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Originally published In Press as doi:10.1074/jbc.M500657200 on February 14, 2005

J. Biol. Chem., Vol. 280, Issue 16, 15587-15594, April 22, 2005
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Dopamine-derived Dopaminochrome Promotes H2O2 Release at Mitochondrial Complex I

STIMULATION BY ROTENONE, CONTROL BY Ca2+, AND RELEVANCE TO PARKINSON DISEASE*

Franco Zoccarato, Paola Toscano, and Adolfo Alexandre{ddagger}

From the Dipartimento di Chimica Biologica and the Istituto di Neuroscienze, Sezione di Biomembrane (Consiglio Nazionale delle Ricerche), Università di Padova, Viale G. Colombo 3, 35121 Padova, Italy

Received for publication, January 19, 2005 , and in revised form, February 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1–0.2 µM)- and respiration-dependent generation of H2O2 at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5–30 nM). The rotenone effect required that contaminating Ca2+ (8–10 µM) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O2, generating and then H2O2. Mitochondrial removal of H2O2 monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca2+ levels. Thus, the H2O2 formed accumulated in the medium if contaminating Ca2+ was present; in the absence of Ca2+, H2O2 was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular release via activation of NADPH oxidase in the microglia. In turn, oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson disease (PD)1 is one of the major human neurodegenerative disorders and is clinically characterized by resting tremors, rigidity, slowness of voluntary movement, and postural instability. The neuropathological hallmark of PD is the progressive loss of the nigrostriatal dopamine (DA)-containing neurons, the cell bodies of which are in the substantia nigra pars compacta and nerve terminals in the striatum (1, 2). Lewy bodies are characteristic aggregates that form in affected cells, and increasing evidence suggests that {alpha}-synuclein, ubiquitin, and iron constitute a major fraction of Lewy body aggregates (3). Defects in mitochondrial Complex I activity and DA deficiency have been detected in patients suffering from the disease (4–6). Mitochondrial Complex I inhibitors such as 1-methyl-4-phenylpyridinium (MPP+), a metabolic product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone have been shown to induce symptoms similar to those of PD in experimental animals and humans (7, 8). Several in vitro studies using MPP+ and rotenone have shown that these compounds can induce oxidative stress, apoptosis, and other biochemical changes similar to those observed in patients afflicted with idiopathic PD (9, 10). The conversion of MPTP to MPP+ is a complex process that is required for toxicity and that is catalyzed by monoamine oxidase (MAO) type B, localized in non-DA glial cells, followed by spontaneous oxidation to MPP+ (11, 12). The latter is taken up by DA neurons via the DA transporter and is concentrated by an active process in mitochondria (13). Rotenone needs no specific transport mechanism. It is the classical Complex I inhibitor that indiscriminately affects respiration in every cell type. However, its low specificity is accompanied by the striking specificity of the pathology following its slow infusion, which includes degeneration of the substantia nigra pars compacta neurons accompanied by the formation of inclusions similar to authentic Lewy bodies (14). It therefore appears that some link must exist between the presence of the neurotransmitter DA and mitochondrial Complex I inhibitors for the development of toxicity.

Oxidative stress appears to contribute to the neurodegeneration observed in PD (8, 15–17). Brains of PD patients have decreased levels of GSH and exhibit oxidative damage to DNA, lipids, and protein (18, 19). Reactive oxygen species responsible for damage are supposed to be produced during DA metabolism or oxidative phosphorylation (20). Normal O2-consuming metabolic processes (e.g. the mitochondrial electron transport chain and the oxidative deamination of DA by MAO) and nonenzymatic autoxidation of DA are believed to result in intraneuronal formation of and H2O2. (21). Like all catecholamines, DA is easily oxidizable. promptly accepts an electron from DA, which is transformed into the o-semiquinone radical; two such radicals disproportionate, generating the o-quinone dopaminoquinone (DAQ) plus DA. The unstable DAQ undergoes spontaneous 1,4-intramolecular cyclization and further oxidation, eventually forming the relatively stable dopaminochrome (DACHR) (see Scheme 1) (21–23). The similar reaction sequence of the catecholamine adrenaline with is extremely fast, and the production of adrenochrome has been used as a method for measuring (24). Also, DA forms DACHR upon interaction with peroxynitrite or other oxidants. Of note, DA oxidation to DAQ and then to DACHR can be performed by the peroxidase component of cyclooxygenase, with the electron acceptor being prostaglandin H2 or also H2O2 (25). When a thiol like GSH is present at the site of DAQ formation, a nucleophilic addition of the thiol at C-5 of DAQ takes place (see Scheme 1). This reaction prevails over the intramolecular cyclization (26). Neuromelanin, the dark pigment that accumulates with age in the substantia nigra, is the product of DA oxidation processes induced primarily by DAQ. Analysis of its composition indicates that it is formed in part from compounds originating from the interaction of DAQ with cellular GSH, leading to 5-S-thiol derivatives (mainly 5-S-cysteine, which is formed by degradation of the GSH adduct) (21). This is what is expected from the facile interaction of DAQ with GSH. However, ~50% of neuromelanin derives from cyclization products (i.e. from DACHR-like species) (27, 28). As noted, such compounds have to originate from a cellular location where GSH is not present at sufficiently high concentrations. This observation is a strong indication that DACHR is formed in vivo.



