Cytochrome c oxidase is decreased in Alzheimer’s disease platelets

Neurobiology of Aging 25 (2004) 105–110

Cytochrome c oxidase is decreased in Alzheimer’s disease platelets Sandra Morais Cardoso a , M. Teresa Proença a,b , Sancha Santos a , Isabel Santana b , Catarina R. Oliveira a,b,c,∗ a

Faculty of Medicine, Center for Neuroscience of Coimbra, University of Coimbra, 3004 517 Coimbra, Portugal b University Hospital, University of Coimbra, 3000 Coimbra, Portugal c Faculty of Medicine of Coimbra, University of Coimbra, 3000 Coimbra, Portugal Received 13 August 2002; received in revised form 27 January 2003; accepted 3 February 2003

Abstract Cytochrome c oxidase (COX) activity reportedly is reduced in Alzheimer’s disease (AD) brain and platelets. The reasons for the defect in either tissue are unknown, but its presence in a non-degenerating tissue suggests it is not simply a consequence of neurodegeneration. We now offer confirmation of the AD platelet COX defect. Compared to age-matched controls, in mitochondria isolated from AD platelets there was a 15% decrease in COX activity despite the fact that COX subunits were present at normal levels. Platelet ATP levels were diminished in AD (from 11.33 ± 0.52 to 9.11 ± 0.72 nmol/mg), while reactive oxygen species (ROS) were increased (from 97.03 ± 25.9 to 338.3 ± 100 K/mg). Platelet membrane fluidity, Vitamin E, and cholesterol content were similar between groups. We conclude that COX catalytic activity is indeed diminished in AD platelet mitochondria, does not result from altered membrane fluidity, and is associated with ROS overproduction and ATP underproduction. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Platelets; Alzheimer’s disease; Cytochrome oxidase

1. Introduction Alzheimer’s disease (AD), the most common neurodegenerative disease of late life [13], is associated with deficient cytochrome c oxidase (COX) activity [3,16,22]. Whether this represents primary or secondary pathology is unclear. Neurons are susceptible to oxidative stress because of their high rate of oxygen consumption, high polyunsaturated fatty acid content, high transition metal ion content, and relatively limited antioxidant defense systems [12]. As the electron transport system consumes 85–90% of cell oxygen, mitochondria are the single greatest source of neuronal reactive oxygen species (ROS) production [28]. Electron transport chain defects can further increase ROS, which often causes [16,20,21] structural and functional cell membrane alterations [36]. Oxidative damage to the mitochondrial inner membrane (MIM) could facilitate further ROS Abbreviations: Complex I, NADH–ubiquinone oxidoreductase; Complex II/III, succinate–cytochrome c oxidoreductase; Complex IV, cytochrome c oxidase; COX, cytochrome c oxidase; DCFH2 , 2 ,7 –dichlorodihydrofluorescin; DPH, diphenylhexatriene; ROS, reactive oxygen species; TMA-DPH, trimethylamino-diphenylhexatriene ∗ Corresponding author. Tel.: +351-239-820190; fax: +351-239-822776. E-mail address: [email protected] (C.R. Oliveira).

production [7], leading to a vicious cycle that culminates in cell death. In the present study, mitochondrial respiratory chain enzyme activities were determined in mitochondria isolated from AD patient platelets. COX activity (but not other electron transport chain activities) was diminished. This defect may be associated with the observed depressed cell ATP and increased ROS. COX dysfunction was not associated with membrane fluidity, Vitamin E, or cholesterol changes.

2. Methods Participation of AD and control subjects was approved by the Institutional Review Board of the University Hospital of Coimbra. AD subjects were recruited from the Neurology Service at the University Hospital of Coimbra and met NINCDS-ADRDA criteria for probable AD. They did not manifest signs or symptoms of an alternative neurodegenerative disorder. Control subjects were free of any neurodegenerative disease. The mean age of the AD group (n = 20) was 68.95 ± 2.06 years, and for the control group (n = 5) it was 60.67 ± 3.3 years. Ten AD subjects were under 60 years of age and were considered part of

