Succinate Oxidation Research Articles

344-OR: Post-translational Modification of SHDA Regulating Macrophage Polarization and Inflammation in Obesity

The succinate dehydrogenase enzyme (SDH) catalyzes the oxidation of succinate into fumarate in the tricarboxylic acid cycle. Among the four subunits, SDHA and SDHB carry the catalytic activity. Mounting evidence implicates the link between the catalytic subunit SDHA expression and mitochondrial energy metabolism. However, the molecular mechanisms regulating SDH subunits stabilization in macrophages remain largely unknown. Here, we showed that AMPK, an energy sensor, could be a key regulator of SDHA ubiquitination through directly binding to sentrin-specific protease in adipose tissue macrophages, thus contributing to obesity-induced inflammation. Our study identified that SDHA expression was significantly downregulated in AMPKα siRNA RAW264.7 cells. Moreover, MG132 could significantly rescue AMPKα knockdown-induced SDHA degradation in cells, indicating that AMPK regulates SDHA stabilization via proteasomal degradation. However, AMPK protein interaction analyses by yeast two-hybrid showed that SDHA is not AMPK downstream target. Instead sentrin-specific protease was identified as a novel, putative AMPK substrate. To further clarify the potential interaction between AMPK, SDHA, and sentrin-specific protease, Co-IP experiment was included, and results indicated that AMPK directly binds with sentrin-specific protease instead of SDHA. As a desumoylating enzyme, SENP catalyzes hydrolysis peptide bond of SUMO. How the SUMOylation of SDHA impacts its ubiquitination still needs more investigations. In our scenario, we identified a new regulatory mechanism of SDHA stabilization, AMPKα-induced SUMOylation, that partially impacts the ubiquitination level of SDHA in activated macrophages. AMPKα-induced SUMOylation level change of SDHA is directly involved in the regulation of macrophage polarization, which may contribute to adipose inflammation. Disclosure R.Wu: None. H.Jiang: None.

Effects of melatonin on cardiac metabolic reprogramming in doxorubicin-induced heart failure rats: A metabolomics study for potential therapeutic targets.

Using mass spectrometry-based targeted metabolomics, we aimed to determine the pattern of cardiac metabolic reprogramming and energetics in doxorubicin-induced heart failure. More importantly, we aimed to identify the potential effects of melatonin on cardiac metabolic reprogramming and energetics in doxorubicin-induced heart failure. Male Wistar rats (n = 18) were randomly divided into three groups (n = 6/group) to receive either (1) normal saline solution as a control, (2) 3 mg/kg/day of doxorubicin on Days 0, 4, 8, 15, 22, and 29, or (3) 3 mg/kg/day of doxorubicin on Days 0, 4, 8, 15, 22, and 29 plus 10 mg/kg/day of melatonin on Days 0-29. On Day 30, echocardiography was carried out and heart rate variability was analyzed for the evaluation of cardiac function. The rats were euthanized on the following day to enable the collection of ventricular cardiac tissue. Compared to the control group, the hearts of rats treated with doxorubicin alone exhibited impaired cardiac function, increased glucose and ketone body utilization, decreased fat utilization, decreased succinate oxidation, and decreased production of adenosine triphosphate. The cotreatment with melatonin could restore cardiac function, glucose and ketone body utilization, and adenosine triphosphate production in the heart. Interestingly, the cotreatment with melatonin led to an increase in cardiac fatty acid oxidation, branched-chain amino acid catabolism, and anaplerosis. All of these findings highlighted the potential efficacy of melatonin with regard to cardiac metabolic reprogramming and energetics. Our findings also suggested that melatonin could be considered as an adjunctive treatment for doxorubicin-induced heart failure in clinical practice.

Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III2-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.

