Browse
Submit Data
Databases
API
Help

Dataset Information

34 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

In Vivo NADH/NAD⁺ Biosensing Reveals the Dynamics of Cytosolic Redox Metabolism in Plants.

ABSTRACT: NADH and NAD⁺ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD⁺ in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.

SUBMITTER: Steinbeck J

PROVIDER: S-EPMC7534465 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor.

Project description:NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.

| S-EPMC3190165 | biostudies-literature

NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro.

Project description:Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

| S-EPMC7738682 | biostudies-literature

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state.

Project description:The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.

| S-EPMC7902596 | biostudies-literature

Redox Dysregulation in Schizophrenia Revealed by in vivo NAD+/NADH Measurement.

Project description:Balance between the redox pair of nicotinamide adenine dinucleotides (oxidized NAD+ and reduced NADH), reflects the oxidative state of cells and the ability of biological systems to carry out energy production. A growing body of evidence suggests that an "immuno-oxidative" pathway including oxidative stress, mitochondrial dysfunction, neuroinflammation, and cell-mediated immune response may contribute to disruptions in brain activity in schizophrenia (SZ). The aim of this study is to assess possible redox imbalance in SZ patients by using a novel in vivo 31P MRS technique. The participants included 40 healthy controls, 21 chronic SZ, 13 first-episode (FE) SZ, and 18 FE bipolar disorder (BD) patients (as a psychiatric control group). All participants initially underwent structural imaging at a 3 Tesla (3 T) and 31P MRS measurements were performed on a 4 T MR scanner. NAD+ and NADH components were determined by nonlinear least-square fitting of the model simulated spectra; these incorporated prior chemical shift and coupling constant information to in vivo resonances obtained from 31P MRS experiments. We found a significant reduction in the NAD+/NADH ratio in chronically ill SZ patients compared to a matched healthy control group, and in FE SZ patients compared to both a matched FE BD patient group and a matched healthy control group. These findings provide evidence for redox imbalance in the brain in all phases of SZ, potentially reflecting oxidative stress.

| S-EPMC5216857 | biostudies-literature

Genetically encoded biosensors for evaluating NAD+/NADH ratio in cytosolic and mitochondrial compartments.

Project description:The ratio of oxidized to reduced NAD (NAD+/NADH) sets intracellular redox balance and antioxidant capacity. Intracellular NAD is compartmentalized and the mitochondrial NAD+/NADH ratio is intricately linked to cellular function. Here, we report the monitoring of the NAD+/NADH ratio in mitochondrial and cytosolic compartments in live cells by using a modified genetic biosensor (SoNar). The fluorescence signal of SoNar targeted to mitochondria (mt-SoNar) or cytosol (ct-SoNar) responded linearly to physiological NAD+/NADH ratios in situ. NAD+/NADH ratios in cytosol versus mitochondria responded rapidly, but differently, to acute metabolic perturbations, indicating distinct NAD pools. Subcellular NAD redox balance regained homeostasis via communications through malate-aspartate shuttle. Mitochondrial and cytosolic NAD+/NADH ratios are influenced by NAD+ precursor levels and are distinctly regulated under pathophysiological conditions. Compartment-targeted biosensors and real-time imaging allow assessment of subcellular NAD+/NADH redox signaling in live cells, enabling future mechanistic research of NAD redox in cell biology and disease development.

| S-EPMC8659198 | biostudies-literature

Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging.

Project description:AimCytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox.ResultsWe show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting.InnovationBecause the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor.ConclusionImaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.

| S-EPMC5041510 | biostudies-literature

The effect of succinate on brain NADH/NAD+ redox state and high energy phosphate metabolism in acute traumatic brain injury.