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  		SCHEME 1
 
The mechanism by which DA metabolites may be involved in reactive oxygen species generation is, however, not fully elucidated, and the molecular basis for facilitation of reactive oxygen species production in DA-containing neurons is not established. In this study, we report that the interaction between DACHR and respiring brain mitochondria generates H2O2. We show that DACHR dose-dependently increases the production of H2O2 constitutively observed at Complex I of the mitochondrial respiratory chain and that the presence of very low, marginally inhibitory concentrations of the Complex I inhibitor rotenone further increase peroxide production. We also report on Ca2+ control of H2O2 generation and removal. The relevance of these results in the pathogenesis of PD is discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) was from Molecular Probes, Inc. Horseradish peroxidase (HRP) (grade I; EC 1.11.1.7 [EC] ) was from Roche Applied Science. Mushroom tyrosinase (EC 1.14.18.1 [EC] ) and were pargyline from Sigma. All other reagents were of analytical grade.

Preparation of Rat Brain Mitochondria—Brain mitochondria were prepared as described (29). Specifically, the cerebral cortices of two 6–7-week-old rats were rapidly removed into 20 ml of ice-cold isolation medium (320 mM sucrose, 5 mM MOPS, 0.5 mM EDTA, and 0.05 mM EGTA, pH 7.3) and homogenized. The homogenate was centrifuged at 900 x g for 5 min at 4 °C. The supernatant was centrifuged at 8500 x g for 10 min, and the resulting pellet was resuspended in 1 ml of isolation medium. This was layered on a discontinuous gradient consisting of 4 ml of 6% Ficoll, 1.5 ml of 9% Ficoll, and 4 ml of 12% Ficoll (all prepared in isolation medium) and centrifuged at 75,000 x g for 30 min. The myelin, synaptosomal, and free mitochondrial fractions formed above the 6% layer, as a doublet within the 9% layer, and as a pellet, respectively. The pellet was resuspended in 250 mM sucrose and 10 mM K-MOPS, pH 7.2, and centrifuged at 8500 x g for 15 min before being resuspended in this last medium to 10–20 mg of protein/ml by the Gornall protein assay. The mitochondria were well coupled as judged by the increase in the oxygen consumption rate upon addition of ADP (respiratory control ratio), which was between 3.5 and 6 with glutamate and malate as substrates.

Oxygen Consumption—Oxygen consumption was monitored with a Clark-type oxygen electrode in a 1.6-ml closed chamber thermostatted at 30 °C with continuous stirring (30).

Standard Incubation Method—Mitochondria (0.4–0.7 mg/ml) were incubated at 30 °C in 125 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 500 µg/ml defatted bovine serum albumin, 20 mM MOPS, pH 7.2 (adjusted with KOH), and 40 µM pargyline (an MAO inhibitor). Further additions were as specified in the figure legends.

DACHR Generation—DACHR was generated in the test cuvette by incubating variable amounts of DA with tyrosinase (44 units/ml) for 5 min prior to the addition of mitochondria (31). The reaction proceeded without the liberation of reactive oxygen species, leading to the quantitative generation of DACHR as determined by the consumption of 2.0 nanoatoms of oxygen/nmol of DA and the appearance of typical absorbance at 470 nm (molar extinction coefficient of 3.24 x 103) (Scheme 1) (28, 32). It is shown that DA is oxidized by tyrosinase to DAQ, which readily undergoes irreversible spontaneous 1,4-intramolecular cyclization to give 5,6-dihydroxyindoline (leuco-DACHR), which in turn is oxidized to DACHR by one molecule of DAQ. Alternatively, the interaction of DA with tyrosinase was induced in the presence of 50 µM GSH. Under these conditions, the high reactivity of DAQ with the thiol led to the quantitative formation of the GSH adduct at C-5 (5-S-glutathionyldopamine) as evidenced by the consumption of only 1 atom of oxygen/molecule of DA and the absence of an absorbance increase at 470 nm (Scheme 1). In our experiments, the formation of DACHR was conducted in the presence of bovine serum albumin, which decreased the yield of DACHR by 20–25%, probably due to interaction of the thiols in bovine serum albumin with DAQ. The concentration of DACHR under each experimental condition was determined by the absorbance at 470 nm.