Direct ammonium oxidation to nitrogen gas (Dirammox) in Alcaligenes strain HO-1: The electrode role

It has been recently suggested that Alcaligenes use a previously unknown pathway to convert ammonium into dinitrogen gas (Dirammox) via hydroxylamine (NH2OH). This fact alone already implies a significant decrease in the aeration requirements for the process, but the process would still be dependent on external aeration. This work studied the potential use of a polarised electrode as an electron acceptor for ammonium oxidation using the recently described Alcaligenes strain HO-1 as a model heterotrophic nitrifier. Results indicated that Alcaligenes strain HO-1 requires aeration for metabolism, a requirement that cannot be replaced for a polarised electrode alone. However, concomitant elimination of succinate and ammonium was observed when operating a previously grown Alcaligenes strain HO-1 culture in the presence of a polarised electrode and without aeration. The usage of a polarised electrode together with aeration did not increase the succinate nor the nitrogen removal rates observed with aeration alone. However, current density generation was observed along a feeding batch test representing an electron share of 3% of the ammonium removed in the presence of aeration and 16% without aeration. Additional tests suggested that hydroxylamine oxidation to dinitrogen gas could have a relevant role in the electron discharge onto the anode. Therefore, the presence of a polarised electrode supported the metabolic functions of Alcaligenes strain HO-1 on the simultaneous oxidation of succinate and ammonium.

Causes and consequences of succinate accumulation during cardiac ischemia.

Ischemia occurs when restricted blood flow prevents oxygen delivery to cells and can result in cell injury or death, especially for organs with high oxygen consumption demand, such as the heart. When oxygen demand exceeds supply, the TCA cycle intermediate succinate accumulates in the myocardium. If blood flow is restored, termed reperfusion, oxidation of the accumulated succinate leads to the production of high levels of reactive oxygen species (ROS) putting oxidative stress on cardiomyocytes. Identification of therapeutic targets for ischemia-reperfusion injury requires determining and quantifying the mechanisms of how succinate is produced and oxidized under these conditions. In vitro experiments on suspensions of purified mitochondria were used to quantify routes of succinate production during hypoxia and succinate oxidation under reperfusion conditions. A computer model of mitochondrial metabolism, accounting for mitochondrial substrate transport, oxidative phosphorylation, and the TCA cycle was developed to aid our research by analyzing data, identifying novel hypotheses, and designing experiments to test identified hypotheses. Results suggest that during hypoxia, succinate is primarily generated by the reversal of succinate dehydrogenase. We also found evidence that following a shift from hypoxic to oxygenated conditions, respiration on succinate causes a rapid accumulation of toxic levels of oxaloacetate (OAA), which in turn inhibits complex II and impairs ATP production. Thus, we hypothesize that, following ischemia and reperfusion in the heart, excess OAA must be cleared away before normal energy metabolism can be restored. Potential pathways of OAA clearance include canonical TCA cycle activity via citrate synthase, as well as via malic enzyme, glutamic oxaloacetic transaminase, and enzymes with oxaloacetate decarboxylase activity. Experiments with and without pharmacological inhibition of these pathways were conducted to quantify their relative contributions to OAA clearance under reperfusion conditions.

Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.

Membrane Potential-requiring Succinate Dehydrogenase Constitutes the Key to Propionate Oxidation and Is Unique to Syntrophic Propionate-oxidizing Bacteria

Propionate oxidation in Pelotomaculum thermopropionicum is performed under a thermodynamic limit. The most energetically unfavorable reaction in the propionate oxidation pathway is succinate oxidation. Based on previous genomic and transcriptomic ana-lyses, succinate oxidation in P. thermopropionicum under propionate-oxidizing conditions is conducted by the membrane-bound forms of two succinate dehydrogenases (SDHs). We herein examined the activity of SDH, the mechanisms underlying the succinate oxidation reaction in P. thermopropionicum, and the importance of the protein sequences of related genes. SDH activity was highly localized to the membrane fraction. An ana-lysis of the soluble fraction revealed that fumarate reductase received electrons from NADH, suggesting the involvement of membrane-bound SDH in propionate oxidation. We utilized an uncoupler and inhibitors of adenosine triphosphate (ATP) synthase and membrane-bound SDH to investigate whether the membrane potential of P. thermopropionicum supports propionate oxidation alongside hydrogen production. These chemicals inhibited hydrogen production, indicating that membrane-bound SDH requires a membrane potential for succinate oxidation, and this membrane potential is maintained by ATP synthase. In addition, the phylogenetic distribution of the flavin adenine dinucleotide-binding subunit and conserved amino acid sequences of the cytochrome b subunit of SDHs in propionate-oxidizing bacteria suggests that membrane-bound SDHs possess specific conserved amino acid residues that are strongly associated with efficient succinate oxidation in syntrophic propionate-oxidizing bacteria.