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an early-onset group. Ten AD subjects were above 60 and comprised a late-onset group. 2.1. Platelet and mitochondria preparation Following informed consent, 60 ml of blood was collected by venipuncture in tubes containing acid-citrate-dextrose as an anticoagulant. Mitochondria were obtained from human platelets according to previously described methods [18]. Platelet and mitochondria protein concentrations were measured by the Sedmak method [26], in which bovine serum albumin was used as the standard. 2.2. Citrate synthase assay Citrate synthase activity was determined by the method of Coore et al., which spectrophotometrically follows the formation of 5-thio-2-nitrobenzoate (412 nm) [9]. The assay was initiated by the addition of 100 ␮M oxaloacetate at 30 ◦ C. Results are expressed as nanomoles per minute per milligram of protein. 2.3. Mitochondrial respiratory chain complexes 2.3.1. NADH–ubiquinone oxidoreductase assay Mitochondria complex I activity was determined by the method of Ragan et al., which follows the decrease in NADH absorbance (340 nm) that occurs when ubiquinone (CoQ1 ) is reduced to form ubiquinol [23]. The reaction was initiated by adding CoQ1 (50 ␮M) to the reaction mixture, at 30 ◦ C. After 5 min, rotenone (10 ␮M) was added and the reaction was registered for a further 5 min. Complex I activities are expressed in nanomoles per minute per milligram of protein and represent the rotenone sensitive rates. 2.3.2. Succinate–cytochrome c oxidoreductase assay Complex II/III activity was measured by determining the formation of reduced cytochrome c at 550 nm [15]. The mitochondrial sample was pre-incubated at 30 ◦ C for 5 min with 20 mM succinate, 1 mM KCN to activate the enzyme, and then added to the reaction mixture. The enzymatic assay was performed at 30 ◦ C. Antimycin A (0.02 mM) was added to the reaction medium to inhibit complex II/III. Activity of complex II/III is expressed as nanomoles per minute per milligram of protein, and represents the antimycin A sensitive rate. 2.3.3. Cytochrome c oxidase assay Complex IV activity was determined using the method of Wharton and Tzagotoff, which measures the oxidation of reduced cytochrome c by cytochrome c oxidase at 550 nm [37]. To prepare reduced cytochrome c, cytochrome c was mixed with a few crystals of ascorbate, placed into a dialysis membrane for 18–24 h against a 0.01 M phosphate buffer, pH 7.0, at 4 ◦ C. Reduced cytochrome c concentration was

then determined with 0.1 M ferricyanide. The pseudo first order rate constant K was calculated, because the reaction is of first order with respect to cytochrome c. Results are expressed as K per minute per milligram of protein. 2.4. Analysis of adenine nucleotides Platelet adenine nucleotides were extracted on ice with 1.2 M perchloric acid, as described by Stocchi et al. [30]. Adenine nucleotides (ATP, ADP and AMP) were assayed by separation using reverse-phase HPLC. The chromatographic apparatus was a Beckman System Gold, consisting of a 126 Binary Pump Model and 166 Variable UV detector, controlled by computer. The column used was a Lichrospher 100 RP-18 (5 ␮M) from Merk (Germany). An isocratic elution with 100 mM KH2 PO4 buffer at pH 7.4 and 1% methanol was performed at a flow rate of 1.2 ml/ml. The adenine nucleotides (ATP, ADP and AMP) were detected at 254 nm for 6 min. 2.5. Membrane fluidity analysis Platelet mitochondrial membrane fluidity was evaluated with the fluorescent probes diphenylhexatriene (DPH) and trimethylamino-diphenylhexatriene (TMA-DPH), according to the method described by Shinitzky and Barenholz [29]. The DPH and TMA-DPH fluorescence anisotropy was determined by the formula: r = I − I⊥ /I + 2I⊥ , where I and I⊥ are the intensities of the emitted light whose plane of polarization is oriented, respectively parallel and perpendicular to the plane of polarization of the excitation beam. An increase in fluorescence anisotropy (r) values reflects a decrease in the probe mobility and an increase in the membrane structural order and/or a decrease in membrane “fluidity” [34]. 2.6. Vitamin E and cholesterol levels determination Vitamin E was analyzed by reversed-phase HPLC in platelet-derived lipid extracts according to the method of Vatassery et al. [35]. Vitamin E levels are expressed as micromloles per gram of protein. Cholesterol was assayed in lipid extracts obtained from platelets, using methanol and chloroform as organic solvents. We used a commercial kit for this (Boehringer, Mannheim). Cholesterol levels are expressed as micromloles per gram of protein. 2.7. Determination of protein levels Protein subunit patterns were analyzed using a bidimensional electrophoresis technique described by Gorg et al. [11]. For the bidimensional method an Immobiline DryStrip Kit in the first dimension and an SDS–PAGE gel in the second dimension was used. Mitochondria were isolated from

