The Interrelation Between Brain Oxidative Metabolism and Extracellular Potassium in the Unanesthetized Gerbil

The unanesthetized gerbil model was used to study the interrelation between metabolic, electrical and ionic activities in the brain. A combined K+DC surface electrode with a fiber optic light guide was implanted above the parietal cortex and cemented to the skull. The subjects were exposed to various pathological and physiological conditions such as anoxia, hypoxia, spreading cortical depression, and ischemia. During anoxia a leakage of cellular K⁺e was detected simultaneously with an increased level of NADH. The recovery phase in a few animals was followed by a spreading depression phenomenon. Exposing the brain to spreading depression led to a typical oxidation cycle of NADH, and the shape of this cycle was affected by hypoxia. Unilateral carotid artery ligation induced localized ischemia that affected cellular and K⁺e responses to spreading depression. Bilateral carotid artery occlusion increased NADH concentration to its maximum level; as a result, K⁺e also accumulated. Complete restoration of NADH and K⁺e to normoxic levels occurred after a few minutes, depending on the duration of the occlusion.     

Simultaneous monitoring of tissue P2 and NADH fluorescence during synaptic stimulation and spreading depression reveals a transient dissociation between oxygen utilization and mitochondrial redox state in rat hippocampal slices

Nicotinamide adenine dinucleotide (NADH) imaging can be used to monitor neuronal activation and ascertain mitochondrial dysfunction, for example during hypoxia. During neuronal stimulation in vitro, NADH normally becomes more oxidized, indicating enhanced oxygen utilization. A subsequent NADH overshoot during activation or on recovery remains controversial and reflects either increased metabolic activity or limited oxygen availability. Tissue P₂ measurements, obtained simultaneously with NADH imaging in area CA1 in hippocampal slices, reveal that during prolonged train stimulation (ST) in 95% O₂, a persistent NADH oxidation is coupled with increased metabolic demand and oxygen utilization, for the duration of the stimulation. However, under conditions of either decreased oxygen supply (ST-50% O₂) or enhanced metabolic demand (K⁺-induced spreading depression (K⁺-SD) 95% O₂) the NADH oxidation is brief and the redox balance shifts early toward reduction, leading to a prolonged NADH overshoot. Yet, oxygen utilization remains elevated and is correlated with metabolic demand. Under these conditions, it appears that the rate of NAD⁺ reduction may transiently exceed oxidation, to maintain an adequate oxygen flux and ATP production. In contrast, during SD in 50% O₂, the oxygen levels dropped to a point at which oxidative metabolism in the electron transport chain is limited and the rate of utilization declined.     

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD⁺ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD⁺ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K⁺, typical correlates of neuronal activity, induced a more reduced NADH/NAD⁺ redox ratio. While application of glutamate decreased [ATP], K⁺ as well as the combination of glutamate and K⁺ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K⁺ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg²⁺ caused a shift of the NADH/NAD⁺ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.     

In situ studies of oxidative energy metabolism during transient cortical ischemia in cats.

Changes in oxidative energy metabolism were monitored noninvasively during periods of transient cortical ischemia in cats by means of fluorometric observation of the redox level of intramitochondrial NADH and by dual wavelength reflectance spectrophotometry of cytochrome a. The latter technique also allowed measurements of the oxygenation state of hemoglobin and of blood volume in the optical field. We report here that incomplete ischemia is accompanied by increased levels of NADH and reduced cytochrome a, an increase in the unloading of O₂ from hemoglobin and a decrease in the blood volume, but these all turn back toward baseline with the establishment of “collateral” circulation. Complete ischemia is accompanied by sustained increased levels of reduced NAD and cytochrome a, the extent of which equaled the reduction levels produced by terminal anoxia. When short ischemic episodes were repeated, the rates of return of NAD and cytochrome a to baseline redox levels were faster after each successive ischemia, while blood volume returned at the same rate and hemoglobin saturation returned more slowly to its original state of oxygenation. We interpret that the primary lesion of short ischemic episodes is the uncoupling of oxidative phosphorylation.     

A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling,thereby reducing energy requirements during ischemia

Polyphenol resveratrol (RSV) has been associated with Silent Information Regulator T1 (SIRT1) and AMP‐activated protein kinase (AMPK) metabolic stress sensors and probably responds to the intracellular energy status. Our aim here was to investigate the neuroprotective effects of RSV and its association with SIRT1 and AMPK signaling in recurrent ischemia models. In this study, elderly male Wistar rats received a combination of two mild transient middle cerebral artery occlusions (tMCAOs) as an in vivo recurrent ischemic model. Primary cultured cortical neuronal cells subjected to combined oxygen–glucose deprivation (OGD) were used as an in vitro recurrent ischemic model. RSV administration significantly reduced infarct volumes, improved behavioral deficits and protected neuronal cells from cell death in recurrent ischemic stroke models in vivo and in vitro. RSV treatments significantly increased the intracellular NAD⁺/NADH ratio, AMPK and SIRT1 activities, decreased energy assumption and restored cell energy ATP level. SIRT1 and AMPK inhibitors and specific small interfering RNA (siRNA) for SIRT1 and AMPK significantly abrogated the neuroprotection induced by RSV. AMPK‐siRNA and inhibitor decreased SIRT1 activities; however, SIRT1‐siRNA and inhibitor had no impact on phospho‐AMPK (p‐AMPK) levels. These results indicated that the neuroprotective effects of RSV increased the intracellular NAD⁺/NADH ratio as well as AMPK and SIRT1 activities, thereby reducing energy ATP requirements during ischemia. SIRT1 is a downstream target of p‐AMPK signaling induced by RSV in the recurrent ischemic stroke model.     

Impaired antioxidant status and reduced energy metabolism in autistic children

Accumulating evidence suggests that oxidative stress induced mechanisms are believed to be associated with the pathophysiology of autism. In this study, we recruited 19 Omani autistic children with age-matched controls to analyze their plasma and serum redox status and the levels of ATP, NAD⁺ and NADH using well established spectrophotometric assays. A significant decrease was observed in the levels of plasma total antioxidants (TA), reduced glutathione (GSH), superoxide and catalase activity in Omani autistic children as compared to their age-matched controls. In contrary, the level of plasma glutathione peroxidase (GSH-Px) was significantly increased in autistic children. Reduced serum NAD⁺ and ATP levels and lower NAD⁺:NADH ratio were observedin patients with autism compared to controls. Finally, a significant inverse correlation was observed between plasma GSH, SOD, catalase activity, and serum NAD⁺ and ATP levels, and autism severity using Childhood Autism Rating Scale (CARS) scores. The levels of plasma GSH-Px and serum NADH correlated strongly with autism severity whilst no significant correlation was observed for plasma TA. Our data suggests that increased vulnerability to oxidative stress in autism may occur as a consequence of alterations in antioxidant enzymes leading to mitochondrial dysfunction.     

The interrelation between brain oxidative metabolism and extracellular potassium in the unanesthetized gerbil

The unanesthetized gerbil model was used to study the interrelation between metabolic, electrical and ionic activities in the brain. A combined K+DC surface electrode with a fiber optic light guide was implanted above the parietal cortex and cemented to the skull. The subjects were exposed to various pathological and physiological conditions such as anoxia, hypoxia, spreading cortical depression, and ischemia. During anoxia a leakage of cellular K+e was detected simultaneously with an increased level of NADH. The recovery phase in a few animals was followed by a spreading depression phenomenon. Exposing the brain to spreading depression led to a typical oxidation cycle of NADH, and the shape of this cycle was affected by hypoxia. Unilateral carotid artery ligation induced localized ischemia that affected cellular and K+e responses to spreading depression. Bilateral carotid artery occlusion increased NADH concentration to its maximum level; as a result, K+e also accumulated. Complete restoration of NADH and K+e to normoxic levels occurred after a few minutes, depending on the duration of the occlusion.     