Effect of Salt Stress on the Activity, Expression, and Promoter Methylation of Succinate Dehydrogenase and Succinic Semialdehyde Dehydrogenase in Maize (Zea mays L.) Leaves

The effect of salt stress on the expression of genes, the methylation of their promoters, and the enzymatic activity of succinate dehydrogenase (SDH) and succinic semialdehyde dehydrogenase (SSADH) was investigated in maize (Zea mays L.). The incubation of maize seedlings in a 150 mM NaCl solution for 24 h led to a several-fold increase in the activity of SSADH that peaked at 6 h of NaCl treatment, which was preceded by an increase in the Ssadh1 gene expression and a decrease in its promoter methylation observed at 3 h of salt stress. The increase in SDH activity and succinate oxidation by mitochondria was slower, developing by 24 h of NaCl treatment, which corresponded to the increase in expression of the genes Sdh1-2 and Sdh2-3 encoding SDH catalytic subunits and of the gene Sdh3-1 encoding the anchoring SDH subunit. The increase in the Sdh2-3 expression was accompanied by the decrease in promoter methylation. It is concluded that salt stress results in the rapid increase in succinate production via SSADH operating in the GABA shunt, which leads to the activation of SDH, the process partially regulated via epigenetic mechanisms. The role of succinate metabolism under the conditions of salt stress is discussed.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

RNA-Seq transcriptomic analysis reveals gene expression profiles of acetic acid bacteria under high-acidity submerged industrial fermentation process.

Acetic acid bacteria (AAB) are Gram-negative obligate aerobics in Acetobacteraceae family. Producing acetic acid and brewing vinegars are one of the most important industrial applications of AAB, attributed to their outstanding ability to tolerate the corresponding stresses. Several unique acid resistance (AR) mechanisms in AAB have been revealed previously. However, their overall AR strategies are still less-comprehensively clarified. Consequently, omics analysis was widely performed for a better understanding of this field. Among them, transcriptome has recently obtained more and more attention. However, most currently reported transcriptomic studies were conducted under lab conditions and even in low-acidity environment, which may be unable to completely reflect the conditions that AAB confront under industrialized vinegar-brewing processes. In this study, we performed an RNA-Seq transcriptomic analysis concerning AAB’s AR mechanisms during a continuous and periodical industrial submerged vinegar fermentation process, where a single AAB strain performed the fermentation and the acetic acid concentration fluctuated between ~8% and ~12%, the highest acidity as far we know for transcriptomic studies. Samples were directly taken from the initial (CK), mid, and final stages of the same period of the on-going fermentation. 16S rRNA sequence analysis indicated the participation of Komagataeibacter europaeus in the fermentation. Transcriptomic results demonstrated that more genes were downregulated than upregulated at both mid and final stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrich analysis reflected that the upregulated genes mainly carried out tricarboxylic acid cycle and oxidative phosphorylation processes, probably implying a considerable role of acetic acid overoxidation in AR during fermentation. Besides, upregulation of riboflavin biosynthesis pathway and two NAD+-dependent succinate-semialdehyde dehydrogenase-coding genes suggested a critical role of succinate oxidation in AR. Meanwhile, downregulated genes were mainly ribosomal protein-coding ones, reflecting that the adverse impact on ribosomes initiates at the transcription level. However, it is ambiguous whether the downregulation is good for stress responding or it actually reflects the stress. Furthermore, we also assumed that the fermentation stages may have a greater effect on gene expression than acidity. Additionally, it is possible that some physiological alterations would affect the AR to a larger extent than changes in gene expression, which suggests the combination of molecular biology and physiology research will provide deeper insight into the AR mechanisms in AAB.

