A Walk in the Memory, from the First Functional Approach up to Its Regulatory Role of Mitochondrial Bioenergetic Flow in Health and Disease: Focus on the Adenine Nucleotide Translocator
From the liver we move to the heart, where maintaining a high ATP supply is critically important for maintaining cardiac performance since the amount of ATP made and used per minute is many times greater than the size of the ATP pool. Furthermore, since ATP availability in cardiac cells is dependent mainly on OXPHOS, which can occur only in intact mitochondria, we investigated the ATP production made in coupled and respiring mitochondria with the aim of ascertaining the process controlling cell ATP availability. Furthermore, there was reason to suspect that cell ATP supply could be modified in the heart of both normotensive and spontaneous hypertensive aging rats. It was prompted us to investigate how ATP availability outside mitochondria is regulated in mitochondria from rat heart left ventricles (MLV) in hypertrophy and hypertrophy/hypertension states and whether and how it changes in aging.
In this as well as in similar cases, to understand the relationships between the different cellular components in a system biology framework, it may be more appropriate to consider functional products rather than genes, in light of their specific expression in different conditions (i.e., tissue, developmental stage, or pathological status) [130]. On the other hand, studies based on a proteomic approach [131,132] are limited by the impossibility of assessing both the activity of the changed proteins and the rate-limiting step of any biochemical pathway. Thus, having resorted to a kinetic approach in which the rate of supply of ATP produced in OXPHOS was continuously measured in MLV, in a first step in this study, we observed that such a rate is dependent on the rate of ADP/ATP exchange via ANT and significantly decreases in spontaneously hypertensive rats (SHRs) as a result of change in both carrier activity and ADP affinity for ANT.
Successively, the ANT rate proved to change in an opposite manner in aging: It decreased in MLV isolated from normotensive Wistar Kyoto rats (WKYRs), but increased in MLV from SHRs due to changes in the ANT function depending on the lifespan, as kinetically shown [133,134,135].
Interestingly, this occurred in two opposite manners: the rate of ATP cytosolic supply decreased in the WKYR, but it increased in the SHRs. Indeed, in aging WKYRs we found a reduced ANT activity in accordance with the altered ANT activity and expression already observed in a variety of human heart failure [136,137]; contrarily, in SHRs the rate of ATP supply outside mitochondria in vitro increased by 300–600% in the physiological ADP concentration range (20–60 μM) [138].
The explanation of the mechanism by which the ANT alteration takes place remains a matter of speculation: the generation of ROS in the electron flow along the mRC [139] and/or the occurrence of reactive aldehydes, already reported to deactivate mitochondrial enzymes, including the ANT [139], were taken into consideration.
ANT damage could also be dependent on a shift in ANT isoform composition, as in [140], as well as on a specific age-dependent decrease in cardiolipin content [141,142].
At present we do not know which is the mechanism by which the Vmax of ANT decreases; however, in light of data in which no difference in ANT expression was found in MLV from four- and eight-month-old rats [137], we assume that a change in the ANT catalytic efficiency occurs in aging.
Our conclusion is not unique: A change in ANT conformation in aging has already been shown via electron microscopy [143], but this paper has the added dimension of showing how such a change relates to ANT function in aging.
Consistently, in transgenic rats overexpressing ANT1 in the heart and crossed with renin-overexpressing rats suffering from hypertension-induced cardiac insufficiency, [137] showed that an accelerated ATP/ADP transport across the mitochondrial membrane improves mitochondrial structure and function, leading to a reduction in fibrosis and an improvement in cardiac tissue architecture, and thus may be a basic principle for new strategies in treating heart disease. In this study, the authors measured the ANT-mediated ADP/ATP exchange rate using ATP D.S., but unlike our original procedure, they preferred to use ATP-loaded mitochondria and then cause ATP efflux following the addition of ADP. What a pity! In fact, it follows that in order for mitochondria to accumulate ATP not only must their metabolism be blocked, but the neo-synthesis of ATP from ADP that has entered the mitochondria must also be blocked. We are facing a situation far from the physiological one!
To describe the specific mechanisms of cell death occurring during neurodegenerative disorders such as Alzheimer’s disease (AD), many investigations, both in vivo and in vitro, have attempted to label the particular pathway of cell death either as apoptosis or as necrosis. Both apoptotic and necrotic cells can be found in AD tissue, and both death types can overlap, sequentially occur under certain conditions, and not be detected unequivocally.
Therefore, although many in vivo and in vitro studies favor apoptosis in AD, there is considerable evidence that a mixture of both events may contribute to neurodegeneration in AD and to its final pathology.
Even though the deposition of β-amyloid and the formation of neurofibrillary tangles due to Tau protein hyperphosphorylation and deposition represent morphological hallmarks of the disease, several impressive lines of evidence suggest that both lesions are not sufficient to cause the AD neurodegenerative process.