Hydrogen Peroxide Measurements—5 µM Amplex Red and 15 µg/ml HRP (3.75 units) were included in the incubations. H2O2 was detected by the formation of the fluorescent Amplex Red oxidation product resorufin using excitation and emission wavelengths of 563 and 587 nm, respectively, on a Shimadzu RL-5000 spectrofluorometer in a stirred cuvette thermostatted at 30 °C (33). The H2O2 calibration scale is linear in the 0–6 µM range, and at the end of each assay, traces were calibrated by the addition of H2O2 (500 pmol).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
As shown in Fig. 1, H2O2 was generated by brain mitochondria respiring on the NAD-linked substrates glutamate and malate. The release rate was low and could be reliably detected only with the high sensitivity reagent Amplex Red. H2O2 production is to be ascribed to autoxidation of some redox component within Complex I (generating , which is then transformed by superoxide dismutase into H2O2) located on the substrate side of the rotenone inhibition site as evidenced by stimulation of peroxide release induced by fully inhibitory rotenone concentrations (data not shown) (34–36). DACHR (3.6 µM) induced a strong long lasting potentiation of H2O2 generation. The DACHR effect was almost completely eliminated in the absence of substrates (and with some malonate to minimize the oxidation of endogenous substrates). With substrates and DACHR, an additional large increase in H2O2 production was induced by a very low concentration of rotenone (15 nM), which only marginally inhibited respiration (see below) and was almost without effect on H2O2 release in the absence of DACHR. The DACHR potentiation of H2O2 release was visible at concentrations as low as 0.1–0.2 µM and increased essentially linearly with DACHR (Fig. 2A). Concentrations in excess of 10 µM were not tested as they were likely of no physiological significance. 15 nM rotenone was stimulatory at all DACHR concentrations tested, and the potentiation of H2O2 output was also linear. The effect of increasing rotenone concentrations (up to 30 nM) at a fixed DACHR concentration is shown in Fig. 2B. In this concentration range, rotenone only very slightly increased H2O2 production in the absence of DACHR. The rotenone potentiation of H2O2 release was visible at <5 nM. Fig. 2C shows the dose-response inhibition by rotenone of mitochondrial respiration. The H2O2 production rate at a fixed DACHR concentration as a function of substrate concentration and the effect of rotenone (15 nM) are shown in Fig. 2D. Rotenone stimulation was slightly more evident at the lower substrate concentrations.



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  		FIG. 1.
Potentiation of H2O2 generation induced by DACHR in respiring mitochondria: effect of low rotenone concentrations. DACHR was generated in each cuvette prior to the addition of mitochondria by a 5-min incubation in standard medium containing Amplex Red/HRP with tyrosinase and 5 µM DA (traces c–e). The concentration of DACHR formed was 3.6 µM. Additions included 0.5 mM glutamate, 0.5 mM malate, 0.5 mM malonate, and 15 nM rotenone. Mitochondria (0.65 mg/1.6 ml) were added at the arrow. Trace a, glutamate/malate; trace b, glutamate/malate and rotenone; trace c, malonate and DACHR; trace d, glutamate/malate and DACHR; trace e, glutamate/malate, rotenone, and DACHR. Traces are representative of duplicate traces from at least five independent experiments.

 



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  		FIG. 2.
Effect of increasing DACHR, rotenone, and substrate concentrations on H2O2 generation. The Ca2+ dependence of the rotenone-induced increase in H2O2 output was investigated. Experimental conditions were as described in the legend for Fig. 1. A, dependence of H2O2 output on the DACHR concentration. {circ}, glutamate/malate; •, glutamate/malate and 15 nM rotenone. B, dependence of H2O2 output on the rotenone concentration. {circ}, glutamate/malate; •, glutamate/malate and 3.6 µM DACHR; {triangleup}, glutamate/malate plus DACHR and 300 µM EGTA. C, dose-response inhibition of mitochondrial respiration by rotenone in the absence ({circ}) and presence ( •) of EGTA. 1.5 mM ADP was added. D, H2O2 production as a function of substrate concentration. {circ}, no addition; •, 15 nM rotenone; {triangleup}, 3.6 µM DACHR; {blacktriangleup}, DACHR and 15 nM rotenone. When necessary, the pH was adjusted to compensate for the H+ released upon metal binding by EGTA. In A, B, and D, data are means ± S.E. from at least five independent experiments. In C, data points from a single experiment are representative of at least four independent experiments.

