of NR in GWI is warranted to determine its potential efficacy in treating GWI.

Introduction

Nearly thirty years have elapsed since the 1991 Gulf War (GW) conflict, but 30% of veterans from this conflict continue to suffer from Gulf War Illness (GWI) and experience debilitating symptoms such as pain, fatigue and memory problems (Binns et al., 2008; White et al., 2016). Both clinical and animal studies suggest that disturbances in bioenergetics may be part of the underlying pathological features associated with the heterogeneous symptoms experienced by veterans with GWI (Abdullah et al., 2016; Shetty et al., 2017; Koslik et al., 2014; Rayhan et al., 2013a). Several imaging studies have shown abnormal levels of bioenergetics markers in the brains of veterans with GWI (Rayhan et al., 2013b; Rayhan et al., 2013c). Therefore, therapies that are aimed at improving mitochondrial bioenergetics in the brain may help with improving the general health and well-being of veterans with GWI.

Energy disturbances in veterans with GWI are characterized by increases in lipid peroxidation products in plasma, changes in mitochondrial plasma lipids, inability to recover phosphocreatine levels in muscle after exercise, and damage to mitochondrial DNA corresponding with reduced activity of mitochondrial enzymes involved in electron transport chain (ETC) and anti-oxidant defense (Abdullah et al., 2016; Koslik et al., 2014; Chen et al., 2017; Joshi et al., 2018). Evidence for central nervous system (CNS) bioenergetics disturbances is suggested by a study showing increased lactate utilization in the prefrontal cortex following an exercise challenge in a subset of veterans with GWI and glucose hypometabolism in the hippocampus of GW veterans with GWI (Rayhan et al., 2013a). In a GWI mouse model, neurobehavioral deficits corresponded with decreases in the Krebs cycle intermediary compounds and downregulation of pathways related to the conversion of pyruvate to lactate and gluconeogenesis (Abdullah et al., 2016). Lipid analysis of this mouse model showed alterations in mitochondria-specific lipids, such as cardiolipin (CL) and acylcarnitine levels in the brains of GWI mice (Abdullah et al., 2016). A decrease in several ETC enzymes was also observed in the brains of GW chemical-exposed mice (Zakirova et al., 2017). A rat model of GW chemical exposure also showed chronic oxidative stress, inflammation, and mitochondrial dysfunction along with memory and mood dysfunction in exposed rats (Shetty et al., 2017; Parihar et al., 2013). These studies suggest that mitochondria dysfunction may be a contributory factor in bioenergetics deficits in GWI, as it has been observed both in veterans with this condition and in mouse models developed using GW-relevant chemical exposures.

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor of many metabolic processes, including glycolysis, fatty acid β-oxidation and the Krebs cycle, while its reduced form, NADH, serves as an electron-rich source that takes part in oxidative phosphorylation (OXPHOS), ultimately contributing to the generation of adenosine triphosphate (ATP) as energy (Berger et al., 2004; Ruggieri et al., 2015). During OXPHOS, NAD+/NADH simply shuttle electrons and are not consumed during this process. However, NAD + is actively consumed by sirtuins during the protein deacetylation process (Sauve, 2010). It is well known that NAD + increases the expression and the activity of sirtuins (Sauve et al., 2006; ichiro and Guarente, 2014), which corresponds with reduced inflammation via nuclear-factor κB (NFκB) mediated pathways and targets mitochondria biogenesis through activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-dependent pathways (Chong et al., 2012; Hou et al., 2018; Hasan-Olive et al., 2019). Although the exact mechanisms remain unknown, depletion of NAD + or its reduced form NADH is considered a hallmark of aging and is also observed in chronic fatigue syndrome (CFS), which has some clinical resemblance to GWI (Alegre et al., 2010; Sun et al., 2016; Camacho-Pereira et al., 2016; Mikirova et al., 2012; Fang et al., 2017).

Given the importance of NAD + in cellular bioenergetics, various approaches have been explored for supplementing NAD+ (Hou et al., 2018; Lee and Yang, 2019; Cantó et al., 2012). Supplementation with the NAD + precursor nicotinamide riboside (NR) appears to be a viable option as this form of NAD + can enter the cell and cross the blood-brain-barrier (BBB) (Spector and Johanson, 2007). In the cell, NR is converted to NAD + by an enzyme nicotinamide riboside kinase (NRK) (Wang et al., 2018). Supplementation with NR in animal models of neurodegeneration reduces inflammation and attenuates neuropathological phenotypes through sirtuin-1 (Sirt1)-mediated deacetylation of the NFκB pathway (Hou et al., 2018; Chen et al., 2018). Others have shown that NR treatment enhances cognitive functioning and reduces glial activation (Hou et al., 2018; Liu et al., 2013). Treatment with NR may be a valuable therapeutic approach due to its high bioavailability and minimal toxicity in humans (Trammell et al., 2016). In a pilot human study, levels of NAD + rose by 3-fold after a single oral dose of 300 mg of NR (Trammell et al., 2016). We therefore hypothesize that NR treatment may reduce disturbances in mitochondrial bioenergetics, oxidative stress and chronic neuroinflammation. We therefore tested NR in a mouse model of GWI that exhibits neurobehavioral and brain bioenergetics deficits and neuroinflammation that are relevant to the etiology of GWI. The studies described below provide preclinical evidence for potential use of NR in treating the bioenergetic deficits in this GWI mouse model and provides translational relevance of these findings to veterans with GWI.