Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism.

The electron transport chain (ETC) of mitochondria, bacteria, and archaea couples electron flow to proton pumping and is adapted to diverse oxygen environments. Remarkably, in mice, neurological disease due to ETC complex I dysfunction is rescued by hypoxia through unknown mechanisms. Here, we show that hypoxia rescue and hyperoxia sensitivity of complex I deficiency are evolutionarily conserved to C. elegans and are specific to mutants that compromise the electron-conducting matrix arm. We show that hypoxia rescue does not involve the hypoxia-inducible factor pathway or attenuation of reactive oxygen species. To discover the mechanism, we use C. elegans genetic screens to identify suppressor mutations in the complex I accessory subunit NDUFA6/nuo-3 that phenocopy hypoxia rescue. We show that NDUFA6/nuo-3(G60D) or hypoxia directly restores complex I forward activity, with downstream rescue of ETC flux and, in some cases, complex I levels. Additional screens identify residues within the ubiquinone binding pocket as being required for the rescue by NDUFA6/nuo-3(G60D) or hypoxia. This reveals oxygen-sensitive coupling between an accessory subunit and the quinone binding pocket of complex I that can restore forward activity in the same manner as hypoxia.

Saturation of the mitochondrial NADH shuttles drives aerobic glycolysis in proliferating cells.

Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4-/- mouse.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4-/- mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.

The NADH Dehydrogenase Nde1 Executes Cell Death after Integrating Signals from Metabolism and Proteostasis on the Mitochondrial Surface.

The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

The tricarboxylic acid (TCA) cycle: a malleable metabolic network to counter cellular stress.

The tricarboxylic acid (TCA) cycle is a primordial metabolic pathway that is conserved from bacteria to humans. Although this network is often viewed primarily as an energy producing engine fueling ATP synthesis via oxidative phosphorylation, mounting evidence reveals that this metabolic hub orchestrates a wide variety of pivotal biological processes. It plays an important part in combatting cellular stress by modulating NADH/NADPH homeostasis, scavenging ROS (reactive oxygen species), producing ATP by substrate-level phosphorylation, signaling and supplying metabolites to quell a range of cellular disruptions. This review elaborates on how the reprogramming of this network prompted by such abiotic stress as metal toxicity, oxidative tension, nutrient challenge and antibiotic insult is critical for countering these conditions in mostly microbial systems. The cross-talk between the stressors and the participants of TCA cycle that results in changes in metabolite and nucleotide concentrations aimed at combatting the abiotic challenge is presented. The fine-tuning of metabolites mediated by disparate enzymes associated with this metabolic hub is discussed. The modulation of enzymatic activities aimed at generating metabolic moieties dedicated to respond to the cellular perturbation is explained. This ancient metabolic network has to be recognized for its ability to execute a plethora of physiological functions beyond its well-established traditional roles.

In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

Multiomics analysis reveals serine catabolism as a potential therapeutic target for MELAS.

Mitochondrial disease is a devastating genetic disorder, with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and m.3243A>G being the most common phenotype and genotype, respectively. The treatment for MELAS patients is still less effective. Here, we performed transcriptomic and proteomic analysis in muscle tissue of MELAS patients, and discovered that the expression of molecules involved in serine catabolism were significantly upregulated, and serine hydroxymethyltransferase 2 (SHMT2) increased significantly in both the mRNA and protein levels. The SHMT2 protein level was also increased in myoblasts with m.3243A>G mutation, which was transdifferentiated from patients derived fibroblasts, accompanying with the decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio and cell viability. After treating with SHMT2 inhibitor (SHIN1), the NAD+/NADH ratio and cell viability in MELAS myoblasts increased significantly. Taken together, our study indicates that enhanced serine catabolism plays an important role in the pathogenesis of MELAS and that SHIN1 can be a potential small molecule for the treatment of this disease.

Reverse electron transfer is activated during aging and contributes to aging and age-related disease.

Mechanisms underlying the depletion of NAD+ and accumulation of reactive oxygen species (ROS) in aging and age-related disorders remain poorly defined. We show that reverse electron transfer (RET) at mitochondrial complex I, which causes increased ROS production and NAD+ to NADH conversion and thus lowered NAD+ /NADH ratio, is active during aging. Genetic or pharmacological inhibition of RET decreases ROS production and increases NAD+ /NADH ratio, extending the lifespan of normal flies. The lifespan-extending effect of RET inhibition is dependent on NAD+ -dependent Sirtuin, highlighting the importance of NAD+ /NADH rebalance, and on longevity-associated Foxo and autophagy pathways. RET and RET-induced ROS and NAD+ /NADH ratio changes are prominent in human induced pluripotent stem cell (iPSC) model and fly models of Alzheimer's disease (AD). Genetic or pharmacological inhibition of RET prevents the accumulation of faulty translation products resulting from inadequate ribosome-mediated quality control, rescues relevant disease phenotypes, and extends the lifespan of Drosophila and mouse AD models. Deregulated RET is therefore a conserved feature of aging, and inhibition of RET may open new therapeutic opportunities in the context of aging and age-related diseases including AD.

