1,721,007 research outputs found
Real-time monitoring of basal and activity-dependent mitochondrial bioenergetics in cultured mouse dopamine neurons.
No full textMitochondria play an essential role in multiple cellular processes including ATP production, intracellular Ca2+ signaling, regulation of reactive oxygen species and apoptotic signalling. Neuronal functions and synaptic activity are strongly dependent on mitochondrial function for ATP supply and Ca2+ buffering. The identification of gene mutations in Parkinson's disease (PD) leading to impaired mitochondrial function and dopamine (DA) neuron death makes it critical to characterize mitochondrial bioenergetics in DA neurons. In the present study we quantified basal and activity-dependent mitochondrial energy metabolism in primary cultures of postnatal mesencephalic mouse DA neurons, grown on an astrocytes monolayer. Using a Seahorse XF24 analyzer, we measured simultaneously the rate of oxygen consumption (OCR), indicative of mitochondrial respiration and the rate of extracellular acidification (ECAR), reflecting glycolysis, thus providing a physiological readout of energetic metabolism. We first compared OCR and ECAR of mesencephalic DA neuron cultures to that of astrocytes cultured alone. We find that DA neuron cultures show considerably higher basal OCR and higher maximal OCR, measured in the presence of CCCP, an uncoupler agent. Quantification of ECAR revealed that cultured DA neurons also show elevated ECAR compared to astrocytes, but the relative difference was considerably less than for OCR, compatible with the known dependence of astrocytes on glycolysis rather than oxydative phosphorylation to produce ATP and the existence of more efficient mitochondrial energetic metabolism in neurons. To gain insight into the energetic metabolism of DA neurons compared to other types of mesencephalic neurons, we performed parallel experiments in purified DA neuron cultures prepared from TH-GFP transgenic mice. Our findings suggest that cultured DA neurons have higher basal OCR compared to other mesencephalic neurons.
Finally, we used veratridine, an agent preventing Na+ channel desensitization and TTX, a Na+ channel blocker, to evaluate the activity-dependence of mitochondrial bioenergetics in cultured DA neurons. Veratridine enhanced basal OCR and inhibited CCCP-stimulated respiration, while TTX had the oppostite effect. These treatments were without effect in astrocyte cultures. Together these data provide a first overview of mitochondrial bioenergetics in DA neurons. Further experiments are now planned to evaluate these parameters in genetic models of PD
Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2 -mediated oxidative insult in H9C2 myocardial cells
No full textMitochondrial calcium drives clock gene-dependent activation of pyruvate dehydrogenase and of oxidative phosphorylation
No full textRegulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In the present study we show that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork
Elevated Mitochondrial Bioenergetics and Axonal Arborization Size Are Key Contributors to the Vulnerability of Dopamine Neurons
No full textAlthough the mechanisms underlying the loss of neurons in Parkinson's disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson's disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP(+) (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP(+) but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson's disease is directly due to their particular bioenergetic and morphological characteristics
Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2-mediated oxidative insult in H9C2 myocardial cells
No full textAbstractCarvedilol, a β-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H2O2 production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H2O2 were investigated. The H2O2-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor.We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I
Autonomous Oscillatory Mitochondrial Respiratory Activity: Results of a Systematic Analysis Show Heterogeneity in Different In Vitro-Synchronized Cancer Cells
No full text: Circadian oscillations of several physiological and behavioral processes are an established process in all the organisms anticipating the geophysical changes recurring during the day. The time-keeping mechanism is controlled by a transcription translation feedback loop involving a set of well-characterized transcription factors. The synchronization of cells, controlled at the organismal level by a brain central clock, can be mimicked in vitro, pointing to the notion that all the cells are endowed with an autonomous time-keeping system. Metabolism undergoes circadian control, including the mitochondrial terminal catabolic pathways, culminating under aerobic conditions in the electron transfer to oxygen through the respiratory chain coupled to the ATP synthesis according to the oxidative phosphorylation chemiosmotic mechanism. In this study, we expanded upon previous isolated observations by utilizing multiple cell types, employing various synchronization protocols and different methodologies to measure mitochondrial oxygen consumption rates under conditions simulating various metabolic stressors. The results obtained clearly demonstrate that mitochondrial respiratory activity undergoes rhythmic oscillations in all tested cell types, regardless of their individual respiratory proficiency, indicating a phenomenon that can be generalized. However, notably, while primary cell types exhibited similar rhythmic respiratory profiles, cancer-derived cell lines displayed highly heterogeneous rhythmic changes. This observation confirms on the one hand the dysregulation of the circadian control of the oxidative metabolism observed in cancer, likely contributing to its development, and on the other hand underscores the necessity of personalized chronotherapy, which necessitates a detailed characterization of the cancer chronotype
cAMP/PKA Signaling Modulates Mitochondrial Supercomplex Organization
Full text linkThe oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production
Effect of Chicken Bone Extracts on Metabolic and Mitochondrial Functions of K562 Cell Line
Full text linkBackground: Tetracyclines’ use in intensive animal farming has raised some concerns regarding the biosafety for humans. Increasing evidences have revealed the presence of these drugs in processed animal by-products, such as bone, throughout the food chain. A potential off-target of tetracyclines is the bacterial-like mitochondrial translational machinery, thereby causing proteostatic alterations in mitochondrial DNA-encoded components of the oxidative phosphorylation system. Methods: The Seahorse methodology, confocal microscopy imaging of mitochondrial potential and reactive oxygen species, and q-RT-PCR analysis of the expression of genes involved in mitochondrial biogenesis and mitophagy were carried out on human lymphoblast derived K562 cell line challenged with bone powder derived from chicken treated with or without oxytetracycline and pure oxytetracycline. Results: A complex dose-dependent profile was attained with a low dosage of bone powder extracts causing a metabolic adaptation hallmarked by stimulation of the mitochondrial respiration and enhanced expression of mitochondriogenic factors in particular in cells challenged with oxytetracycline-free bone extract. Conversely, a higher dosage of bone powder extracts, regardless of their source, caused a progressive inhibition of mitochondrial respiration and glycolysis, ultimately leading to cell death. No significant effects of the pure oxytetracycline were observed. Conclusion: Bone powder, regardless of chicken treatment, contains and releases factors/chemicals responsible for the observed effects on energy metabolism. Quantitative differential effects appear to depend on biochemical alterations in the bone matrix caused by antibiotics rather than antibiotics themselves
- …
