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.     

Quinolinic acid induces neuritogenesis in SH‐SY5Y neuroblastoma cells independently of NMDA receptor activation

Glutamate and nicotinamide adenine dinucleotide (NAD⁺) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N‐methyl‐D‐aspartate (NMDA) receptors and also a precursor for the formation of NAD⁺. The effect of QA on cell survival and differentiation has therefore been examined on SH‐SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 μm ) induced differentiation of SH‐SY5Y cells into a neuronal phenotype showing neurite growth. QA (50–150 nm ) also caused a concentration‐dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker β3‐tubulin in whole‐cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA‐induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene‐iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK‐801) did not prevent quinolinate‐induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of β3‐tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD⁺, independently of NMDA receptors.     

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.     

Sirtuin 5 is Anti-apoptotic and Anti-oxidative in Cultured SH-EP Neuroblastoma Cells

As a nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase, demalonylase, and desuccinylase, sirtuin 5 (SIRT5) in host cells has been reportedly observed in the mitochondria, in the cytosol/cytoplasm or in the nucleus. Various functional roles of SIRT5 have also been described in cellular metabolism, energy production, detoxification, oxidative stress, and apoptosis, but some of the reported results are seemingly inconsistent or even contradictory to one another. Using immunocytochemistry, molecular biology, gene transfection, and flow cytometry, we investigated the expression, subcellular distribution, and possible functional roles of SIRT5 in regulating apoptosis and oxidative stress of cultured SH-EP neuroblastoma cells. Both endogenous and transfected exogenous SIRT5 were observed in mitochondria of host SH-EP cells. Overexpression of SIRT5 markedly protected SH-EP cells from apoptosis induced by staurosporine or by incubation in Hank’s balanced salt solution. SIRT5 also lowered the level of oxidative stress and countered the toxicity of hydrogen peroxide to SH-EP cells. It was suggested that the anti-apoptotic role of SIRT5 was mediated, at least in part, by its anti-oxidative effect in SH-EP neuroblastoma cells although the involved molecular mechanisms remain to be elucidated in details.     

Iptakalim ameliorates MPP+‐induced astrocyte mitochondrial dysfunction by increasing mitochondrial complex activity besides opening mitoKATP channels

In addition to the established role of the mitochondrion in energy metabolism, regulation of cell death has been regarded as a major function of this organelle. Our previous studies have demonstrated that iptakalim (IPT), a novel ATP‐sensitive potassium channel (K_ATP channel) opener, protects against 1‐methyl‐4‐phenyl‐pyridinium ion (MPP⁺)–induced astrocyte apoptosis via mitochondria and mitogen‐activated protein kinase signal pathways. The present study aimed to investigate whether IPT can protect astrocyte mitochondria against MPP⁺‐induced mitochondrial dysfunction. We showed that treatment with IPT could ameliorate the inhibitory effect of MPP⁺ on mitochondrial respiration and ATP production by using mitochondrial complex I–supported substrates. IPT could also inhibit the increased production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria induced by MPP⁺. However, mitochondrial ATP‐sensitive potassium (mitoK_ATP) channel blocker 5‐hydroxydecanoate (5‐HD) could partly abolish all of the above effects of IPT. Because mitochondrial complex dysfunction impairs mitochondrial respiration and ATP production, a further experiment was undertaken to study the effects of IPT on the activity of mitochondrial complex (COX) I and COX IV. It was found that IPT inhibited the decrease in mitochondrial COX I and COX IV activity induced by MPP⁺, but 5‐HD failed to abolish these effects. Taken together, these findings suggest that IPT may protect astrocyte mitochondrial function by regulating complex activity in addition to opening mitoK_ATP channels. © 2008 Wiley‐Liss, Inc.     