Factors affecting the oxygen balance in the awake cerebral cortex exposed to spreading depression

Oxygen balance was evaluated in the cerebral cortex, using the surface fluorometry technique of the intramitochondrial NADH redox state, exposed to various physiological and pathological situations. Using flexible fiber optic light guide, connected to the brain surface via a cemented holder, the measurements were done continuously from the awake rat and gerbil. In few experiments the NADH redox state was correlated with the electrical and ionic activity (measured by surface K⁺ and DC electrodes). Three different animal models were used in the study: the adult rat, the very young rat (20 g) and the adult gerbil. Those 3 models were used in studying the effect of hypoxia, partial ischemia, and anesthesia on the metabolic and ionic activities measured from the awake brain. Spreading cortical depression (elicited by topical KCl solution) was used as a standard stimulation of the ionic and metabolic activities of the cerebral cortex. Two typical metabolic responses to spreading depression (SD) were recorded, namely ‘oxidation cycle’ and ‘reduction cycle’ depending upon the ability of the tissue to compensate for the extra amount of oxygen needed for the higher mitochondrial activity.It was found that the adult rat brain showed oxidation cycles in most conditions (besides partial ischemia), while the young rat and the gerbil brains were much more sensitive to the various perturbations of the brain and exhibited reduction cycle (as a response to SD) under all pathological situations tested.We conclude from our detailed studies that the type of response to SD, as measured by NADH surface fluorometry, represents the oxygen balance which exists in the tissue under various conditions.     

Sequential alterations of cerebral carbohydrate metabolism associated with gamma-hydroxybutyrate

The cerebral metabolic effects of intravenous administration of 1000 mg/kg gamma-hydroxybutyrate (GHB) were studied by sequential measurement of the cerebral contents of selected glycolytic-critic cycle intermediates and energy phosphates in lightly anesthetized rats. The initial change in the glycolytic pathway occured by 2.5 min, with increases of tissues glucose-6-phosphate and decreases of fructose-1,6-diphosphate which indicated an inhibition of phosphofructokinase. This pattern was transient and was replaced at 5–15 min by increasing tissue glucose and decreasing glucose-6-phosphate which indicated an inhibition of hexokinase. The initial inhibition of phosphofructokinase was associated with functional depression, an isoelectric EEG and an increase of the tissue phosphocreatine which suggested that the observed metabolic pattern was an adaptation to the reduced energy needs of neuronal depression.Within 2.5 min of GHB injection tissue α-ketoglutarate and aspartate showed significant increases which suggested a shift in the aspartate aminotransferase reaction. Preliminary calculations indicated that the probable cause of this shift was an increase in oxaloacetate content due to GHB oxidation.The cytoplasmic NADH/NAD⁺ ratio remained unchanged throughout the entire exposure to GHB (2.5–180 min) and thus gave no support for the hypothesis that GHB interfers with glycolysis via the restriction of free cytoplasmic NAD⁺ required for the glyceraldehyde phosphate dehydrogenase step.     

The calculation of the mitochondrial free [NAD]/[NADH][H] ratio in brain: Effect of electroconvulsive seizure

This study is an investigation into the validity of calculating the mitochondrial redox state in brain in vivo using models of seizure and anoxia in rats. At six intervals following electroconvulsive seizure (0.5–10 min) and after 5 min of complete anoxia, multiple metabolites were measured in freeze-blown or freeze-clamped brain. From substrate ratios, the apparent changes in the mitochondrial free [NAD⁺]/[NADH] [H⁺] ratio were calculated from thel-glutamate dehydrogenase reaction [EC 1.4.1.3] and compared with shifts in the oxidized to reduced ratio of total ubiquinone (a component of the mitochondrial phosphorylation chain). During complete anoxia the calculated mitochondrial free [NAD⁺]/[NADH][H⁺] ratio and the ubiquinone redox ratio both became more reduced by a factor of approximately 7. In contrast, following seizure the two indicators of the mitochondrial redox state moved in opposite directions. Mainly because of a large increase in tissue NH_4⁺, the calculated mitochondrial free [NAD⁺]/[NADH][H⁺] ratio paradoxically became more oxidized, plateauing between 2 and 10 min post seizure at a value approximately douhle that of the control. At the same time, however, the ubiquinone redox state fell to one-half the control value at two min and moved back towards normal between 5 and 10 min after the onset of the seizure. The results have been taken to be evidence against the applicability of the calculation of the mitochondrial free [NAD⁺]/[NADH][H⁺] ratio from thel-glutamate dehydrogenase reaction in brain at least under conditions of rapid change. The results also suggest the possibility that the NH_4⁺ produced during seizure is extra-mitochondrial and has relatively little tendency to diffuse into the matrix.     