NMRK2 is an efficient diagnostic indicator for Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinomas (tRCC) are a rare and highly malignant type of renal cancer, lacking efficient diagnostic indicators and therapeutic targets. Through the analysis of public databases and our cohort, we identified NMRK2 as a potential diagnostic marker for distinguishing Xp11.2 tRCC from kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) due to its specific upregulation in Xp11.2 tRCC tissues. Mechanistically, we discovered that TFE3 fusion protein binds to the promoter of the NMRK2 gene, leading to its upregulation. Importantly, we established RNA- and protein-based diagnostic methods for identifying Xp11.2 tRCC based on NMRK2 expression levels, and the diagnostic performance of our methods was comparable to a dual-color break-apart fluorescence in situ hybridization assay. Moreover, we successfully identified fresh Xp11.2 tRCC tissues after surgical excision using our diagnostic methods and established an immortalized Xp11.2 tRCC cell line for further research purposes. Functional studies revealed that NMRK2 promotes the progression of Xp11.2 tRCC by upregulating the NAD+/NADH ratio, and supplementation with ß-nicotinamide mononucleotide (NMN) or nicotinamide riboside chloride (NR), effectively rescued the phenotypes induced by the knockdown of NMRK2 in Xp11.2 tRCC. Taken together, these data introduce a new diagnostic indicator capable of accurately distinguishing Xp11.2 tRCC and highlight the possibility of developing novel targeted therapeutics. © 2024 The Pathological Society of Great Britain and Ireland.

Photoredox catalysis may be a general mechanism in photodynamic therapy.

Elucidating the underlying photochemical mechanisms of action (MoA) of photodynamic therapy (PDT) may allow its efficacy to be improved and could set the stage for the development of new classes of PDT photosensitizers. Here, we provide evidence that "photoredox catalysis in cells," wherein key electron transport pathways are disrupted, could constitute a general MoA associated with PDT. Taking the cellular electron donor nicotinamide adenine dinucleotide as an example, we have found that well-known photosensitizers, such as Rose Bengal, BODIPY, phenoselenazinium, phthalocyanine, and porphyrin derivatives, are able to catalyze its conversion to NAD+. This MoA stands in contrast to conventional type I and type II photoactivation mechanisms involving electron and energy transfer, respectively. A newly designed molecular targeting photocatalyst (termed CatER) was designed to test the utility of this mechanism-based approach to photosensitizer development. Photoexcitation of CatER induces cell pyroptosis via the caspase 3/GSDME pathway. Specific epidermal growth factor receptor positive cancer cell recognition, high signal-to-background ratio tumor imaging (SBRTI = 12.2), and good tumor growth inhibition (TGI = 77.1%) are all hallmarks of CatER. CatER thus constitutes an effective near-infrared pyroptotic cell death photo-inducer. We believe the present results will provide the foundation for the synthesis of yet-improved phototherapeutic agents that incorporate photocatalytic chemistry into their molecular design.

Respiratory complex I with charge symmetry in the membrane arm pumps protons.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Mated-Atom Nanozymes with Efficient Assisted NAD+ Replenishment for Skin Regeneration.

Excessive accumulation of reduced nicotinamide adenine dinucleotide (NADH) within biological organisms is closely associated with many diseases. It remains a challenge to efficiently convert superfluous and detrimental NADH to NAD+. NADH oxidase (NOX) is a crucial oxidoreductase that catalyzes the oxidation of NADH to NAD+. Herein, M1M2 (Mi=V/Mn/Fe/Co/Cu/Mo/Rh/Ru/Pd, i = 1 or 2) mated-atom nanozymes (MANs) are designed by mimicking natural enzymes with polymetallic active centers. Excitingly, RhCo MAN possesses excellent and sustainable NOX-like activity, with Km-NADH (16.11 µM) being lower than that of NOX-mimics reported so far. Thus, RhCo MAN can significantly promote the regeneration of NAD+ and regulate macrophage polarization toward the M2 phenotype through down-regulation of TLR4 expression, which may help to recover skin regeneration. However, RhRu MAN with peroxidase-like activity and RhMn MAN with superoxide dismutase-like activity exhibit little modulating effects on eczema. This work provides a new strategy to inhibit skin inflammation and promote skin regeneration.