 
In the experiments described above, the Ca2+ contamination of the incubation mixture (8–10 µM) was not removed. When such contaminating Ca2+ was removed by EGTA, the constitutive H2O2 production and the stimulated release induced by DACHR were essentially unchanged. However, rotenone stimulation was less evident (Fig. 2B). The decreased rotenone effect in the presence of EGTA was not due to a variation of the inhibitory potency of rotenone on respiration (Fig. 2C) and appeared to depend solely on the removal of free Ca2+, which does not mimic the heavy metal chelator TPEN (15–30 µM) or the iron chelator deferoxamine (15 µM) (data not shown). Adding small amounts of Ca2+ to the incubation medium (with EGTA omitted) did not increase the rotenone effect further (data not shown).

DACHR- and MAO-dependent Production of Peroxide and Its Removal by Mitochondria: Effect of Ca2+—The results presented above were obtained by monitoring H2O2 as soon as it formed with the high affinity trap Amplex Red/HRP. Such a situation does not exist in vivo, where mitochondrially produced H2O2 has access to cellular peroxidases, notably the mitochondrial glutathione peroxidase. Regeneration of GSH is via glutathione reductase, which utilizes NADPH produced by the energy-dependent transhydrogenase. We recently reported that this system is under the control of intramitochondrial Ca2+, which acts as an inhibitor of both glutathione reductase (maximal inhibition of 70%, EC50 = 0.9 µM Ca2+) and glutathione peroxidase (maximum inhibition of 40%, EC50 = 0.9 µM Ca2+) (29). Such Ca2+-mediated control of H2O2 removal was evident when we analyzed the ability of mitochondria to dispose of H2O2 generated by MAO (which is located at the outer mitochondrial membrane) using DA as the substrate (Table I). First, H2O2 production was measured for 5 min in the continuous presence of the Amplex Red/HRP trap. It was unaffected by the presence of respiratory substrates or EGTA. The accumulation of H2O2 in the medium, which represents the balance between H2O2 production and removal, was then measured using the H2O2 detection system after a 5-min incubation. The highest levels of accumulated H2O2 were observed in the absence of respiratory substrates. With substrates, residual H2O2 greatly decreased. However, substantial H2O2 was still detected in the suspension medium if EGTA was omitted, whereas no residual H2O2 was measurable with EGTA, in line with the notion that Ca2+ negatively controls H2O2 removal.


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  		TABLE I
MAO-dependent production of H2O2 and its Ca2+-controlled removal by glutathione reductase/glutathione peroxidase

Mitochondria (0.7 mg/1.6 ml) were incubated in standard medium (without pargyline) in the presence of Amplex Red/HRP and DA (75 µM). Alternatively, HRP was added at 5 min of incubation (30 s after Amplex Red addition) to monitor net H2O2 accumulation. Results were corrected for a small nonspecific deflection upon the addition of HRP. No fluorescent signal was detected upon the addition of Amplex Red. Other additions were 300 µM EGTA, 1 mM glutamate, 1 mM malate, and 0.5 mM malonate. Values are the means ± S.E. from at least four independent experiments.

 
To address the relationship between production and removal of H2O2 when the latter originates from DACHR redox cycling, we performed a series of experiments with DACHR in which we monitored the intrinsic rate of H2O2 production using the Amplex Red/HRP trap and compared it with the accumulated H2O2 in the suspension medium as determined by adding the Amplex Red/HRP detection system at a fixed time (5 min) of the reaction (Table II). As described above (Fig. 2), trap measurements with glutamate/malate showed a strong stimulation of peroxide production by DACHR, which was further increased by rotenone in the absence of EGTA. Residual H2O2 accumulation in the suspension medium was relatively high in the absence of EGTA without and especially with rotenone, suggesting that the peroxidase activity was not sufficient to dispose of all the H2O2 produced. In the presence of EGTA, accumulated H2O2 was greatly reduced. However, a small measurable accumulation of residual H2O2 was consistently detectable, suggesting that the H2O2 removal was not as efficient as with MAO-derived H2O2 (Table I). In the presence of ADP, which decreases the mitochondrial membrane potential ({Delta}{psi}) (State 3), the DACHR-induced authentic peroxide production (measured with the trap) was generally lower. This was particularly evident when EGTA was present: under these conditions, H2O2 production was the lowest, and no H2O2 accumulation was detected. Without EGTA, H2O2 production rates in State 3 were closer to those measured with no ADP (State 4) in both the absence and presence of rotenone. Under these conditions, H2O2 accumulation was evident as expected; it was lower than without ADP.


