Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for ...freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
Synopsis
Freeze‐thawing events cause mitochondrial membrane permeabilization and disrupt mitochondrial functionality in cells and tissues. Reconstitution of maximal mitochondrial respiration allows the analysis of mitochondrial bioenergetics in frozen and thawed crude samples, thus overcoming limitations associated with the current methods.
Following reconstitution, mitochondrial maximal respiration can be assessed in frozen isolated mitochondria or total tissue lysate.
Respiratory rate measurements from tissue lysates reduce by an order of magnitude the minimal mass of tissue required.
Maximal respiration rates in reconstituted frozen samples are comparable to those in fresh ones.
Respiratory rates can be normalized per cell, per total protein or per mitochondrial content in frozen specimens.
Respiratory rates can be measured in stored samples from clinical and animal studies, and drug toxicity assays.
Reconstitution of maximal mitochondrial respiration circumvents the limitations associated with current methods for assessing mitochondrial bioenergetics in frozen clinical samples.
Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce ...Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.
Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic ...fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.
Redox networks in the cell integrate signaling pathways that control metabolism, energetics, cell survival, and death. The physiological second messengers that modulate these pathways include nitric ...oxide, hydrogen peroxide, and electrophiles. Electrophiles are produced in the cell via both enzymatic and nonenzymatic lipid peroxidation and are also relatively abundant constituents of the diet. These compounds bind covalently to families of cysteine-containing, redox-sensing proteins that constitute the electrophile-responsive proteome, the subproteomes of which are found in localized intracellular domains. These include those proteins controlling responses to oxidative stress in the cytosol—notably the Keap1-Nrf2 pathway, the autophagy-lysosomal pathway, and proteins in other compartments including mitochondria and endoplasmic reticulum. The signaling pathways through which electrophiles function have unique characteristics that could be exploited for novel therapeutic interventions; however, development of such therapeutic strategies has been challenging due to a lack of basic understanding of the mechanisms controlling this form of redox signaling. In this review, we discuss current knowledge of the basic mechanisms of thiol-electrophile signaling and its potential impact on the translation of this important field of redox biology to the clinic. Emerging understanding of thiol-electrophile interactions and redox signaling suggests replacement of the oxidative stress hypothesis with a new redox biology paradigm, which provides an exciting and influential framework for guiding translational research.
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•The oxidative stress paradigm cannot explain the mechanisms of action of reactive species in redox signaling.•The redox biology paradigm offers a new explanation for the lack of efficacy of lipid radical scavenging antioxidants such as α-tocopherol.•The redox biology paradigm proposes that loss of control of specific redox signaling leads to pathology.•This model allows for the development of specific molecular therapeutics for diseases of redox signaling dysfunction.
The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic ...in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.
Parkinson's disease is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, ...exposure to the environmental neurotoxin rotenone increases the probability of developing Parkinson's disease. We previously reported that in differentiated SH‐SY5Y cells, rotenone‐induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 h, caused cell death at 24 h with an LD50 of 10 nM. Overall, autophagic flux was decreased by 10 nM rotenone at both 2 and 24 h, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 h, suggesting that a mitochondrial‐specific lysosomal degradation pathway may be activated. Up‐regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3‐methyladenine exacerbated cell death. Interestingly, while 3‐methyladenine exacerbated the rotenone‐dependent effects on bioenergetics, rapamycin did not prevent rotenone‐induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor.
Exposure to the neurotoxin rotenone is a risk factor for Parkinson's disease. We tested the hypothesis that autophagy is protective against rotenone toxicity in primary neurons. Exposure to nanomolar concentrations of rotenone caused immediate mitochondrial dysfunction, associated with a suppression of macroautophagy. However, mitophagy occurred that was independent of LC3II accumulation, and the surviving neurons exhibited adaptations to their cellular bioenergetic profiles. Cotreatment with the autophagy enhancer rapamycin was protective, whereas further inhibition of autophagy with 3‐methyladenine (3‐MA) exacerbated cell death, resulting in additional bioenergetic adaptations in the surviving neurons.