Combined Action of Melatonin and Water Deficiency on Growth and MDA Content of Hypocotyls and Roots of Lupine and Respiration of Mitochondria Isolated from These Organs

Melatonin is known to improve the tolerance of plants to adverse abiotic factors (AAF), including dehydration. Nevertheless, details of the action of this phytohormone are not sufficiently comprehended. In the current study, the effects of melatonin on the growth, water status, and content of malonic dialdehyde were evaluated in etiolated seedlings of narrow-leaved lupine (Lupinus angustifolius L.) subjected to dehydration. The respiration of mitochondria isolated from different organs of these plants was also analyzed. Here, the 4-day-old seedlings have been deprived of water for 1 day. This entailed the increased water deficit up to 22% in the roots and 6% in the hypocotyls. Such desiccation of the seedlings retarded the growth of their hypocotyls but increased that of the roots by 13% in comparison with the undesiccated control. Both root and hypocotyl tissues accumulated MDA under these conditions. The dehydration of the seedlings negatively affected the respiration of the mitochondria isolated from these organs. The rate of oxidation of the substrates, especially malate, was diminished at the metabolic State 3 mainly due to an inhibition of the cytochrome respiration pathway (CP). For example, upon oxidation of malate, the activity of CP decreased 1.8 times in the mitochondria from hypocotyls and four to five times in those from roots. Pretreatment of the seedlings with 0.1 µM melatonin prevented the dehydration-induced rise in the MDA level in the hypocotyls but, by contrast, stimulated this parameter by 27% in the roots. Under drought conditions combined with exogenous melatonin, the growth of hypocotyls was close to that in the undesiccated control, while the root growth was lower than that in the control. In the seedlings normally supplied with water, treatment with melatonin did not significantly influence the respiration of the mitochondria prepared from the hypocotyls or roots. However, under water shortage, melatonin completely blocked the inhibition of a substrate oxidation in the hypocotyl mitochondria, chiefly because of sustaining the CP activity. As a result, exogenous melatonin increased the rate of malate oxidation by 87% and succinate oxidation by 26% in metabolic state 3 in the mitochondria from the hypocotyls. Application of this hormone to the plants only partially reversed the dehydration-induced inhibition of the oxidative phosphorilation in the root mitochondria. In this case, the oxidation of respiratory substrates, especially succinate, and activity of CP were still inhibited. Possible causes of the differences between the hypocotyl and root cells of the lupine seedling in their responses to melatonin and water deficit, namely, an extent of oxidative stress and functions of mitochondria, are discussed.

How an assembly factor enhances covalent FAD attachment to the flavoprotein subunit of complex II

The membrane-bound complex II family of proteins is composed of enzymes that catalyze succinate and fumarate interconversion coupled with reduction or oxidation of quinones within the membrane domain. The majority of complex II enzymes are protein heterotetramers with the different subunits harboring a variety of redox centers. These redox centers are used to transfer electrons between the site of succinate–fumarate oxidation/reduction and the membrane domain harboring the quinone. A covalently bound FAD cofactor is present in the flavoprotein subunit, and the covalent flavin linkage is absolutely required to enable the enzyme to oxidize succinate. Assembly of the covalent flavin linkage in eukaryotic cells and many bacteria requires additional protein assembly factors. Here, we provide mechanistic details for how the assembly factors work to enhance covalent flavinylation. Both prokaryotic SdhE and mammalian SDHAF2 enhance FAD binding to their respective apoprotein of complex II. These assembly factors also increase the affinity for dicarboxylates to the apoprotein–noncovalent FAD complex and stabilize the preassembly complex. These findings are corroborated by previous investigations of the roles of SdhE in enhancing covalent flavinylation in both bacterial succinate dehydrogenase and fumarate reductase flavoprotein subunits and of SDHAF2 in performing the same function for the human mitochondrial succinate dehydrogenase flavoprotein. In conclusion, we provide further insight into assembly factor involvement in building complex II flavoprotein subunit active site required for succinate oxidation.