Starting from this awareness, with the aim of shedding light on the molecular mechanisms responsible for the onset of AD, for about 30 years our study essentially has made use of an AD cell model, which consists of cerebellar granule cells (CGCs) undergoing apoptosis due to potassium deprivation, but we also used the characteristic AD hallmarks, in particular the smaller and more potent NH226–44-tau peptide combined or not with Aβ 1-42, the major pathogenic species in AD [144], for our ex vivo experiments on mitochondria function, observing that they cooperate by potentiating the ANT-1 impairment ex vivo in coupled neuronal mitochondria (see below).
Deliberately leaving aside the studies carried out on events that progressively cause death, here we report only those studies in which ANT has been studied, highlighting how its role and functionality in the dying cell vary (Figure 3).
One of the outstanding problems about the role of mitochondria in apoptosis concerns ATP production and utilization in cytosol, where ATP is used in a variety of processes, including caspase activation [154]. Thus, the role of ANT in apoptosis is crucial. Although the mPTP is suggested to be an important mediator of apoptosis (see Section 3.6.2), we measured ADP/ATP exchange and mitochondrial permeability transition (mPT) as a function of time after the induction of apoptosis and we concluded that the function of ANT in cells en route to apoptosis is as follows: In early apoptosis a decrease in the transport efficiency of ANT occurs, caused by a ROS-mediated post-translational modification as shown by its prevention by superoxide dismutase (SOD) and resulting in reduced affinity for ADP. This finding was concomitant with that of Nakagawa [155], who suggested that impairment of ANT is due to ROS-dependent cardiolipin oxidation. In late apoptosis, together with cyclosporin A-sensitive mPTP opening, a further progressive decrease in ANT transport function, i.e., of ADP/ATP exchange, which can be prevented by the caspase inhibitor z-VAD, occurred. Consistently, Xia et al. [156] found that recombinant caspase-3 directly induced mPTP opening and the associated mitochondrial apoptotic phenotype.
It is clear that in this experimental context, ATP D.S. was well suited to measuring the rate of ATP production, obviously reflecting the mitochondrial impairment that occurs during apoptosis [157], which is fitting given that the system measures the exchange in conditions close to real. However, the usefulness of the type of experiments carried out lay precisely in verifying from time to time that the ultimate effect of advancing apoptosis-dependent cellular deterioration as well as that operated by the molecules used, i.e., SOD and z-VAD, safeguard the limiting step of the global process, i.e., the ADP/ATP exchange.
We believe that in this context it is appropriate to dwell on an elegant experiment in which both ADP/ATP exchange and mPTP opening, measured as ADK release into the extramitochondrial phase, were investigated as a function of time (0–8 h) of apoptosis [145]. In early apoptosis (up to 3 h), the rate of ADP/ATP exchange was found to decrease with respect to the control, in which it remained constant at all times, with inhibition increasing with the progression of apoptosis; in the same time range, no or negligible mPTP opening was found. In late apoptosis, a further decrease in ADP/ATP exchange was found, whereas mPTP opening occurred over the 3–8 h time range in apoptotic cells, showing that during apoptosis ANT functionality gives way to mPTP opening.
In order to further refine this type of experiment and to obtain some insight into the mechanism by which these processes occur as well as to determine whether the two processes are really linked to one another, we treated cells undergoing apoptosis with a variety of compounds designed to inhibit certain activities that participate in the processes leading to apoptosis.
Then, in cells incubated with SOD, i.e., under No-ROS conditions, almost complete prevention of ANT impairment was found in early apoptosis, showing that the decrease in ANT transport efficiency depends on ROS production. In late apoptosis, in spite of the negligible change in ROS production [145], progressive impairment in ANT transport was found but to a lesser extent than with apoptotic cells. No mPTP opening was found in early apoptosis, showing that ROS cannot themselves cause the mPT, which occurred only in the 3–8-h time range, but to a lesser extent than the control, i.e., in the absence of SOD.
Interestingly, in apoptotic cells treated with z-VAD, a molecule that prevents caspase activity, no opening of the mPTP occurred either in early or late apoptosis, showing conclusively that mPTP opening requires caspase action. Furthermore, the time-dependent decrease in ANT activity in early apoptosis paralleled that found in control cells, but in late apoptosis no significant further reduction was found, showing that only the late ANT impairment is caspase dependent. As expected in light of the above results, when both ROS and caspase were put out of action, i.e., in the presence of SOD and z-VAD added simultaneously, ANT activity remained largely unaffected with respect to normal cells, and no mPT occurred at all [145].
In order to summarize the data, ANT is damaged in two phases as far as ADP/ATP exchange is concerned, with initial damage due to ROS and further additional damage owing to caspase. Conversely, even if damaged by ROS, ANT can still translocate adenine nucleotides, but cannot participate in mPTP formation. Later, as a result of caspase action, ANT begins to participate in mPTP opening.