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AD platelets and 200 ␮g were solubilized in a lysis solution containing SDS 0.25%. After overnight rehydration of the Immobiline DryStrip, samples were loaded and an isoelectric focusing (IEF) was run for 7 h. After equilibration in the second dimension, electrophoresis was initiated using 0.025 M Tris (pH 8.3), 0.192 M glycine, and 0.1% SDS as the running buffer. 2.8. Free radical determinations Reactive oxygen species were measured following the oxidation of 2 ,7 -dichlorodihydrofluorescin (DCFH2 -DA) to fluorescent DCF, which detects the formation of intracellular peroxides [2]. After isolation, platelets were loaded in darkness with 5 ␮M DCFH2 -DA for 20 min in a Na+ -medium. After washing, DCF fluorescence was measured in the same medium using excitation and the emission wavelengths of 502 and 550 nm, respectively. 2.9. Data analysis Data are expressed as means ± S.E.M. of the indicated number of determinations. There were at least three independent experiments performed for each parameter. Statistical analyses were performed using Student’s t-test (two-way, P < 0.05). 3. Results 3.1. Mitochondrial respiratory chain complex activities and platelet ATP levels Table 1 shows respiratory chain enzyme activities for mitochondria isolated from AD and age-matched control platelets. Rates are reported as absolute values and as a ratio to citrate synthase. Expressing electron transport chain enzyme activities as a ratio to citrate synthase is intended to act as a safeguard for potential differences in mitochondrial enrichment. As shown in Fig. 1, citrate synthase activity was comparable between groups (257.14 ± 11.42 nmol/min/mg in controls, 250.02 ± 10.67 nmol/min/mg in early-onset AD and 255.28 ± 69.00 nmol/min/mg in late-onset AD). Differences in mitochondrial mass are therefore unlikely to account for the observed decrease in complex IV activity.

Fig. 1. Citrate synthase activity in platelet mitochondria. Data represent the mean ± S.E.M. of 5–10 independent experiments.

Table 2 ATP levels in human platelets mitochondria ATP levels (nmol/mg) 11.33 ± 0.52 9.11 ± 0.72∗

Control AD

The results are the means ± S.E.M. of duplicate determinations in 5–20 different experiments. ∗ P < 0.05, significantly different as compared to control platelet.

AD cytochrome c oxidase (complex IV) activity from both early and early-onset groups was decreased relative to that of controls (Table 1). There were no differences for either AD group in NADH–ubiquinone oxidoreductase (complex I) or in succinate dehydrogenase-cytochrome c reductase (complex II/III) electron transfer assay. Thus, electron transport chain dysfunction in AD specifically relates to complex IV and is indeed present in a non-degenerating tissue. ATP levels in AD platelet mitochondria were decreased by about 20% (11.33 ± 0.52 nmol/mg in controls and 9.11 ± 0.72 nmol/mg in AD) (Table 2), while the levels of ADP and AMP were not significantly affected (data not shown). 3.2. Membrane fluidity, Vitamin E and cholesterol levels in AD platelets Platelet membrane fluidity analysis is possible with the fluorescent probes TMA-DPH and DPH. TMA-DPH evaluates the fluidity of the inner mitochondrial membrane outer leaflet, and DPH evaluates the fluidity of the inner mitochondrial membrane inner leaflet. The results of this

Table 1 Activity of mitochondrial respiratory chain complexes in AD platelet mitochondria corrected for citrate synthase activity Control Complex Complex Complex Complex Complex Complex

I/CS II/III/CS IV/CS I (nmol/min/mg) II/III (nmol/min/mg) IV (K/min/mg)

0.0954 0.0953 1.1800 27.36 27.38 302.33

AD early-onset ± ± ± ± ± ±

0.0019 0.0121 0.0630 0.54 3.48 16.38

0.1049 0.1069 1.008 26.25 26.72 257.61

± ± ± ± ± ±

AD late-onset

0.0047 0.0097 0.0600∗ 1.17 2.43 18.43∗

The results are the means ± S.E.M. of duplicate determinations in 5–20 different experiments. ∗ P < 0.05 and compared to control platelet mitochondria.