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  		TABLE II
DACHR-dependent production of H2O2 and its Ca2+ -controlled removal by glutathione reductase/glutathione peroxidase

Conditions were as described in the legend for Fig. 1. Glutamate and malate were each present at 1 mM, and succinate was used at 0.5 mM. When present, the concentration of ADP was 1.5 mM, that of DACHR was 3.6 µM, and that of rotenone was 15 nM. Alternatively, HRP was added at 5 min of incubation (30 s after Amplex Red addition) to monitor net H2O2 accumulation. Values are the means ± S.E. from seven independent experiments. ND, not determined.

 
Succinate is a powerful generator of in mitochondria under some very specific metabolic conditions. H2O2 production with succinate, which depends on reverse electron transfer from Complex II (the electron carrier from succinate to coenzyme Q) back to Complex I, requires the {Delta}{psi} to be high and is extremely sensitive to even small decreases in the potential (it is completely removed in State 3) (29). Rotenone at a high concentration acts as an inhibitor, an indication that the autoxidizable carrier is located uphill of the rotenone inhibition site (29, 35, 36). Furthermore, H2O2 release is inhibited by modestly increasing intramitochondrial Ca2+ (i.e. in the presence of contaminating Ca2+) and is partially inhibited by the contemporary oxidation of NAD-linked substrates (29). In vivo, succinate is never oxidized alone by the mitochondria, and its concentration is dictated by the concentration of other tricarboxylic acid cycle substrates, which are NAD-dependent. Some of the experiments in Table II were performed with succinate together with glutamate and malate to try to reproduce a possible in vivo situation. In the absence of DACHR, the constitutive H2O2 release in State 4 with the three substrates was quite high when contaminating Ca2+ was removed by EGTA (this allowed a large contribution from succinate to the overall H2O2 output). Without EGTA, constitutive H2O2 generation was greatly decreased because contaminating Ca2+ depresses the succinate component of the H2O2 release (29). Despite the high production rate, no H2O2 accumulation in the medium was visible with EGTA because the glutathione reductase/glutathione peroxidase system was fully functional under these conditions. In the presence of ADP (State 3), the succinate-dependent contribution to the overall constitutive H2O2 production both without and with EGTA was eliminated because of the decreased membrane potential. When DACHR was included, H2O2 output was invariably increased. The highest values were obtained in State 4 and with EGTA (i.e. under conditions in which the succinate-dependent component of peroxide release was maximal); without EGTA, the release was greatly reduced. The accumulation of H2O2 was relatively high, as expected, without EGTA (i.e. when the glutathione reductase/glutathione peroxidase system was inhibited). However, relatively large amounts of H2O2 accumulated in the suspension medium also when contaminating Ca2+ was removed by EGTA. This is clearly at variance with the results of Table I, where a similar rate of H2O2 generation by MAO activity was accompanied by no H2O2 accumulation when EGTA was present. Thus, the peroxide-removing activity appears to be compromised when a high H2O2 production rate originates from the interaction of DACHR with the mitochondria. In all cases, 15 nM rotenone resulted in the potentiation of H2O2 production and accumulation, most evident in the absence of EGTA. The presence of ADP (State 3) depressed H2O2 output, particularly with EGTA. A low level of rotenone promoted the increased production as well as the increased accumulation of H2O2 when EGTA was omitted.

In general, these results indicate that DACHR extracts electrons from Complex I, apparently from the same site that constitutively generates and with a similar control system. A main difference is that, with DACHR, contaminating Ca2+ induces a substantial potentiation of the H2O2 release in the presence of low rotenone levels. The peroxide released following the interaction of DACHR with Complex I is removed by the mitochondrial glutathione reductase/glutathione peroxidase, with its normal control by Ca2+. However, with DACHR, the maximal peroxide-scavenging activity, as observed in the absence of contaminating Ca2+ (i.e. with EGTA), appears to be somewhat compromised.

Exogenous superoxide dismutase was reported to increase the mitochondrial production of H2O2 when it derives from generated on the cytosolic face of the inner membrane (36), but not when it derives from internally produced . The former situation applies to non-physiological antimycin-stimulated H2O2 production (37), which originates from generated on the cytosolic face of Complex III, the latter to Complex I-derived H2O2, which is from intramitochondrially formed (29). The DACHR-stimulated H2O2 release was not increased by exogenous superoxide dismutase (data not shown), additional proof that the site of electron leakage to DACHR is internal and likely the same that undergoes constitutive autoxidation.

The GSH Adduct of DAQ Does Not Promote H2O2 Generation, but DACHR-dependent Peroxide Production Is Not Prevented by GSH—When the tyrosinase-catalyzed oxidation of DA was performed in the presence of GSH, no DACHR formation took place, and 5-S-glutathionyldopamine was formed instead (see "Experimental Procedures" and Scheme 1). This is explained by the high reactivity of tyrosinase-produced DAQ with thiols, taking precedence over cyclization of DAQ to leuco-DACHR. When tested during respiration-dependent H2O2 production, the GSH adduct exhibited no activity both without and with rotenone (Fig. 3). However, if DACHR was first produced (i.e. in the absence of GSH), and GSH was supplied later together with mitochondria, the respiration-promoted H2O2 release and stimulation by rotenone were unmodified. Accordingly, the DACHR absorbance at 470 nm was not altered by GSH (data not shown). Thus, once formed, DACHR is relatively stable and is capable of performing redox cycling also in the presence of GSH.



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  		FIG. 3.
The GSH adduct 5-S-glutathionyl-DA does not promote mitochondrial H2O2 release, but DACHR-dependent peroxide production is not prevented by GSH. Experimental conditions were as described in the legend for Fig. 1. Where indicated, 5-S-glutathionyl-DA (5-S-G-DA) was formed by including 50 µM GSH during preincubation with tyrosinase and 5 µM DA (see "Experimental Procedures"). Alternatively, GSH was supplied after preincubation together with mitochondria. Additions included 1 mM glutamate, 1 mM malate, and 15 nM rotenone. Where indicated, DACHR was present at 3.6 µM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Reactive oxygen species originating from DA oxidation products have long been suspected to participate in the pathogenesis of PD. DA oxidation to DAQ depends on its interaction with , peroxynitrite, and other reactive species such as ·OH originating via Fenton chemistry from H2O2 and metals such as iron, which is elevated in PD substantia nigra. DAQ is unstable and readily undergoes additional reactions with thiols such as GSH or cyclization to leuco-DACHR, followed by oxidation to DACHR. Furthermore, DA can be transformed into DAQ by cyclooxygenase, being used as an electron donor in the peroxidase component of the cyclooxygenase reaction, where prostaglandin G2 (but also H2O2) is the electron acceptor (25). Although cyclooxygenase-1 is constitutively present in the microglia, cyclooxygenase-2 has recently been shown to be present in DAergic neurons of the substantia nigra of PD patients (and to be induced upon MPTP treatment), and this may represent a strong contribution to DA oxidation (38).

The involvement of Complex I of the respiratory chain in the pathogenesis of PD has long been suspected because Complex I inhibitors such as MPTP-derived MPP+ and, more recently, rotenone have been shown to promote PD-like symptoms (7, 8) and because the activity of Complex I has been reported to be decreased in affected individuals (4–6). Particularly impressive is the finding that a slow infusion of low concentrations of rotenone, a very specific, highly hydrophobic inhibitor, induces typical PD-like lesions, including the formation of Lewy bodies (14).

In the absence of respiratory chain inhibitors, Complex I is the main site where and H2O2 are generated (29, 36). H2O2 is formed from both NAD-linked substrates and succinate (in this case, via reverse electron flow to Complex I). In the first case, the production is very slow and is increased by fully inhibitory concentrations of the Complex I inhibitor rotenone, which acts distal to the autoxidizable component. Succinate-dependent H2O2 production is much faster. It is inhibited by rotenone (which interrupts the backwards electron transfer), by decreasing {Delta}{psi} (such as during ADP-stimulated respiration), and by small amounts of Ca2+ (such as present as a contaminant in saline medium) (29).

We have shown in this study that H2O2 is formed following the interaction of DACHR with respiring mitochondria, apparently exclusively at the level of Complex I, with properties that are complex and very similar to those of constitutive H2O2 generation, but at higher rates. Stimulation of H2O2 release above basal levels was detected at DACHR concentrations as low as 0.1–0.2 µM, and the rate increased strongly and essentially linearly with increasing DACHR concentrations. In essence, it appears that the site within Complex I responsible for the monoelectronic reduction of O2 to is also capable of transferring an electron to the o-quinone DACHR, with the likely generation of a highly reactive semiquinone radical, which in turn transfers an electron to O2, forming and regenerating DACHR, which operates redox cycling. Electron transfer from Complex I to DACHR takes precedence over transfer to O2, given that its rate is much faster at much lower concentrations. A similar mode of interaction has previously been proposed for adrenochrome interaction with Complex I (39). Furthermore, a low concentration of rotenone, which marginally inhibits respiration and induces a negligible increase in the constitutive H2O2 output, induced a strong potentiation of peroxide release by DACHR. The rotenone-induced extra electron leakage to DACHR was largely prevented by removing contaminating Ca2+.

