The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

Naked mole‐rats (NMRs) are mouse‐sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging‐related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long‐lived species produce less ROS than do mitochondria of short‐lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long‐lived species from short‐lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H₂O₂; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H₂O₂ generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.     

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

Low hydrogen peroxide production in mitochondria of the long‐lived Arctica islandica: underlying mechanisms for slow aging

The observation of an inverse relationship between lifespan and mitochondrial H₂O₂ production rate would represent strong evidence for the disputed oxidative stress theory of aging. Studies on this subject using invertebrates are surprisingly lacking, despite their significance in both taxonomic richness and biomass. Bivalve mollusks represent an interesting taxonomic group to challenge this relationship. They are exposed to environmental constraints such as microbial H₂S, anoxia/reoxygenation, and temperature variations known to elicit oxidative stress. Their mitochondrial electron transport system is also connected to an alternative oxidase that might improve their ability to modulate reactive oxygen species (ROS) yield. Here, we compared H₂O₂ production rates in isolated mantle mitochondria between the longest‐living metazoan—the bivalve Arctica islandica—and two taxonomically related species of comparable size. In an attempt to test mechanisms previously proposed to account for a reduction of ROS production in long‐lived species, we compared oxygen consumption of isolated mitochondria and enzymatic activity of different complexes of the electron transport system in the two species with the greatest difference in longevity. We found that A. islandica mitochondria produced significantly less H₂O₂ than those of the two short‐lived species in nearly all conditions of mitochondrial respiration tested, including forward, reverse, and convergent electron flow. Alternative oxidase activity does not seem to explain these differences. However, our data suggest that reduced complex I and III activity can contribute to the lower ROS production of A. islandica mitochondria, in accordance with previous studies. We further propose that a lower complex II activity could also be involved.     

Reverse electron flow-mediated ROS generation in ischemia-damaged mitochondria: Role of complex I inhibition vs. depolarization of inner mitochondrial membrane

Background

The reverse electron flow-induced ROS generation (RFIR) is decreased in ischemia-damaged mitochondria. Cardiac ischemia leads to decreased complex I activity and depolarized inner mitochondrial membrane potential (ΔΨ) that are two key factors to affect the RFIR in isolated mitochondria. We asked if a partial inhibition of complex I activity without alteration of the ΔΨ is able to decrease the RFIR.

Methods

Cardiac mitochondria were isolated from mouse heart (C57BL/6) with and without ischemia. The rate of H₂O₂ production from mitochondria was determined using amplex red coupled with horseradish peroxidase. Mitochondria were isolated from the mitochondrial-targeted STAT3 overexpressing mouse (MLS-STAT3E) to clarify the role of partial complex I inhibition in RFIR production.

Results

The RFIR was decreased in ischemia-damaged mouse heart mitochondria with decreased complex I activity and depolarized ΔΨ. However, the RFIR was not altered in the MLS-STAT3E heart mitochondria with complex I defect but without depolarization of the ΔΨ. A slight depolarization of the ΔΨ in wild type mitochondria completely eliminated the RFIR.

Conclusions

The mild uncoupling but not the partially decreased complex I activity contributes to the observed decrease in RFIR in ischemia-damaged mitochondria.

General significance

The RFIR is less likely to be a key source of cardiac injury during reperfusion.     

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen speciesgeneration

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca²⁺ uptake. Here we sought to elucidate the effects of extramitochondrial Ca²⁺ (e[Ca²⁺]) on ROS production (measured as H₂O₂ release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl₂ and then energized with the complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. Mitochondrial H₂O₂ release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca²⁺] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H₂O₂ (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca²⁺], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H₂O₂ release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca²⁺] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H₂O₂ emission from complex III during ischemia.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Mitochondrial generation of reactive oxygen species is enhanced at the Q<Subscript>o</Subscript> site of the complex III in the myocardium of <Emphasis Type="Italic">Trypanosoma cruzi-</Emphasis>infected mice: beneficial effects of an antioxidant

In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O₂ ^•−) and amplex red (detects H₂O₂), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q_Nf-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q_o center that was different from the Q_i center of O₂ ^•− generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q_o site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-α-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q_o site of complex III resulted in increased electron leakage and O₂ ^•− formation in infected myocardium, and was controlled by PBN.     

The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide

Neurodegenerative diseases, a subset of age-driven diseases, have been known to exhibit increased oxidative stress. The resultant increase in reactive oxygen species (ROS) has long been viewed as a detrimental byproduct of many cellular processes. Despite this, therapeutic approaches using antioxidants were deemed unsuccessful in circumventing neurodegenerative diseases. In recent times, it is widely accepted that these toxic by-products could act as secondary messengers, such as hydrogen peroxide (H₂O₂), to drive important signaling pathways. Notably, mitochondria are considered one of the major producers of ROS, especially in the production of mitochondrial H₂O₂. As a secondary messenger, cellular H₂O₂ can initiate redox signaling through oxidative post-translational modifications (oxPTMs) on the thiol group of the amino acid cysteine. With the current consensus that cellular ROS could drive important biological signaling pathways through redox signaling, researchers have started to investigate the role of cellular ROS in the pathogenesis of neurodegenerative diseases. Moreover, mitochondrial dysfunction has been linked to various neurodegenerative diseases, and recent studies have started to focus on the implications of mitochondrial ROS from dysfunctional mitochondria on the dysregulation of redox signaling. Henceforth, in this review, we will focus our attention on the redox signaling of mitochondrial ROS, particularly on mitochondrial H₂O₂, and its potential implications with neurodegenerative diseases.Subject terms: Post-translational modifications, Neurodegenerative diseases     

The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H₂O₂ in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.     

Caloric restriction influences hydrogen peroxide generation in mitochondrial sub-populations from mouse liver

Calorie restriction (CR) has been shown to decrease H₂O₂ production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H₂O₂ production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H₂O₂ production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H₂O₂ production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H₂O₂ outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H₂O₂ further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H₂O₂ to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.     

Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo

There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H₂O₂) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H₂O₂ content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H₂O₂ into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H₂O₂ in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H₂O₂ content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H₂O₂ content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H₂O₂ content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H₂O₂. The mechanism underlying this observed relationship between SMR and in vivo H₂O₂ content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H₂O₂.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H₂O₂)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H₂O₂ produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H₂O₂. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.     

Moderate Dependence of ROS Formation on ΔΨm in Isolated Brain Mitochondria Supported by NADH-linked Substrates

The membrane potential (ΔΨm) dependence of the generation of reactive oxygen species (ROS) in isolated guinea-pig brain mitochondria respiring on NADH-linked substrates (glutamate plus malate) was addressed. Depolarization by FCCP was without effect on H₂O₂ formation in the absence of bovine serum albumin (BSA). Addition of BSA (0.025%) to the assay medium hyperpolarized mitochondria by 6.1 ± 0.9 mV (from 169 ± 3 to 175.1 ± 2.1 mV) and increased the rate of H₂O₂ formation from 207 ± 4.5 to 312 ± 12 pmol/min/mg protein. Depolarization by FCCP (5–250 nM) in the presence of BSA decreased H₂O₂ formation but only to the level observed in the absence of BSA. Rotenone stimulated the formation of H₂O₂ both in the absence and presence of BSA. It is suggested that H₂O₂ formation in mitochondria supported by NADH-linked substrates is sensitive to changes in ΔΨm only when mitochondria are highly polarized and even then, 60% of ROS generation is independent of ΔΨm. This is in contrast to earlier reports on the highly ΔΨm sensitive ROS formation related to reverse electron flow observed in well-coupled succinate-supported mitochondria. Special issue dedicated to John P. Blass.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.