Responses of electrical potential, potassium levels, and oxidative metabolic activity of the cerebral neocortex of cats.

We measured simultaneously the oxidative metabolic activity, monitored as the tissue fluorescence attribute to intramitochondrial NADH, the extracellular potassium level with ion-selective microelectrodes, and the focal extracellular electrical potential, of one site in intact cerebral cortex of cats. When the cerebral was stimulated by trains of repeated electric pulses applied either directly to its surface or to an afferent pathway, the corrected cortical fluorescence (F-R) declined indicating oxidation of NADH, the activity of extracellular potassium [K+]o increased, and the extracellular potential (Vec) shifted in the negative direction. When mild to moderate stimuli not exceeding 10-15 sec in duration were used, a 3-fold correlation was found between these three variables. The regression of F-R over either Vec, or over log [K+]o had a positive ordinal intercept. The results are in agreement with earlier suggestions 4,24,25,43,45,46 that (a) much but not all the oxidative metabolic response of cortex to electrical stimulation is expended in restoring disturbed ion balance; and (b) that sustained shifts of potential (SP) in response to repetitive electrical stimulation are generated by glia cells depolarized by excess potassium. The magnitude of SP shifts associated with a given elevation of [k+]o are smaller in cerebral cortex than in spinal cord48,49. The correlation of F-R with [K+]o breaks down when pathologic processes of either seizure activity or spreading depression set in. During paroxysmal activity [K+]o tends to remain confined below 10-12 mM, a level observed in non-convulsing cortex as well, but oxidation of NADH progresses beyond that seen in non-convulsing cortex as well, but oxidation of NADH progresses beyond that seen in non-convulsing tissue. This observation is hard to reconcile with the suggestion that excess potassium is a factor in the generation of seizures, at least of the type observed in this study. When [K+]o levels exceeded 10-12 mM, spreading depression invariably followed at least under the unanesthetized condition in these experiments. During spreading depression [K+]o levels rose to exceed 30 mM, sometimes 80 mM. NADH was oxidized during spreading depression to a level comparable to that seen in seizures. The observations are compatible with the suggestion13 that spreading depression occurs whenever the release of potassium into extracellular fluid is overloading its clearance therefrom.     