When some succinate was present together with glutamate/malate, which may mimic a more physiological situation, H2O2 release was generally higher than with glutamate/malate alone also when DACHR was omitted. In particular, the succinate potentiation of constitutive H2O2 release was highest with EGTA because the succinate component of the overall H2O2 production was Ca2+-inhibited. In this situation of higher constitutive H2O2 production, the inclusion of DACHR promoted an additional large increase, as may be expected if the sites of O2 and DACHR reduction are the same (and if the reaction with DACHR takes precedence over that with O2). The succinate-dependent increased constitutive H2O2 production was largely eliminated when {Delta}{psi} was decreased by ADP. Accordingly, also the high rate of H2O2 output induced by DACHR with the three substrates decreased to values similar to the no-succinate situation in the presence of ADP. In summary, the high peroxide output obtained with succinate plus glutamate/malate in the presence of EGTA with (and, to a lesser extent, also without) DACHR was readily eliminated as soon as {Delta}{psi} was decreased. It is likely that, in vivo, {Delta}{psi} is not at its maximum and that these high levels of H2O2 released are not normally observed. When EGTA was omitted, the succinate-dependent component of H2O2 release was largely eliminated, and peroxide production was closer to that observed without succinate both without and with DACHR. The site of production with DACHR is located on the inner face of the inner mitochondrial membrane.

An important point that has been addressed in this study is how the mitochondrial peroxidase(s) deals with mitochondrially produced H2O2. Allowing H2O2 formed at a known rate to accumulate in the suspension medium for some minutes before introducing the detection system provided information about how H2O2 is handled. We found recently that the glutathione reductase/glutathione peroxidase system, responsible for much of the peroxide removal in mitochondria, is inhibited by low intramitochondrial Ca2+ levels (29). At 75 µM DA, a relatively high concentration that may, however, be of physiological significance, MAO activity monitored by immediately measuring H2O2 release was unaffected by mitochondrial respiration or by EGTA. In contrast, H2O2 that accumulated in the medium was highest without respiration; it was greatly decreased with substrates and no EGTA, but was totally absent if EGTA was also present. Also the DACHR-dependent accumulation of H2O2 was under the control of intramitochondrial Ca2+ and was at its lowest when contaminating Ca2+ was removed by EGTA. However, there are indications that the glutathione reductase/glutathione peroxidase system is partially inhibited in the presence of DACHR. In fact, when the DACHR-induced peroxide production was high, i.e. in State 4 with succinate and EGTA (a rate that was very similar to the MAO activity at 75 µM DA), accumulated peroxide was still relatively high, whereas it was undetectable when it originated from MAO (compare Tables I and II). Some H2O2 accumulated under similar conditions even in the absence of succinate. A possible explanation for the decreased H2O2-removing activity with DACHR-derived peroxide is that, with DACHR, the primary product formed is the intramitochondrial DACHR semiquinone, followed by electron transfer to O2, forming . It is likely that some intramitochondrial DACHR semiquinone accumulates, escaping reaction with O2, and that such a species or its derivatives interact with mitochondrial proteins, altering their activity. Decreased glutathione reductase/glutathione peroxidase activity in the presence of intramitochondrial DACHR semiquinone would likely secondarily affect also the removal of MAO-derived H2O2. It is important to emphasize that the duration of the experiments in this study was rather short. It follows that this aspect may likely become of great importance in longer time frames.

The control of both MAO- and DACHR-induced H2O2 production by intramitochondrial Ca2+ (an increase in which prevents efficient peroxide removal, leading to a higher H2O2 steady state) may be an important new aspect of cell physiology. The Ca2+ increase required for the inhibition of H2O2 removal is small (semimaximal effect at 0.9 µM (29)) and readily achieved in vivo. It is the same order of increase required for the activation of pyruvate, isocitrate, and {alpha}-oxoglutarate dehydrogenases (40). Increased intracellular Ca2+ levels in PD have been repeatedly suggested. Recently, it was shown that the inhibition of Ca2+-activated proteases (calpains) attenuates MPTP toxicity, directly involving increased Ca2+ levels in the pathogenesis of PD (41). An increase in cytosolic Ca2+ levels is readily followed by increased mitochondrial Ca2+ levels (42).