0.1004 0.1175 0.9346 25.63 30.00 239.87 ∗∗ P

± ± ± ± ± ±

0.0023 0.0196 0.085∗∗ 0.58 5.00 20.10∗∗

< 0.01, significantly different as

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Fig. 4. Platelet reactive oxygen species production. Data are expressed as the increasing slope per milligram of protein (K/mg), and as the mean±S.E.M. for 5–10 independent experiments. ∗ P < 0.05, significantly different as compared to control platelets.

3.3. Protein subunit pattern in AD platelet mitochondria

Fig. 2. Platelet mitochondrial inner membrane fluidity. (A) DPH-TMA determined fluidity of the exterior inner membrane leaflet; (B) DPH determined fluidity of the interior inner membrane leaflet. Data represent the mean ± S.E.M. of 5–10 independent experiments.

AD and control platelet mitochondria were tested for protein subunit patterns using a bidimensional electrophoretic technique. Fig. 3A–C shows that protein subunit patterns were comparable between AD and control mitochondria. 3.4. ROS production in AD platelets

Table 3 Levels of Vitamin E and cholesterol in human platelets membranes

Control AD

Vitamin E (␮mol/g)

Cholesterol (nmol/g)

0.81 ± 0.08 1.56 ± 0.41

0.21 ± 0.04 0.16 ± 0.02

The results are the means ± S.E.M. of duplicate determinations in 10 different experiments.

analysis are shown in Fig. 2. Fluorescence for both probes was comparable between all groups and does not suggest mitochondrial inner membrane fluidity is different between AD and control platelet mitochondria. Vitamin E levels in AD platelets were similar to those of control platelets (Table 3) (P > 0.05). Cholesterol levels between AD and control platelets were equivalent (Table 3).

DCF fluorescence was increased in AD platelets as compared to controls (338.3 ± 100 and 97.0 ± 26 K/mg, respectively) (Fig. 4). This indicates that ROS production was increased in AD platelets.

4. Discussion We found that COX activity is reduced in both early and early-onset sporadic AD subjects. This represents a focal electron transport chain deficit and is present in a non-degenerating tissue. This defect is not due to a change in membrane lipid fluidity or to a protein subunit loss. Although the magnitude of the complex IV defect of AD platelets is small when measured in crude mitochondrial fractions, and that an increase in ROS production could lead

Fig. 3. Protein subunit pattern in mitochondria. Control mitochondria (A); early-onset AD mitochondria (B); late-onset AD mitochondria (C). The results were obtained from three independent experiments.