Exposure to the neurotoxin rotenone is a risk factor for Parkinson's disease. We tested the hypothesis that autophagy is protective against rotenone toxicity in primary neurons. Exposure to nanomolar concentrations of rotenone caused immediate mitochondrial dysfunction, associated with a suppression of macroautophagy. However, mitophagy occurred that was independent of LC3II accumulation, and the surviving neurons exhibited adaptations to their cellular bioenergetic profiles. Cotreatment with the autophagy enhancer rapamycin was protective, whereas further inhibition of autophagy with 3‐methyladenine (3‐MA) exacerbated cell death, resulting in additional bioenergetic adaptations in the surviving neurons.
Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, ...diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.
Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subjected to stress, ...mitochondria are capable of drawing upon a 'reserve capacity' which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular context. Therefore the goal of the present study was to determine whether 'bioenergetic reserve capacity' does indeed exist in the intact myocyte and whether it is utilized in response to stress induced by the pathologically relevant reactive lipid species HNE (4-hydroxynonenal). We found that intact rat neonatal ventricular myocytes exhibit a substantial bioenergetic reserve capacity under basal conditions; however, on exposure to pathologically relevant concentrations of HNE, oxygen consumption was increased until this reserve capacity was depleted. Exhaustion of the reserve capacity by HNE treatment resulted in inhibition of respiration concomitant with protein modification and cell death. These data suggest that oxidized lipids could contribute to myocyte injury by decreasing the bioenergetic reserve capacity. Furthermore, these studies demonstrate the utility of measuring the bioenergetic reserve capacity for assessing or predicting the response of cells to stress.
Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative ...phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.
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•CD4 T cells rapidly increase mitochondrial respiration during Th17 differentiation•OXPHOS is essential for Th17 cell pathogenicity in a mouse model of MS•Mitochondrial respiration shapes the Th17 and Treg cell fate decision•OXPHOS facilitates TCR and mTOR signaling, which in turn support BATF induction
Shin et al. report that ATP-linked mitochondrial respiration controls the Th17 and Treg cell fate decision by supporting TCR signaling and Th17-associated molecular events. Inhibition of mitochondrial OXPHOS ablates Th17 pathogenicity in a mouse model of MS and results in generation of functionally suppressive Treg cells under Th17 conditions.
Parkinson's disease (PD) is a movement disorder with widespread neurodegeneration in the brain. Significant oxidative, reductive, metabolic, and proteotoxic alterations have been observed in PD ...postmortem brains. The alterations of mitochondrial function resulting in decreased bioenergetic health is important and needs to be further examined to help develop biomarkers for PD severity and prognosis. It is now becoming clear that multiple hits on metabolic and signaling pathways are likely to exacerbate PD pathogenesis. Indeed, data obtained from genetic and genome association studies have implicated interactive contributions of genes controlling protein quality control and metabolism. For example, loss of key proteins that are responsible for clearance of dysfunctional mitochondria through a process called mitophagy has been found to cause PD, and a significant proportion of genes associated with PD encode proteins involved in the autophagy‐lysosomal pathway. In this review, we highlight the evidence for the targeting of mitochondria by proteotoxic, redox and metabolic stress, and the role autophagic surveillance in maintenance of mitochondrial quality. Furthermore, we summarize the role of α‐synuclein, leucine‐rich repeat kinase 2, and tau in modulating mitochondrial function and autophagy. Among the stressors that can overwhelm the mitochondrial quality control mechanisms, we will discuss 4‐hydroxynonenal and nitric oxide. The impact of autophagy is context depend and as such can have both beneficial and detrimental effects. Furthermore, we highlight the potential of targeting mitochondria and autophagic function as an integrated therapeutic strategy and the emerging contribution of the microbiome to PD susceptibility.
This review provides highlights on recent observations regarding the multifacet impact of pathological proteins, endogenously produced reactive species, environmental toxins, and metabolism including glucose and fatty acid metabolism, on mitochondria – autophagy function, in the context of Parkinson's disease. The review also discusses future studies of designing targeted strategies to aid the treatment of Parkinson's disease.
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