Both in vitro and in vivo studies have established that the primary lesions in rotenone-induced toxicity are not in DAergic neurons, but in the microglia (43–45). Extremely low rotenone concentrations promote the extracellular release of NADPH oxidase-derived from the microglia (43). The importance of NADPH oxidase in the pathogenesis of PD has been well documented (44, 46). Such activation of glial derived may indeed be the primary event in rotenone toxicity (and likely also in MPP+ toxicity because MPP+ is produced from MPTP in the microglia and is actively taken up first by the mitochondria in these cells). Although it seems unlikely that significant DA levels are present in the microglia, it is conceivable that microglial released initiates DA oxidation by acting on extracellular DA in the synaptic cleft or its vicinity via monoelectronic DA oxidation to the DA semiquinone radical, followed by dismutation of the semiquinone to produce DAQ. The low concentration of GSH in the extracellular environment may favor the transformation into DACHR of the unstable DAQ, the interaction of which with thiols would otherwise be strongly favored. Extracellularly formed DACHR would be taken up by neuronal DA transporters to initiate the redox cycling described above, further amplified by the rotenone effect on neuronal mitochondria, in particular in situations in which mitochondrial Ca2+ levels are elevated. The hypothesis that DACHR formation is primarily an extracellular event is supported by the finding that ~50% of DA oxidation products in neuromelanin are derived from DAQ cyclization to leuco-DACHR (27, 28), which requires low GSH concentrations. Once formed and transferred inside the DAergic neurons, DACHR is resistant to the interaction with GSH and can promote long lasting redox cycling. Intracellularly formed DAQ, via cyclooxygenase-2 expression in PD DAergic neurons (38) or by other means, may also originate DACHR, probably at a later time given the decreased GSH in PD; it may be easily transformed into thiol derivatives, which themselves are toxic (47).

The elevation of neuronal intracellular Ca2+ levels could itself depend on the formation of extracellular -derived H2O2. In fact, low extracellular H2O2 levels were recently reported to facilitate Ca2+ influx by promoting the opening of voltage-gated Ca2+ channels (48–50). H2O2 also promotes iron uptake via the transferrin receptor (51). DACHR-dependent oxidative stress, potentiated by increased intracellular Ca2+ levels and further amplified by the increased iron availability, may secondarily promote the production and toxicity of {alpha}-synuclein by favoring its polymerization (52–54), cyclooxygenase-2 generation, etc. The suggested sequence of events may be important in the pathogenesis of PD, also independently of the toxicological situations represented by MPTP or rotenone intoxication. In fact, a glial inflammatory component involving NADPH oxidase activation in the generation of extracellular is likely an obligatory step in the induction of PD (44, 55). Data on the efficacy of anti-inflammatory agents in PD are accumulating (55, 56).

In summary, we have shown that the DA oxidation product DACHR promotes respiration-dependent H2O2 production in mitochondria. H2O2 was released at the same site in Complex I responsible for H2O2 release in the absence of DACHR. DACHR-induced H2O2 was visible at submicromolar concentrations and was strongly stimulated by extremely low rotenone concentrations. The rotenone effect required that contaminating Ca2+ (8–10 µM) was not removed from the incubation medium. H2O2 derived from DACHR or from MAO activity was subject to removal by mitochondrial glutathione reductase/glutathione peroxidase. In turn, the activity of these enzymes was under the inhibitory control of intramitochondrial Ca2+, which controlled peroxide accumulation in the suspension medium. In the presence of contaminating Ca2+, H2O2 (from MAO as well as DACHR) accumulated to a relatively large extent in the medium, whereas in the absence of Ca2+, MAO-derived H2O2 was completely removed. Also DACHR-derived peroxide was largely eliminated, but the removal was less efficient when the DACHR-dependent production rate was high. Based on the finding that very low rotenone concentrations promote the activation of NADPH oxidase in the microglia, we propose that, in rotenone-dependent parkinsonism, NADPH oxidase-derived superoxide triggers the extracellular oxidation of DA to DAQ and DACHR and that this species enters neurons to initiate the (rotenone-potentiated) peroxide production, supported by the increase in intramitochondrial Ca2+ levels (also possibly dependent on NADPH oxidase-derived H2O2 activation of voltage-dependent Ca2+ channels).


   FOOTNOTES
 
* This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca Progetti di Ricerca di rilevante Interesse Nazionale 2004 "Apoptosis and Mitochondria: New Targets in Neoplastic, Degenerative, and Immunologic Diseases." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 39-49-827-6146; Fax: 39-49-807-3310; E-mail: adolfo.alexandre{at}unipd.it.

1 The abbreviations used are: PD, Parkinson disease; DA, dopamine; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MAO, monoamine oxidase; DAQ, dopaminoquinone; DACHR, dopaminochrome; HRP, horseradish peroxidase; MOPS, 4-morpholinepropanesulfonic acid; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alberto Bindoli for helpful discussion.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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