Influence of Microbial Metabolites and Itaconic Acid Involved in Bacterial Inflammation on the Activity of Mitochondrial Enzymes and the Protective Role of Alkalization.

Human microbiota produces metabolites that may enter the bloodstream and exert systemic influence on various functions including mitochondrial. Mitochondria are not only a target for microbial metabolites, but also themselves, due to the inhibition of several enzymes, produce metabolites involved in infectious processes and immune response. The influence of indolic acids, microbial derivatives of tryptophan, as well as itaconic acid, formed in the tricarboxylic acid cycle under the action of bacterial lipopolysaccharides, on the activity of mitochondrial enzymes was studied by methyl thiazolyl tetrazolium (MTT), dichlorophenolindophenol (DCPIP) and pyridine nucleotide fluorescence assays. Thus, it was found that indolic acids suppressed succinate and glutamate oxidation, shifting the redox potential of pyridine nucleotides to a more oxidized state. Itaconic acid, in addition to the well-known inhibition of succinate oxidation, also decreased NAD reduction in reactions with glutamate as a substrate. Unlike itaconic acid, indolic acids are not direct inhibitors of succinate dehydrogenase and glutamate dehydrogenase as their effects could be partially eliminated by the thiol antioxidant dithiothreitol (DTT) and the scavenger of lipid radicals butyl-hydroxytoluene (BHT). Alkalization turned out to be the most effective means to decrease the action of these metabolites, including itaconic acid, which is due to the protective influence on redox-dependent processes. Thus, among mitochondrial oxidative enzymes, the most accessible targets of these microbial-related metabolites are succinate dehydrogenase and glutamate dehydrogenase. These are important in the context of the shifting of metabolic pathways involved in bacterial inflammation and sepsis as well as the detection of new markers of these pathologies.

Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium tuberculosis.

ABSTRACTSuccinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the tricarboxylic acid (TCA) cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bidirectional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 and sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 and sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of cell wall-targeting antibiotics and bioenergetic inhibitors against M. tuberculosis. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.

Tumor necrosis factor induces pathogenic mitochondrial ROS in tuberculosis through reverse electron transport.

Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. Here, using the zebrafish, we identify the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake increases the Krebs cycle intermediate succinate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor, metformin, a widely used anti-diabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis-infected zebrafish and human macrophages, suggesting its utility in tuberculosis therapy.

DIPG-07. Preclinical and case study results underpinning the phase II clinical trial testing the combination of ONC201 and paxalisib for the treatment of patients with diffuse midline glioma (NCT05009992)

Abstract Diffuse midline gliomas (DMG), including those of the brainstem (diffuse intrinsic pontine glioma - DIPG), are pediatric CNS tumors recognized as the most lethal of all children’s cancers. Palliative radiotherapy is the only approved treatment, with survival just 9-11–months post-diagnosis. ONC201 shows preclinical and emerging clinical efficacy in early-stage clinical trials, extending survival of DIPG patients by ~9-11–months compared to historic controls. However, patients invariably develop resistance, with some patients completely refractory to treatment. Using a multi-omics approach, including pharmacology, proteomics, genomics, epigenetics, in vitro and in vivo modeling, across ten international laboratories, we have uncovered the inherent mechanisms of resistance to ONC201. We find ONC201 elicits antagonism of the Dopamine receptor D2 (DRD2), whilst also causing mitochondrial degradation through potent agonism of the Mitochondrial protease CLPP, that drives proteolysis of the electron transport chain (ETC) protein Succinate dehydrogenase A (SDHA) and degradation of critical mitochondrial tricarboxylic acid (TCA) cycle regulator Isocitrate dehydrogenase 3B (IDH3B). Loss mitochondrial respiration increased hypoxia and reduced α-ketoglutarate, inhibiting lysine demethylation, increasing methylation of H3K4me3 and H3K27me3, thus altering the epigenome of primary DIPG cells. Loss of SDHA caused oxidation of succinate forming superoxide driving redox regulated PI3K/AKT signaling, counteracted using the PI3K/AKT inhibitor paxalisib. The combination of ONC201 and paxalisib synergically extended survival of two aggressive DIPG PDX models (SU-SIPG-VI vehicle=73 vs. combination=100-days, p=0.0027; SF8626 vehicle=36 vs. combination=43-days, p=0.0002). Compassionate access to this combination (n=2 patients; immediately post-RT and following re-RT) resulted in reductions in tumor volume and complete resolution of disease symptoms, extending overall survival (e.g., diagnosis patient MR axial scan=1554 mm2 , following eight months on the combination, current tumor volume=464 mm2 (<70%), patient remains on treatment). Our findings harness the powerful anti-DMG/DIPG pharmacokinetic/dynamic properties of ONC201 and paxalisib, a combination that is currently in clinical trials (NCT05009992).

Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis.

SUMMARYFumarate can be a surrogate for O2 as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O2 to H2O. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous 13C4-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O2 consumption by uncoupling adjacent RPE mitochondria.

Single Cell Assessment of Mitochondrial Function

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

Computational Modeling of Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics in the Heart and Kidney Cortex and Outer Medulla

RationaleIt is recognized that the kinetics and efficiency of mitochondrial O2 consumption for ATP production depend on the choice of respiratory substrates. Various substrates differentially generate the reducing equivalents NADH and FADH2 via the tricarboxylic acid (TCA) cycle that feed electrons to the electron transport chain (ETC) driving oxidative phosphorylation (OxPhos). However, the contributions of these substrates and their combinations have not been systematically characterized in either the heart or the kidney, the two major energy consuming organs in the body.MethodBioenergetic responses (respiration and membrane potential) of mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Drawly rats were characterized with different substrate combinations and different ADP perturbations. To better understand these distinct substrate and tissue‐specific mitochondrial bioenergetics, thermodynamically‐constrained mechanistic computational models were developed by integrating the kinetics and regulation of mitochondrial substrate transport and oxidation, TCA cycle, and ETC/OxPhos, incorporating published data and the novel mitochondrial bioenergetics data from our laboratory. Intrinsic model parameters such as Michaelis‐Menten constants were assumed identical and fixed for the heart and kidney, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on experimental data for the heart and kidney mitochondrial bioenergetics.ResultsConsistent with the data, model simulations of the ADP‐induced state 3 responses of the heart and kidney cortex and OM mitochondria yielded dramatically different values, which for a given organ were also very distinct for different substrates. In addition, heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturated [ADP] failed to produce a robust state 3 response. This was in contrast to kidney cortex and OM mitochondria for which responses to succinate±rotenone were similar, indicating that reverse electron transfer via complex I played less prominent regulatory role in the kidney than the heart. In addition, oxaloacetate, a TCA cycle intermediate, was shown to accumulate faster in the heart compared to the kidney, thereby potently inhibiting succinate oxidation more in the heart than the kidney. As such, the model was able to quantitatively characterize how various substrate combinations account for the differential contributions of different substrate oxidation pathways within the mitochondria towards OxPhos and ATP synthesis in the heart and kidney cortex and OM.ConclusionModeling of these data provided a deeper quantitative understanding of key determinants of kinetic and molecular mechanisms involved in the differential regulation of substrate‐dependent mitochondrial bioenergetics in the heart and kidney cortex and OM.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 μM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e− ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.