Serum or target deprivation‐induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+

Trophic deprivation‐mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant‐induced intracellular Zn²⁺ release ([Zn²⁺]_i) from metallothionein‐3 (MT‐III), mitochondria or ‘protein Zn²⁺’, was implicated in trophic deprivation neurotoxicity. We have previously shown that neurotoxicity of extracellular Zn²⁺ required entry, increased [Zn²⁺]_i, and reduction of NAD⁺ and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD⁺ and sirtuin inhibition attenuated Zn²⁺ neurotoxicity. Here we show that: (1) Zn²⁺ is released intracellularly after oxidant and SD injuries, and that sensitivity to these injuries is proportional to neuronal Zn²⁺ content; (2) NAD⁺ loss is involved – restoration of NAD⁺ using exogenous NAD⁺, pyruvate or nicotinamide attenuated these injuries, and potentiation of NAD⁺ loss potentiated injury; (3) neurons from genetically modified mouse strains which reduce intracellular Zn²⁺ content (MT‐III knockout), reduce NAD⁺ catabolism (PARP‐1 knockout) or increase expression of an NAD⁺ synthetic enzyme (Wld^s) each had attenuated SD and oxidant neurotoxicities; (4) sirtuin inhibitors attenuated and sirtuin activators potentiated these neurotoxicities; (5) visual cortex ablation (VCA) induces Zn²⁺ staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn²⁺ diet attenuated injury; and finally (6) NAD⁺ synthesis and levels are involved given that LGNd neuronal death after VCA was dramatically reduced in Wld^s animals, and by intraperitoneal pyruvate or nicotinamide. Zn²⁺ toxicity is involved in serum and trophic deprivation‐induced neuronal death.     

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)‐scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O₂^– to H₂O₂ and molecular oxygen (O₂). Among the three forms of SOD identified, manganese‐containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2^?/+) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole‐brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53^?/? × SOD2^?/+ and p53^?/? × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole‐brain homogenate. The analysis of well‐known antioxidant systems such as thioredoxin‐1 and Nrf2/HO‐1/BVR‐A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels. © 2015 Wiley Periodicals, Inc.     

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.     

Mitochondrial Uncoupling Protein-2 (UCP2) Mediates Leptin Protection Against MPP+ Toxicity in Neuronal Cells

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP⁺ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP⁺-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP⁺ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP⁺-induced toxicity.     

Control mechanisms in mitochondrial oxidative phosphorylation

Distribution and activity of mitochondria are key factors in neuronal development,synaptic plasticity and axogenesis.The majority of energy sources,necessary for cellular functions,originate from oxidative phosphorylation located in the inner mitochondrial membrane.The adenosine-5’triphosphate production is regulated by many control mechanism-firstly by oxygen,substrate level,adenosine-5’-diphosphate level,mitochondrial membrane potential,and rate of coupling and proton leak.Recently,these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes,allosteric inhibition of cytochrome c oxidase,thyroid hormones,effects of fatty acids and uncoupling proteins.Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia.Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands.Large amounts of reactive oxygen species are released by defective mitochondria,similarly,decline of antioxidative enzyme activities(e.g.in the elderly) enhances reactive oxygen species production.We reviewed data concerning neuroplasticity,physiology,and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.     

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 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.     

Unregulated mitochondrial GSK3β activity results in NADH:Ubiquinone oxidoreductase deficiency

GSK3β is prominent for its role in apoptosis signaling and has been shown to be involved in Parkinson’s disease (PD) pathogenesis. The overall effects of GSK3β activity on cell fate are well-established, but the effects of mitochondrial GSK3β activity on mitochondrial function and cell fate are unknown. Here we selectively expressed constitutively active GSK3β within the mitochondria and found that this enhanced the apoptosis signaling activated by the PD-mimetic NADH:ubiquinone oxidoreductase (complex I) inhibitors 1-methyl-4-phenylpyri-dinium ion (MPP⁺) and rotenone. Additionally, expression of GSK3β in the mitochondria itself caused a significant decrease in complex I activity and ATP production. Increased mitochondrial GSK3β activity also increased reactive oxygen species production and perturbed the mitochondrial morphology. Conversely, chemical inhibitors of GSK3β inhibited MPP⁺- and rotenone-induced apoptosis, and attenuated the mitochondrial GSK3β-mediated impairment in complex I. These results indicate that unregulated mitochondrial GSK3β activity can mimic some of the mitochondrial insufficiencies found in PD pathology.     