NAD metabolism and heart failure: Mechanisms and therapeutic potentials.

Nicotinamide adenine dinucleotide provides the critical redox pair, NAD+ and NADH, for cellular energy metabolism. In addition, NAD+ is the precursor for de novo NADP+ synthesis as well as the co-substrates for CD38, poly(ADP-ribose) polymerase and sirtuins, thus, playing a central role in the regulation of oxidative stress and cell signaling. Declines of the NAD+ level and altered NAD+/NADH redox states have been observed in cardiometabolic diseases of various etiologies. NAD based therapies have emerged as a promising strategy to treat cardiovascular disease. Strategies that reduce NAD+ consumption or promote NAD+ production have repleted intracellular NAD+ or normalized NAD+/NADH redox in preclinical studies. These interventions have shown cardioprotective effects in multiple models suggesting a great promise of the NAD+ elevating therapy. Mechanisms for the benefit of boosting NAD+ level, however, remain incompletely understood. Moreover, despite the robust pre-clinical studies there are still challenges to translate the therapy to clinic. Here, we review the most up to date literature on mechanisms underlying the NAD+ elevating interventions and discuss the progress of human studies. We also aim to provide a better understanding of how NAD metabolism is changed in failing hearts with a particular emphasis on types of strategies employed and methods to target these pathways. Finally, we conclude with a comprehensive assessment of the challenges in developing NAD-based therapies for heart diseases, and to provide a perspective on the future of the targeting strategies.

Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1.

Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the Kd value for NADH compared to the WT enzyme. However, we determined that the kred value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the kcat/KNADH value. Moreover, there is no significant difference in the kcat/KBQ (∼1 × 106 M-1s-1) and kcat (∼24 s-1) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin.

Involvement of SlSTOP1 regulated SlFDH expression in aluminum tolerance by reducing NAD+ to NADH in the tomato root apex.

Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria-localized FDH with Km values of 2.087 mm formate and 29.1 µm NAD+ . Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase-polymerase chain reaction. CRISPR/Cas9-generated SlFDH knockout lines were more sensitive to Al stress and formate than wild-type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD+ accumulated in response to Al stress. Co-expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual-luciferase reporter assay and electrophoretic mobility shift assay. The Al-induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato.

Valacyclovir and Acyclovir Are Substrates of the Guanine Deaminase Cytosolic PSD-95 Interactor (Cypin).

Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither Km nor KD values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy.

Evaluation of safety and effectiveness of NAD in different clinical conditions: a systematic review.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide cofactor that is present in cells and in several important biological processes, including oxidative phosphorylation and production of adenosine triphosphate, DNA repair, calcium-dependent secondary messenger and gene expression. The purpose of this systematic review is to examine whether the coenzyme formulae NAD+ and NADH are safe and effective when acting as a supplement to humans. This systematic review of randomized clinical trials performed a search in six electronic databases: PubMed, MEDLINE (ovid), Embase, Cochrane CENTRAL (clinical trials), Web of Science, and Scopus. Secondary search included the databases (e.g., Clinical trials.gov, Rebec, Google Scholar - advance). Two reviewers assessed and extracted the studies independently. The risk of bias in studies was performed using version 2 of the Cochrane risk of bias tool for randomized trials. This review includes 10 studies, with a total of 489 participants. The studies included different clinical conditions, such as chronic fatigue syndrome (CFS), older adults, Parkinson's disease, overweight, postmenopausal prediabetes, and Alzheimer's disease. Based on studies, the supplementation with NADH and precursors was well tolerated and observed clinical results such as, a decrease in anxiety conditions and maximum heart rate was observed after a stress test, increased muscle insulin sensitivity, insulin signaling. Quality of life, fatigue intensity, and sleep quality among others were evaluated on patients with CFS. All studies showed some side effects, thus, the most common associated with NADs use are muscle pain, nervous disorders, fatigue, sleep disturbance, and headaches. All adverse events cataloged by the studies did not present a serious risk to the health of the participants. Overall, these findings support that the oral administration of NADH can be associated to an increase in general quality of life and improvement on health parameters (e.g., a decrease in anxiety, maximum heart rate, inflammatory cytokines in serum, and cerebrospinal fluid). NADH supplementation is safe and has a low incidence of side effects. Future investigations are needed to evidence the clinical benefits regarding specific diseases and doses administered.

Metabolic response of auditory brainstem neurons to their broad physiological activity range.

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5-24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand.