The Dual Function of Reactive Oxygen/Nitrogen Species in Bioenergetics and Cell Death: The Role of ATP Synthase

Oxidation and S-nitrosylation have also been shown to occur at the level of ATP synthase in a canine model of dyssynchronous heart failure (DHF) [26]. Indeed, DHF was correlated with disulfide bond formation in the ATP synthase complex occurring between either cysteine 294 of the neighboring α subunits or cysteine 294 of the α subunit and cysteine 103 of the γ subunit. This cross-linking was negatively correlated with ATP hydrolytic activity. Furthermore, cysteine 294 of the ATP synthase α subunit was also S-glutathionylated in DHF, another modification that correlated with a reduction in ATP synthase activity [104]. Interestingly, during chronic resynchronization therapy (CRT, the only clinically effective therapy for DHF) the disulfide bond at cysteine 294 was found to be reversed and partially replaced by S-nitrosylation, resulting in the recovery of ATPase activity. Indeed, the same group has shown that cysteine 294 is critical for ATP synthase function in vitro and that it may play a major role in redox regulation of ATP production acting as a redox-sensor [160]. Nevertheless, the S-nitrosylation of the α subunit and improvement in ATPase activity observed in this study are in contrast with the previously mentioned results from Sun et al. [29], showing that S-nitrosylation of ATP synthase α subunit in IPC or after GSNO treatment leads to a reduction of its hydrolytic activity. One cannot exclude that different cysteine residues were modified in the two aforementioned studies, a fact that could explain this discrepancy. Another possible explanation is that cross-link reversal and recovery of ATPase activity observed in CRT occur at a higher level compared to the extent of S-nitrosylation, suggesting that the majority of cysteine residues are actually free in CRT hearts [26].

On the other hand, the distances between the two cysteines 294 of neighboring α subunits or between α cysteine 294 and γ cysteine 103 are more than 5 Å (corresponding to α-Cys251 and γ-Cys78, respectively, in Figures 1 and 2), making it unlikely that these disulfide bonds actually occur in the assembled complex. Rather, these bonds could form in the misfolded/aggregated enzyme, suggesting that the reversal of cysteine cross-links observed in CRT may promote the correct assembly of the complex, thus resulting in improved ATPase activity. Of note, CuCl2 treatment of isolated ATP synthase complex from rat mitochondria in vitro showed that disulfide bonds can form also between α and OSCP subunits as well as between the two γ subunits and γ and OSCP [26]. The effect of these disulfide crossbridges on ATP synthase activity was not addressed, and whether these modifications could also occur in vivo still needs to be assessed. Further efforts should also be put in addressing the effect of these oxidative posttranslational modifications on PTP formation.

Mitochondria have a central role in aging-related neurodegenerative diseases. Oxidative stress generated by mitochondria has been inversely correlated with longevity in model organisms [161, 162] and defects in mitochondrial bioenergetics have been implicated in a number of neurodegenerative diseases [163].

In vitro, ATP synthase is susceptible to ROS [103, 164, 165] and to oxidative/nitrosative stress associated with disorders of the central nervous system [166, 167] and aging [35, 168]. The fact that ROS/RNS modifications can alter the mitochondrial oxidative phosphorylation efficiency may explain the mitochondrial involvement in neurological diseases.

Moreover, the involvement of PTP in neurodegenerative diseases has been demonstrated by the use of CsA both in vitro and in vivo and the ablation in mouse models of its target, CyPD [17, 18]. CyPD plays an important dual function on the modulation of ATP synthase; on the one hand, it sensitizes PTP to matrix calcium, while, on the other hand, it inhibits both ATP hydrolysis and synthesis by 30% [11]. In cellular mechanisms of neurodegenerative diseases, when mitochondria are hydrolyzing ATP, CyPD binding and inhibition of ATP synthase can be an advantage avoiding ATP consumption and cell death, or a disadvantage in cases in which PTP is sensitized to calcium and the displacement of CyPD would promote cell survival. This is only one of the complex relationships between ATP synthase and PTP in neurodegeneration, and in this paragraph we attempt to summarize possible mechanisms linking ATP synthase/PTP and ROS/RNS modifications.

Amyotrophic lateral sclerosis (ALS) is a progressive degeneration of motor neurons. In familial ALS, mutations have been found in Cu,Zn-SOD that are suggested to increase generation of ROS [169, 170], RNS, and nitrosylation [171]. Furthermore, Beal et al. [172] detected increased levels of nitrotyrosine staining in motor neurons of both sporadic ALS and familial ALS, suggesting that ONOO− mediated oxidative damage may play a role in both forms of the disease. Of note, as already mentioned, tyrosine modifications at the level of the β subunit of ATP synthase have been described to be modulatory of the catalytic ATPase activity in the presence of ROS/RNS [108, 109], even if their direct involvement in ALS remains to be addressed.

In light of the susceptibility of the mitochondrial respiratory chain to nitrosative stress, it is not surprising that mitochondrial function is impaired in ALS [173]. Spinal cord mitochondria in ALS mouse model display decreased calcium retention capacity long before the onset of motor weakness and neuronal death [174], and this was corrected by ablation of the Ppif gene which encodes CyPD [175]. In these mice, an improved mitochondrial ATP synthesis was matched by PTP inhibition and significant suppression of motor neuron death throughout disease, although survival was not improved confirming the role of ATP synthesis and permeability transition in ALS neuron cell death [176, 177]. Increased ATP synthesis in this mouse ALS model lacking CyPD could also be explained by our observation that ablation of CyPD increases the catalysis of ATP synthase [11].

AD, the most common form of dementia in aged people, is characterized by deposition of amyloid plaques formed by the amyloid β peptide [178]. Amyloid β can be imported in mitochondria [179]. Several studies have observed activity changes in key mitochondrial enzymes in AD. While the exact mechanism for this loss of activity is unclear, evidence suggests that ROS/RNS production is increased in AD. Nitrotyrosine residues have been detected in postmortem Alzheimer's tissue but not in age-matched controls [180], indicating the presence of ONOO−. The induction of iNOS in cultured rat astrocytes causes NO-mediated neuronal death in a coculture system [181].

Amyloid β is also reported to stimulate glial NO production [182–184] and it has been shown to inhibit both purified complex IV [185] and complex IV in isolated brain mitochondria [186]. Additionally, in neuronal cultures amyloid β caused a loss of activity of all the mitochondrial complexes and a loss of mitochondrial integrity, due to PTP opening [187]. It has been shown that amyloid β oligomers alter calcium homeostasis [188].

Upon import in mitochondria, amyloid β interacts with CyPD and enhances PTP induction, since neurons from CyPD KO mice are protected from cell death induced by amyloid β dependent PTP opening [189]. Interestingly, a novel association with AD risk has been recently identified in the ATP synthase ATP5H locus, which encodes subunit d of the lateral stalk [190]. Intriguingly, in a proteomic analysis of human heart mitochondria in normal condition, a tryptophan residue at the level of d subunit was identified as a “hot spot” for oxidation [36], the latter being another important pathophysiological factor in AD. Moreover, in the filamentous fungus Podospora anserina, a model with a clear defined mitochondrial etiology of aging, another tryptophan residue on the α subunit of ATP synthase (Trp503) has been described to be crucial for the selective targeting of oxidative damage [107]. Although this residue is not conserved in unicellular fungi and higher eukaryotes, the example of P. anserina is instructive because the authors proposed that oxidized cysteine and methionine can be efficiently reduced by repair mechanisms, whereas tryptophan oxidation products are irreversible and have the potential to form markers detected by the mitochondrial quality control system.

In multiple sclerosis (MS), the myelin sheath of neurons in the central nervous system is destroyed leading to axonal degeneration [191]. This is associated with mitochondrial calcium overload and bioenergetic dysfunction [192]. CyPD KO mice with autoimmune encephalomyelitis display a marked protection from axonal degeneration and a milder clinical picture despite a normal inflammatory response, thus suggesting that PTP might be involved in disease pathogenesis [193], even if other effects on bioenergetics caused by CyPD ablation cannot be excluded. A further indication of the important role played by the PTP in MS and its activation by ROS is provided by the observation that axonal damage of mice undergoing experimental autoimmune encephalomyelitis is reduced by genetic ablation of p66Shc [194]. Importantly, a proteomic study of experimental autoimmune encephalomyelitis identified the ATP synthase dimer-forming subunit e differentially expressed [195]. Moreover, mutations at the level of the genes encoding subunits a and A6L were observed in MS patients [196].

A large body of evidence exists implicating increased generation of RNS in MS. The observation that the concentration of NO2 − plus NO3 − in cerebrospinal fluid is elevated by 70% in MS patients supports this hypothesis [197]. Furthermore, increased iNOS activity and iNOS mRNA have been demonstrated in astrocytes associated with demyelinating lesions in postmortem MS brain [198] and in experimental models of demyelination [199, 200]. Nitrotyrosine residues indicating the presence of ONOO− have also been detected in MS brain [201]. Whether NO-mediated mitochondrial damage is the cause of the disease remains to be established; however, the first direct evidence of altered mitochondrial function in MS came from a study by Lu et al. [202]. These authors described a decrease of respiratory complex I activity and a compensatory increase in complex IV in MS. Major targets for nitrosative stress in rat liver under in vivo conditions are tyrosine 345 and tyrosine 368 in the β subunit of the mitochondrial ATP synthase that is suggested as an early marker of nitrosative stress and aging [35]. A comparison between α and β subunits allowed the speculation that the latter is more accessible for RNS modification and that the catalytic conformation of this subunit also affects tyrosine residues exposure to nitration (higher exposure to RNS modifications in the absence of bound nucleotide). Moreover, Ding et al. [203] demonstrated in ND4 mice (an in vivo model for MS) an impaired transport of ATP5b mRNA (which generates subunit β) to mitochondria. This caused decreased ATP synthesis in MS mice due to lower levels of this subunit and not to impaired ATP synthase assembly, as suggested by the authors. However, the possibility that tyrosine modifications in this subunit might affect enzyme activity cannot be excluded.

PD is caused by death of dopaminergic neurons in the substantia nigra pars compacta. A sensitization to PTP opening has been proposed as a major cause of neurodegeneration in several models of the disease characterized by altered homeostasis of intracellular calcium [204–206], including the forms caused by complex I inhibition [207, 208] and by inactivation of the Ser/Thr kinase PINK1 [209], in which changes in calcium storage capacity [210, 211], impairment of respiratory complex I [212], and altered mitophagy [213] are observed. Moreover, the PTP can be induced in dopaminergic neurons because of the inability to buffer increased intracellular ROS levels [214]. In a PD mouse model induced by parkin deficiency, state 3 and state 4 respiration rates were both affected indicating a more likely direct modulation of the respiratory chain compared to an effect on ATP synthase [215]. On the other hand, a mouse model lacking chaperone protein HtrA2 and showing a parkinsonian phenotype displays mitochondrial uncoupling at the level of ATP synthase and a truncated α subunit that might be involved in the neurodegeneration observed in these mice [33].

Postsynaptic density protein 95 (PSD-95) binds to neuronal nNOS and the neuroprotective effects of small-molecule inhibitors of this interaction were tested in an in vitro PD model. The observed protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased ROS generation, preserved ATP synthesis, and PTP inhibition [216]. Moreover, Darios et al. [217] demonstrated that in PC12 cells the overexpression of parkin protects from ceramide-induced swelling suggesting that parkin may act directly to prevent PTP mediated cell death, but the exact mechanism remains to be addressed.

Posttranslational modifications of ATP synthase due to ROS/RNS generation might play a dual role by promoting cell death or survival depending on their relative effects on mitochondrial ATP synthase catalysis and PTP. We have illustrated how ATP synthase is the target for oxidative/nitrosative modifications that affect its activity and might promote formation of the PTP. Identification of specific residues involved in the latter event is still lacking and will help to elucidate the mechanisms that mediate the role of ATP synthase in modulating cell survival or death. Finally, we discussed how changes in the ATP synthase activity regulate mitochondrial ROS formation and thus may represent an attractive strategy for the treatment of pathologies such as cancer.