High-concentration homocysteine inhibits mitochondrial respiration function and production of reactive oxygen species in neuron cells

ObjectiveHomocysteine plays critical roles in cellular redox homeostasis, and hyperhomocysteinemia has been associated with multiple diseases, including neurological disorders involving reactive oxygen species-inducing and pro-inflammatory effects of homocysteine that are related to mitochondria. This study investigated the role of homocysteine in regulating mitochondria of neuron cell lines.MethodsNeuron cells were pre-treated with homocysteine, and then flow cytometry was used to detect reactive oxygen species production and mitochondrial membrane potential, while Seahorse XFp Mito stress assay was used to comprehensively analyze mitochondrial function.ResultsThe experimental results showed that high-concentration homocysteine diminished carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone-stimulated oxygen consumption rate and mitochondrial spare respiration capacity in a time- and concentration-dependent manner, and homocysteine also reduced reactive oxygen species in cultured neuron cell lines while no changes in mitochondrial membrane potential were observed.ConclusionThese results indicate that homocysteine diminished mitochondrial respiration function in neuron cell lines mediated by its reactive oxygen species-reducing effects, which may underlie the association between hyperhomocysteinemia and human diseases.     

Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak,but to respiratory chain defects

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1^?/?) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H₂O₂ production rate by PINK1^?/? mitochondria was lower than PINK1^+/+ mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H₂O₂ production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.     

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.     

Pharmacological evidence for a role of peroxynitrite in the pathophysiology of spinal cord injury

Evidence suggests that the reactive oxygen species peroxynitrite (PN) is an important player in the pathophysiology of acute spinal cord injury (SCI). In the present study, we examined the ability of tempol, a catalytic scavenger of PN-derived free radicals, to alleviate oxidative damage, mitochondrial dysfunction and cytoskeletal degradation following a severe contusion (200 kdyn force) SCI in female Sprague-Dawley rats. PN-mediated oxidative damage in spinal cord tissue, including protein nitration, protein oxidation and lipid peroxidation was significantly reduced by acute tempol treatment (300 mg/kg, i.p. within 5 min post-injury). Injury-induced mitochondrial respiratory dysfunction, measured after 24 h in isolated mitochondria, was partially reversed by tempol along with an attenuation of oxidative damage to mitochondrial proteins. Mitochondrial dysfunction disrupts intracellular Ca²⁺ homeostasis contributing to calpain-mediated axonal cytoskeletal protein (α-spectrin, 280 kD) degradation. Increased levels of α-spectrin breakdown proteins (SBDP 145 kD and 150 kD) were significantly decreased at 24 h in tempol-treated rats indicative of spinal axonal protection. However, a therapeutic window analysis showed that the axonal cytoskeletal protective effects require tempol dosing within the first hour after injury. Nevertheless, these findings are the first to support the concept that PN is an important neuroprotective target in early secondary SCI, and that there is a mechanistic link between PN-mediated oxidative compromise of spinal cord mitochondrial function, loss of intracellular Ca²⁺ homeostasis and calpain-mediated proteolytic axonal damage.     

Monitoring of glutamate‐induced excitotoxicity by mitochondrial oxygen consumption

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na⁺ influx increases intracellular Ca²⁺ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na⁺ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na⁺/K⁺‐ATPase exchanges intracellular Na⁺ with extracellular K⁺ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate‐mediated excitotoxicity. Under a pyruvate‐restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 μM. The OCR monitoring also revealed the contribution of the N‐methyl‐D‐aspartate receptor and Na⁺/K⁺‐ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte‐dependent protection of neurons from glutamate‐mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.     

Differential release of β‐NAD+ and ATP upon activation of enteric motor neurons in primate and murine colons

Background The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β‐nicotinamide adenine dinucleotide (β‐NAD⁺) and ADP‐ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. Methods High‐performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5‐HT₃ receptors (5‐HT₃R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. Key Results Nicotinic acetylcholine receptor or 5‐HT₃R agonists increased overflow of ATP and β‐NAD⁺ from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist‐evoked overflow of β‐NAD⁺, but not ATP, was inhibited by tetrodotoxin (0.5 μmol L^?1) or ω‐conotoxin GVIA (50 nmol L^?1), suggesting that β‐NAD⁺ release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. Conclusions & Inferences These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β‐NAD⁺, or its metabolites, serve as the purinergic inhibitory neurotransmitter.