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to a deficit in COX activity and induce a decrease in ATP levels due to oxidative damage to other mitochondrial component, we argue that this COX defect is associated with increased ROS generation and decreased ATP. A previous study of AD platelet bioenergetics using crude homogenates failed to find a complex IV defect [33] while one study that assessed complex IV activity in purified mitochondrial fractions found an ∼50% defect [20]. The purity of the assayed mitochondrial fraction therefore appears relevant to determining the magnitude of the defect, and appears to account for discrepancies between laboratories. It is not known whether the complex IV defect of AD is etiologically important or simply a secondary or epiphenomenal consequence of neurodegeneration. Our confirmation of the findings of Parker et al. [20,21] that complex IV activity is reduced in platelets, a non-degenerating tissue, is perhaps relevant to this issue. Further, our data suggest that modest reductions of COX activity can lead to ROS production, which could also potentially serve as a toxic mediator. Also data obtained by Bennett et al. [4] indicate that a decrease in the activity of cytochrome oxidase induced with azide impaired both spatial and non-spatial learning in rats, being the behavioral deficits not secondary to a sensory or motor impairment. Another possible consequence of the AD platelet COX defect is diminished ATP. If this is also true in AD brain, it would perhaps represent another COX-related pathologic event that is relevant to AD neurodegeneration or to AD protein aggregation pathology. Regarding the latter point, Roder and Ingram [25] found in bovine brain that low ATP levels may contribute to aberrant protein phosphorylation, such as the type that precedes neurofibrillary tangle formation. ROS can induce lipid peroxidation through the degradation of polyunsaturated fatty acyl side chains of membrane phospholipids. This alters membrane fluidity and composition [5,19,36]. We therefore studied platelet membrane fluidity using two different fluorescent probes, TMA-DPH (as a fluidity probe for the outer leaflet of the inner mitochondrial membrane) and DPH (as a fluidity probe for the inner leaflet of the inner mitochondrial membrane lipid bilayer) [29,34]. Despite an elevation of ROS production in AD platelets, we detected no differences in membrane fluidity, suggesting that down COX activity is not driven through peroxidation of the mitochondrial inner membrane. Cells use endogenous antioxidants to defend themselves against ROS. One natural antioxidant is Vitamin E. Vitamin E is localized to the hydrocarbon core of the phospholipid bilayer, where it protects polyunsaturated fatty acids and membrane protein thiol groups from oxidation [32]. To assess whether Vitamin E levels were potentially responsible for preserving mitochondrial membrane fluidity despite the observed increase in ROS, we measured Vitamin E levels and found a non-significant trend towards increased Vitamin E in AD platelets. Further studies, however, would be required before concluding that this represents a compensatory response to COX-derived oxidative stress.

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In addition to membrane lipid peroxidative damage, in vitro studies indicate membrane proteins themselves are potentially damaged by free radicals [10,24]. To evaluate whether decreased COX activity in AD platelets was explainable by a loss of protein subunit(s), a bidimensional electrophoretic study of mitochondrial electron transport chain enzymes was performed. Our results indicate no change in COX subunits occurred in AD platelet mitochondria. Our findings are compatible with those of Parker et al., who found that cytochrome aa3 levels in highly purified AD brain mitochondria were normal [22], and concluded since COX levels are preserved in AD brain and that AD brain COX is kinetically abnormal this dysfunction must arise from a catalytic defect rather than an underproduction of COX-related proteins. This is not without controversy, however, as Kish et al. [17] recently reported from an immunohistochemistry study that COX protein subunit levels were moderately reduced in AD brain. These two previous studies and our current study all used different techniques for evaluating this issue, and so it is difficult to directly compare results. Nevertheless, our finding that COX protein levels are normal in AD platelets argues that the AD COX deficit is catalytic in nature and does not arise as a consequence of tissue degeneration, or altered membrane fluidity. One type of radical-mediated damage we did not assess for was damage to mitochondrial DNA. Mitochondrial DNA encodes three COX protein subunits [1], and so mtDNA aberration could conceivably account for the AD COX defect we observed. Consistent with this possibility are data from Swerdlow et al., who found that cybrid cell lines expressing mitochondrial genes from AD subject platelets are complex IV deficient [31]. It was also proved that AD cybrids generated increased amounts of A␤ peptides (1–40 and 1–42) [14], have decreased mitochondrial membrane potential [8], increase ROS [6,31] and changes in calcium homeostasis [27]. Furthermore, our data support the conclusions of Swerdlow et al. [31], since we were unable to find an alternative explanation for the observed AD platelet complex IV dysfunction. In conclusion, cytochrome c oxidase activity is reduced in AD platelets. This defect seems not to be a consequence of tissue degeneration, ROS-induced membrane damage, deficient antioxidant defenses, or decreased enzyme synthesis rates. This apparent COX catalytic defect can be sufficient to reduce AD platelet ATP levels and increase ROS levels. According to previous reports [20,21,31] our data also suggest that AD platelets recapitulate bioenergetic-related pathology that is observed in AD brain. COX dysfunction appears to represent a primary or at least non-amyloid dependent phenomenon in AD.

Acknowledgments We are grateful to Dr. Russell H. Swerdlow for the critical review of the manuscript and to Prof. Moradas Ferreira and Dr. Victor Costa for practical advisement.

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