Project description:A key pathophysiological process and therapeutic target in the critical early post-injury period of traumatic brain injury (TBI) is cell mitochondrial dysfunction; characterised by elevation of brain lactate/pyruvate (L/P) ratio in the absence of hypoxia. We previously showed that succinate can improve brain extracellular chemistry in acute TBI, but it was not clear if this translates to a change in downstream energy metabolism. We studied the effect of microdialysis-delivered succinate on brain energy state (phosphocreatine/ATP ratio (PCr/ATP)) with 31P MRS at 3T, and tissue NADH/NAD+ redox state using microdialysis (L/P ratio) in eight patients with acute major TBI (mean 7 days). Succinate perfusion was associated with increased extracellular pyruvate (+26%, p < 0.0001) and decreased L/P ratio (-13%, p < 0.0001) in patients overall (baseline-vs-supplementation over time), but no clear-cut change in 31P MRS PCr/ATP existed in our cohort (p > 0.4, supplemented-voxel-vs-contralateral voxel). However, the percentage decrease in L/P ratio for each patient following succinate perfusion correlated significantly with their percentage increase in PCr/ATP ratio (Spearman's rank correlation, r = -0.86, p = 0.024). Our findings support the interpretation that L/P ratio is linked to brain energy state, and that succinate may support brain energy metabolism in select TBI patients suffering from mitochondrial dysfunction.

| S-EPMC6057963 | biostudies-literature

Mitochondrial DNA mutation exacerbates female reproductive aging via impairment of the NADH/NAD+ redox.

Project description:Mammals' aging is correlated with the accumulation of somatic heteroplasmic mitochondrial DNA (mtDNA) mutations. Whether and how aging accumulated mtDNA mutations modulate fertility remains unknown. Here, we analyzed oocyte quality of young (≤30 years old) and elder (≥38 years old) female patients and show the elder group had lower blastocyst formation rate and more mtDNA point mutations in oocytes. To test the causal role of mtDNA point mutations on infertility, we used polymerase gamma (POLG) mutator mice. We show that mtDNA mutation levels inversely correlate with fertility, interestingly mainly affecting not male but female fertility. mtDNA mutations decrease female mice's fertility by reducing ovarian primordial and mature follicles. Mechanistically, accumulation of mtDNA mutations decreases fertility by impairing oocyte's NADH/NAD+ redox state, which could be rescued by nicotinamide mononucleotide treatment. For the first time, we answer the fundamental question of the causal effect of age-accumulated mtDNA mutations on fertility and its sex dependence, and show its distinct metabolic controlling mechanism.

| S-EPMC7511885 | biostudies-literature

Biochemical issues in estimation of cytosolic free NAD/NADH ratio.

Project description:Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.

| S-EPMC3343042 | biostudies-literature

Mitochondrial NAD+/NADH Redox State and Diabetic Cardiomyopathy.

Project description:Diabetic cardiomyopathy (DCM) is a frequent complication occurring even in well-controlled asymptomatic diabetic patients, and it may advance to heart failure (HF). Recent Advances: The diabetic heart is characterized by a state of "metabolic rigidity" involving enhanced rates of fatty acid uptake and mitochondrial oxidation as the predominant energy source, and it exhibits mitochondrial electron transport chain defects. These alterations promote redox state changes evidenced by a decreased NAD+/NADH ratio associated with an increase in acetyl-CoA/CoA ratio. NAD+ is a co-substrate for deacetylases, sirtuins, and a critical molecule in metabolism and redox signaling; whereas acetyl-CoA promotes protein lysine acetylation, affecting mitochondrial integrity and causing epigenetic changes.DCM lacks specific therapies with treatment only in later disease stages using standard, palliative HF interventions. Traditional therapy targeting neurohormonal signaling and hemodynamics failed to improve mortality rates. Though mitochondrial redox state changes occur in the heart with obesity and diabetes, how the mitochondrial NAD+/NADH redox couple connects the remodeled energy metabolism with mitochondrial and cytosolic antioxidant defense and nuclear epigenetic changes remains to be determined. Mitochondrial therapies targeting the mitochondrial NAD+/NADH redox ratio may alleviate cardiac dysfunction.Specific therapies must be supported by an optimal understanding of changes in mitochondrial redox state and how it influences other cellular compartments; this field has begun to surface as a therapeutic target for the diabetic heart. We propose an approach based on an alternate mitochondrial electron transport that normalizes the mitochondrial redox state and improves cardiac function in diabetes. Antioxid. Redox Signal. 00, 000-000.

| S-EPMC6306679 | biostudies-other