Critical Role of Astrocyte NAD+ Glycohydrolase in Myelin Injury and Regeneration

Western-style diets cause disruptions in myelinating cells and astrocytes within the mouse CNS. Increased CD38 expression is present in the cuprizone and experimental autoimmune encephalomyelitis models of demyelination and CD38 is the main nicotinamide adenine dinucleotide (NAD⁺)-depleting enzyme in the CNS. Altered NAD⁺ metabolism is linked to both high fat consumption and multiple sclerosis (MS). Here, we identify increased CD38 expression in the male mouse spinal cord following chronic high fat consumption, after focal toxin [lysolecithin (LL)]-mediated demyelinating injury, and in reactive astrocytes within active MS lesions. We demonstrate that CD38 catalytically inactive mice are substantially protected from high fat-induced NAD⁺ depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. A CD38 inhibitor, 78c, increased NAD⁺ and attenuated neuroinflammatory changes induced by saturated fat applied to astrocyte cultures. Conditioned media from saturated fat-exposed astrocytes applied to oligodendrocyte cultures impaired myelin protein production, suggesting astrocyte-driven indirect mechanisms of oligodendrogliopathy. In cerebellar organotypic slice cultures subject to LL-demyelination, saturated fat impaired signs of remyelination effects that were mitigated by concomitant 78c treatment. Significantly, oral 78c increased counts of oligodendrocytes and remyelinated axons after focal LL-induced spinal cord demyelination. Using a RiboTag approach, we identified a unique in vivo brain astrocyte translatome profile induced by 78c-mediated CD38 inhibition in mice, including decreased expression of proinflammatory astrocyte markers and increased growth factors. Our findings suggest that a high-fat diet impairs oligodendrocyte survival and differentiation through astrocyte-linked mechanisms mediated by the NAD⁺ase CD38 and highlights CD38 inhibitors as potential therapeutic candidates to improve myelin regeneration.SIGNIFICANCE STATEMENT Myelin disturbances and oligodendrocyte loss can leave axons vulnerable, leading to permanent neurologic deficits. The results of this study suggest that metabolic disturbances, triggered by consumption of a diet high in fat, promote oligodendrogliopathy and impair myelin regeneration through astrocyte-linked indirect nicotinamide adenine dinucleotide (NAD⁺)-dependent mechanisms. We demonstrate that restoring NAD⁺ levels via genetic inactivation of CD38 can overcome these effects. Moreover, we show that therapeutic inactivation of CD38 can enhance myelin regeneration. Together, these findings point to a new metabolic targeting strategy positioned to improve disease course in multiple sclerosis and other conditions in which the integrity of myelin is a key concern.     

Mitochondrial mechanisms of cell death and neuroprotection in pediatric ischemic and traumatic brain injury

There are several forms of acute pediatric brain injury, including neonatal asphyxia, pediatric cardiac arrest with global ischemia, and head trauma, that result in devastating, lifelong neurologic impairment. The only clinical intervention that appears neuroprotective is hypothermia initiated soon after the initial injury. Evidence indicates that oxidative stress, mitochondrial dysfunction, and impaired cerebral energy metabolism contribute to the brain cell death that is responsible for much of the poor neurologic outcome from these events. Recent results obtained from both in vitro and animal models of neuronal death in the immature brain point toward several molecular mechanisms that are either induced or promoted by oxidative modification of macromolecules, including consumption of cytosolic and mitochondrial NAD⁺ by poly-ADP ribose polymerase, opening of the mitochondrial inner membrane permeability transition pore, and inactivation of key, rate-limiting metabolic enzymes, e.g., the pyruvate dehydrogenase complex. In addition, the relative abundance of pro-apoptotic proteins in immature brains and neurons, and particularly within their mitochondria, predisposes these cells to the intrinsic, mitochondrial pathway of apoptosis, mediated by Bax- or Bak-triggered release of proteins into the cytosol through the mitochondrial outer membrane. Based on these pathways of cell dysfunction and death, several approaches toward neuroprotection are being investigated that show promise toward clinical translation. These strategies include minimizing oxidative stress by avoiding unnecessary hyperoxia, promoting aerobic energy metabolism by repletion of NAD⁺ and by providing alternative oxidative fuels, e.g., ketone bodies, directly interfering with apoptotic pathways at the mitochondrial level, and pharmacologic induction of antioxidant and anti-inflammatory gene expression.     

Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer’s disease. Here we studied the roles of NAD⁺ bioenergetic state, and the NAD⁺-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD⁺ levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD⁺ precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD⁺ levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD⁺ depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD⁺ levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD⁺ levels and reduced ischemic infarct size. Our findings demonstrate that the NAD⁺ bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD⁺ levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD⁺ to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.     

In vivo NADH fluorescence imaging indicates effect of aquaporin-4 deletion on oxygen microdistribution in cortical spreading depression

Using in vivo two-photon imaging, we show that mice deficient in aquaporin-4 (AQP4) display increased fluorescence of nicotinamide adenine dinucleotide (NADH) when subjected to cortical spreading depression. The increased NADH signal, a proxy of tissue hypoxia, was restricted to microwatershed areas remote from the vasculature. Aqp4 deletion had no effects on the hyperemia response, but slowed [K⁺]_o recovery. These observations suggest that K⁺ uptake is suppressed in Aqp4^−/− mice as a consequence of decreased oxygen delivery to tissue located furthest away from the vascular source of oxygen, although increased oxygen consumption may also contribute to our observations.     

Cortical oxygen consumption and NADH flourescence during metrazol seizures in normotensive and hypotensive cats.

The relationship between the oxidation of intramitochondrial NADH and increased oxygen utilization previously described in various preparations has not yet been directly studied in large areas of the cerebral cortex during generalized seizures. In the present study, changes in the NADH fluorescence of one or both cortical hemispheres were measured with a television fluorometer during Metrazol seizures in normotensive and hypotensive states. Fluorescein fluorescence was used as a reference signal. Combined with measurements of sagittal sinus blood flow and oxygen saturation, this technique demonstrated generalized ictal decreases in NADH fluorescence, the time integrals of which linearly related to the integrals of simultaneous increases in relative cortical O₂ consumption during seizures occurring at normal blood pressures. Seizures observed during systemic hypotension and probable cerebral hypoperfusion, however, were characterized by generalized increases in NADH fluorescence, the integrals of which were not related to the integrals of relatively delayed increases in cortical O₂ consumption. Similar anomalous NADH increases during seizures were also occasionally seen in certain cortical areas in normotensive animals, suggesting local ischemia. In addition. NADH fluorescence and cortical O₂ consumption were found to be affected by changes in blood pressure in the absence of ictal activity. These observations suggest that cortical NADH fluorescence is dependent both on metabolic demand and O₂ availability, and consequently may be validly utilized as an indicator of activated cortical oxidative metabolism during seizures only under conditions of adequate cortical oxygenation.     

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice

Nicotinamide adenine dinucleotide (NAD⁺) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD⁺ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD⁺ results in impaired mitochondrial and cellular functions. Administration of NAD⁺ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD⁺ degradation and cellular death. In this study, we identified a novel link between NAD⁺ metabolism and mitochondrial dynamics. A single dose (62.5 mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD⁺ pools for up to 24 hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD⁺ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.     

NAD+ Availability and Proteotoxicity

It has been shown that NAD⁺ availability is important for neuronal survival following ischemia (Liu et al., Neuromolecular Med 11:28–42, 2009). It is proposed here that NAD⁺ may also control proteotoxicity by influencing both formation and catabolism of altered proteins. It is suggested that low NAD⁺ availability promotes synthesis of methylglyoxal (MG) which can induce formation of glycated proteins, ROS, and dysfunctional mitochondria. That glyoxalase overexpression and carnosine are both protective against MG and ischemic injury support this proposal. Recognition and elimination of altered proteins is enhanced by NAD⁺ through effects on stress protein expression and autophagy.     

Cortical spreading depression impairs oxygen delivery and metabolism in mice

Cortical spreading depression (CSD) is associated with severe hypoperfusion in mice. Using minimally invasive multimodal optical imaging, we show that severe flow reductions during and after spreading depression are associated with a steep decline in cerebral metabolic rate of oxygen. Concurrent severe hemoglobin desaturation suggests that the oxygen metabolism becomes at least in part supply limited, and the decrease in cortical blood volume implicates vasoconstriction as the mechanism. In support of oxygen supply-demand mismatch, cortical nicotinamide adenine dinucleotide (NADH) fluorescence increases during spreading depression for at least 5 minutes, particularly away from parenchymal arterioles. However, modeling of tissue oxygen delivery shows that cerebral metabolic rate of oxygen drops more than predicted by a purely supply-limited model, raising the possibility of a concurrent reduction in oxygen demand during spreading depression. Importantly, a subsequent spreading depression triggered within 15 minutes evokes a monophasic flow increase superimposed on the oligemic baseline, which markedly differs from the response to the preceding spreading depression triggered in naive cortex. Altogether, these data suggest that CSD is associated with long-lasting oxygen supply-demand mismatch linked to severe vasoconstriction in mice.