Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice
Abstract
Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In aging increased oxidative stress and cerebromicrovascular endothelial dysfunction impair NVC, contributing to cognitive decline. There is increasing evidence showing that a decrease in NAD+ availability with age plays a critical role in a range of age-related cellular impairments but its role in impaired NVC responses remains unexplored. The present study was designed to test the hypothesis that restoring NAD+ concentration may exert beneficial effects on NVC responses in aging. To test this hypothesis 24-month-old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, for 2 weeks. NVC was assessed by measuring CBF responses (laser Doppler flowmetry) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. NMN supplementation rescued NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory and gait coordination. These findings are paralleled by the sirtuin-dependent protective effects of NMN on mitochondrial production of reactive oxygen species and mitochondrial bioenergetics in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, a decrease in NAD+ availability contributes to age-related cerebromicrovascular dysfunction, exacerbating cognitive decline. The cerebromicrovascular protective effects of NMN highlight the preventive and therapeutic potential of NAD+ intermediates as effective interventions in patients at risk for vascular cognitive impairment (VCI).
Keywords: Oxidative stress, ROS, Endothelial dysfunction, Functional hyperemia, Microcirculation
Graphical abstract
1. Introduction
Maintenance of cerebral homeostasis requires a tightly-controlled supply of oxygen and nutrients as well as washout of harmful metabolites through uninterrupted cerebral blood flow (CBF), which represents 15% of cardiac output [1]. The human brain comprises only 2% of the body's mass, yet it accounts for 20% of the resting total body O2 consumption. The brain has limited energy reserves and cerebral oxygen content can sustain unimpeded neuronal function for only a short time if CBF decreases [2]. Thus, during periods of intense neuronal activity there is a requirement for rapid adjustment of regional oxygen and glucose delivery to metabolic demand through spatially localized adaptive increases in CBF. This is ensured by an evolutionarily conserved physiological mechanism known as neurovascular coupling (NVC) or functional hyperemia [1]. The cellular mechanisms of NVC include release of vasodilator NO from the microvascular endothelium, in response to increased neuronal and astrocytic activation [3,4].
It is now increasingly recognized that (micro)vascular contributions to cognitive impairment and dementia (VCID) play a critical role in elderly patients [1]. There is growing evidence that NVC responses are compromised both in elderly subjects [[5], [6], [7], [8]] and aged laboratory animals [4,9], which may importantly contribute to the age-related decline in higher cortical function, including cognition [10] and gait performance [11]. This concept is supported by recent findings that pharmacologically induced neurovascular dysfunction in mice mimics important aspects of age-related cognitive impairment [12]. Further, our recent studies demonstrate that rescue of NVC responses by treatment with the mitochondria-targeted antioxidative peptide SS-31 [13] or pharmacological SIRT1 activators [4,14] mitigates cognitive impairment in aged mice. These successful preclinical studies provide proof-of-concept that development of translationally relevant therapeutic interventions that target molecular/cellular mechanisms contributing to age-related neurovascular dysfunction is feasible for prevention/treatment of cognitive impairment in elderly patients [4].
NAD+ is a rate-limiting co-substrate for sirtuin enzymes, which are key regulators of pro-survival pathways and mitochondrial function in the endothelial cells [[15], [16], [17], [18]]. There is increasing evidence that with age cellular NAD+ availability decreases [19,20], which is a critical driving force in aging processes. In support of this theory it was demonstrated that enhancing NAD+ biosynthesis extends lifespan in lower organisms [21] and improves health-span in mouse models of aging [22]. There is particularly strong evidence that in aged mice enhancing NAD+ biosynthesis by treatment with nicotinamide mononucleotide (NMN; a key NAD+ intermediate) [23] reverses age-related dysfunction in multiple organs, including the eye [24], skeletal muscle [19] and peripheral arteries [15,25]. A key mechanism underlying the anti-aging action of NMN treatment is reversing age-related decline in mitochondrial function [24]. Although there is strong evidence that mitochondrial dysfunction and increased mitochondrial oxidative stress contribute to cardiovascular dysfunction [[26], [27], [28]] and neurovascular impairment in aging [13], the potential protective effects of NMN on the aged cerebral microvasculature and NVC responses have not been investigated.
The present study was designed to test the hypothesis that NMN supplementation can rescue neurovascular coupling responses in aged mice by attenuating mitochondrial oxidative stress in cerebromicrovascular endothelial cells. To achieve this goal, aged mice were treated with NMN for two weeks. Mice were behaviorally evaluated on a battery of tests for characterization of cognitive function and motor coordination, which are sensitive to alterations in NVC responses. Then, functional tests for NVC responses and cerebromicrovascular endothelial function were performed. Markers of oxidative stress and expression of genes regulating neurovascular coupling responses, antioxidant defenses and mitochondrial function were assessed. To substantiate the in vivo findings the effects of NMN on mitochondrial ROS production and mitochondrial bioenergetics in cerebromicrovascular endothelial cells derived from aged animals were obtained in vitro.