Furthermore, the application of a mathematical model showed that loss of ANT transport function and mPTP opening are inversely correlated in late but not in early apoptosis. Namely, the loss of ANT transport function and onset of mPTP are independent during early apoptosis, but the situation changes over the period of 3–5 h, when they proceed in parallel. That is, in the 5–8-h phase of apoptosis, the progressive decrease in the ANT-dependent transport function occurs simultaneously with mPTP opening.
That a metabolic shift from oxidative phosphorylation to glycolysis (i.e., the Warburg effect) occurs in AD accompanied by an increase of both activity and level of HK-I was discovered some years later [146]. These findings proved that in the early phase of apoptosis VDAC1 activity progressively decreases in concomitance with its physical interaction with HK-I. Instead, in the late phase of apoptosis, compelling evidence suggests that glucose-6-phosphate disrupts the physical interaction between the two proteins, then determining re-opening of the channel and the recovery of VDAC1 function, resulting in a reawakening of the mitochondrial function—which, as reported in [146], was apparently numb, thus inevitably leading to cell death [146].
This study was particularly laborious because for the first time ANT1 and VDAC1 activities were estimated in the same experiment by following the ADP/ATP exchange across mitochondrial membranes measured as in [145], in the absence or in the presence of either ATR or 4,40-diisothiocyanostilbene-2,20-disulfonic acid (DIDS), i.e., cell permeable blockers of ANT and VDAC1 [147,158,159], respectively.
As usual, in a typical experiment, cell homogenate was treated with Ap5A, thus preventing mitochondrial ATP synthesis in a manner not dependent on oxidative phosphorylation, and then incubated in the presence of the ATP detecting system. As a result of ADP addition, the appearance of ATP in the extramitochondrial phase was observed as an increase in absorbance due to the formation of NADPH due to (i) ADP uptake into mitochondria in exchange for endogenous ATP, (ii) ATP synthesis from imported ADP via ATP synthase, and (iii) efflux of the newly synthesized ATP from the mitochondria in exchange for further ADP. Up to this point everything went according to the common experimental fees already adopted to measure ADP/ATP exchange.
The decrease in NADPH formation occurring in the presence of DIDS or ATR showed that (i) the exchange of ADPext with ATPint exclusively occurs through the outer and inner mitochondrial membranes, respectively; (ii) both the membranes are intact; and (iii) the exchange is mediated by protein/s. Therefore, as already done in the previous work in order to establish the limiting step of the global process, applying the control-strength criterion and using DIDS at different concentrations, we observed that the rate of absorbance increase, i.e., ADP/ATP exchange, in the absence of an inhibitor was lower than the value corresponding to the straight line that intercepts the Y-axis at zero inhibitor concentration—obtained by interpolating the experimental points of absorbance increase rate in the presence of DIDS—thus showing that (i) the inhibited step of measured processes, i.e., the ADP/ATP exchange across MOM via VDAC1, is not the limiting step and (ii) the reciprocal value of the intercepts to the ordinate axis is a measure of the inhibited process, i.e., VDAC1 activity. In contrast, the intercept of the Y-axis in the case of the ADP/ATP exchange, inhibited by ATR, coincides with the rate value in the absence of an inhibitor, according to [145].
That ANT is a target of ROS in apoptosis was further confirmed by the use of certain dietary flavonoids known to exert beneficial effects on the central nervous system and to affect neuronal apoptosis [160,161]. The flavonoids genistein and daidzein, which are present in soy, but not catechin or epicatechin, which are present in cocoa, all used in a dietary blood concentration range, were found to prevent apoptosis in a manner consistent with their antioxidant activity [148], as depicted in Figure 3. A detailed investigation of the effect of these compounds on certain mitochondrial events that occur in cells en route to apoptosis showed that genistein and daidzein prevented the impairment of glucose oxidation and mitochondrial coupling, reduced cytochrome c release, and prevented both impairment of ANT and opening of the mitochondrial permeability transition pore, strongly suggesting that the prevention of apoptosis depends mainly on the antioxidant properties.
Furthermore, and for the first time, we demonstrated that extracellular ADP prevents the impairment of mitochondrial ANT-1 [147]. ADP, released into the extracellular space in the brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine.
ADP, released into the extracellular space in the brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine, which is subsequently taken up via the ADO transporter [147]. Consistent with this hypothesis, the internalization process is sensitive to the antagonist of purine receptor and the inhibitors of ectonucleotidase and nucleoside transporter [147], as depicted in Figure 3.
Our findings demonstrate that extracellular ADP inhibits the proapoptotic stimulus supposedly via (i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s, a conclusion validated by the increase in cell AOX system; and (ii) safeguarding of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), an effect mediated by its plausible internalization into cells occurring as such or after its hydrolysis by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell.