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Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer’s disease
Neurobiology of Aging 23 (2002) 371–376
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Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer’s disease Francesca Bosettia,b, Francesca Brizzia, Silvia Barogia, Michelangelo Mancusoc, Gabriele Sicilianoc, Elisabetta A. Tendib, Luigi Murric, Stanley I. Rapoportb, Giancarlo Solainia,* a
Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Via G. Carducci 40, 56127 Pisa, Italy b Brain Physiology and Metabolism Section, National Institute on Aging, NIH; Bethesda, MD 20892, USA c Dipartimento di Neuroscienze, Clinica Neurologica, University of Pisa, Italy Received 8 May 2001; received in revised form 24 August 2001; accepted 24 August 2001
Abstract Evidence suggests that mitochondrial dysfunction is prominent in Alzheimer’s disease (AD). A failure of one or more of the mitochondrial electron transport chain enzymes or of F1F0-ATPase (ATP synthase) could compromise brain energy stores, generate damaging reactive oxygen species (ROS), and lead to neuronal death. In the present study, cytochrome c oxidase (COX) and F1F0-ATPase activities of isolated mitochondria from platelets and postmortem motor cortex and hippocampus from AD patients and age-matched control subjects were assayed. Compared with controls, COX activity was decreased significantly in platelets (-30%, P ⬍ 0.01, n ⫽ 20) and hippocampus (-35 to -40%, P ⬍ 0.05, n ⫽ 6), but not in motor cortex from the AD patients. In contrast, in AD platelets and brain tissues, F1F0-ATP hydrolysis activity was not significantly changed. Moreover, the ATP synthesis rate was similar in mitochondria of platelets from AD patients and controls. These results demonstrate that COX but not F1F0-ATPase is a mitochondrial target in AD, in both a brain association area and in platelets. A reduced COX activity may make the tissue vulnerable to excitotoxicity or reduced oxygen availability. Abbreviations: A: amyloid beta; AD: Alzheimer’s disease; COX: cytochrome oxidase; OS-ATPase: oligomycin-sensitive ATPase; ROS: reactive oxygen species. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer; Mitochondria; F1F0-ATPase; Cytochrome c oxidase; Platelets; Brain
1. Introduction Alzheimer’s disease (AD) affects 5–15% of the population over the age of 65 years [28]. Causes involve multiple genetic and environmental factors. Recently, attention has been directed to the possible contribution of mitochondrial dysfunction and oxidative damage in late-onset neurodegenerative disorders, including the familial and sporadic forms of AD [5,46,56]. The pathology of AD is characterized by progressive accumulation of intraneuronal fibrillary tangles with abnormally phosphorylated tau protein, and by amyloidogenic peptide (A) condensed within extracellular neuritic plaques, often surrounded by proliferating activated micro* Corresponding author. Tel.: ⫹39-050-883-320; fax: ⫹39-050-883215. E-mail address: [email protected] (G. Solaini).
glia and astrocytes [48]. Abnormal proteolytic processing of amyloid precursor protein (APP) has been shown to contribute to A deposition in the cerebrovasculature of AD patients [1,16,18]. Incubation with aggregated A also has been shown to decrease the redox activity and consequently the viability of cultured primary neurons, astrocytes, HeLa and PC12 cells, suggesting that A impairs mitochondrial function and initiates a neurotoxic cascade [26,27,47]. However, the basis of A toxicity is poorly understood, but likely involves free radical damage [6]. It has been suggested that sporadic AD involves mutations in the mitochondrial genome of genes encoding complex IV (COX, cytochrome c oxidase) of the electron transport chain [39,52]. How this would translate into loss of specific neuronal populations, including cholinergic neurons in the forebrain, hippocampus, and neocortex is unclear. One possibility (oxidative stress hypothesis) is that as yet unknown factors cause an imbalance that favors the
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generation of reactive oxygen species (ROS), leading to oxidative damage of cell lipids, proteins and DNA [32]. Neuronal factors that may increase brain sensitivity to oxidative stress include a high oxygen consumption rate, an abundant polyunsaturated fatty acid content and a short half-life of mitochondrial DNA. Reduced activity of one or more of the mitochondrial electron transport chain enzymes, or of F1F0-ATPase (ATP synthase, complex V) could compromise brain ATP synthesis and induce damaging ROS production, and if severe could lead to neuronal death [7]. Deficient COX activity has been reported by several authors in different brain regions [29,33,37,50,59], as well as in platelets [39] and fibroblasts [17]. Moreover, COX activity and mRNA levels of COX subunits I and III have been shown to be decreased in association cortex from AD patients [15]. It is unknown whether reduced COX subunit mRNA is part of a pathological cascade or reflects physiological downregulation due to reduced energy need. The toxic A fragment 25–35 has been reported to selectively decrease COX activity in rat brain mitochondria, without affecting other components of the respiratory chain [12]. However, almost a normal amount of COX was found in mitochondria of the postmortem AD hippocampus [45]. Involvement of F1F0-ATPase in AD is even more controversial. Hydrolysis of ATP has been reported to be slightly increased in both frontal and occipital areas in AD [37]. These data do not correspond to the marked decrease (50 – 60%) of the mRNA level of ATP synthase subunit  found in temporal cortex of AD patients [14], or to the reduced amount of ATP synthase observed in hippocampal extracts from patients [45]. To our knowledge, the ATP synthesis rate of F1F0-ATPase of mitochondria from platelets of AD patients has not been reported. In the present study, we investigated rates of ATP synthesis and hydrolysis catalyzed by F1F0-ATPase, as well as COX activity, in mitochondria from platelets of AD patients and age-matched control subjects. We also assayed ATPase and COX enzyme activities in mitochondria from two regions of autopsied brain from pathologically-confirmed AD patients and age-matched controls. We chose the hippocampus because of its high densities of senile plaques and neurofibrillary tangles [2], and the motor cortex, which usually is little affected by AD pathology.
2. Methods Patients with a clinical diagnosis of senile dementia of the Alzheimer’s type (n ⫽ 20, 9 men and 11 women, 65.2 ⫾ 8.5 (SD) years, range 53–77 years) and age-matched controls (n ⫽ 20, 10 men and 10 women, 63.4 ⫾ 9.1 (SD) years, range 50 – 81 years) without evidence of neuropsychiatric diseases were recruited. The diagnosis of AD was based on DSM IV [19] and NINCDS-ADRDA criteria [34]. All AD patients underwent a battery of neuropsychological
tests, including Mini Mental State Evaluation [22] (mean values 17.7 ⫾ 4.8, range 11–27), and Global Deterioration Scale (mean values 4.4 ⫾ 1.1, range 2– 6) [43]. Patients scored 4 or less on the Hachinski scale [23], indicating that they likely did not include subjects with vascular dementia. No participant in the study had a history of any other neurological, psychiatric or drug abuse disorder, or was taking antioxidant therapy or medication that might interfere with cognitive function. The protocol was approved by our Institution’s Committee on Human Experimentation of the University of Pisa. Informed consent was obtained from each subject or from a relative, after the purposes and procedures of the study had been explained. 2.1. Brain tissue samples Post-mortem samples from the hippocampus and primary motor cortex were obtained from 6 clinically diagnosed and pathologically confirmed AD patients as well as from 6 neurologically and cognitively normal controls matched for age, gender, and post-mortem interval. Brains were obtained from the Brain Physiology and Metabolism Section of the National Institute on Aging, National Institutes of Health, Bethesda, MD. Autopsies were performed within 24 h of death. The brain was frozen at – 80°C immediately following autopsy. A clinical diagnosis of AD was confirmed neuropathologically according to the multifocal distribution of senile plaques and neurofibrillary tangles [60]. The motor cortex and the hippocampus were dissected from these brains for examination. 2.2. Isolation of brain mitochondria All chemicals, of reagent grade, were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified and were used without further purification. The brain sections were put in an ice-cold homogenization buffer (0.32 M sucrose, 1 mM K⫹EDTA, and 10 mM Tris-Cl, pH 7.4), containing 2.5 mg/ml of fatty acid-free bovine serum albumin (BSA) and 0.3 mM phenylmethylsulfonyl fluoride (PMSF, a protease inhibitor). The sections were minced and homogenized with 2 ml of homogenization buffer per gram of tissue, using a Teflon-glass homogenizer. The homogenate was centrifuged at 500 ⫻ g at 4°C for 5 min. The pellet was washed with the homogenization buffer and centrifuged at 500 ⫻ g for 5 min (4°C). The supernatant was centrifuged at 10,000 ⫻ g at 4°C for 15 min. The mitochondrial pellet was washed once and then resuspended in 500 l of homogenization buffer. Samples were stored on ice until enzyme activity was measured. Protein concentration in the mitochondrial suspension was determined by the Bradford method using BSA Fraction V as standard [9].
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2.3. Isolation of platelet mitochondria Human platelets were isolated and purified from 40 ml of venous blood, as previously reported [3]. Mitochondria were resuspended at a concentration of 10 mg/ml in 0.25 M sucrose and 2 mM EDTA, pH 8.0. The protein concentration of the mitochondrial suspension was determined by the Bradford method [9]. 2.4. Enzymatic activities Enzyme activity was determined as nmol/min/mg. In mitochondria from both brain and platelets, COX activity was determined spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm [57] using an extinction coefficient ⌬⑀red-ox ⫽ 19.1 mM⫺1 cm⫺1. Citrate synthase activity, a marker of the total number of mitochondria, was determined as previously reported [49]. 5⬘-Nucleotidase activity, a marker of mitochondrial contaminants, was assayed spectrophotometrically as previously described [4]. In mitochondrial preparations isolated from brain, ATPase activity was determined at 30°C with an ATP-regenerating system by following the decrease of NADH absorption at 340 nm in a Perkin Elmer spectrophotometer, as previously reported [8]. The time-dependent ATPase activity was determined both in the presence and absence of 1 g/ml oligomycin, a specific inhibitor, in order to exclude other enzymatic and non-enzymatic oxidation of NADH and to measure the oligomycin sensitive-ATPase (OS-ATPase). At least three assays of enzyme activity were carried out for each mitochondrial preparation. Intrasample variability was less than 5%. 2.5. Chemiluminescent methods for monitoring ATP synthesis and hydrolysis In mitochondria isolated from platelets, ATP synthesis and hydrolysis rates were assayed as previously described [3]. ATP was determined by the luciferin/luciferase chemiluminescent method [51]. All the assays were carried out at least in triplicate. 2.6. Statistical analysis Mean values were compared by an unpaired t test analysis (two-tailed). P ⬍ 0.05 was considered statistically significant. The data were expressed as the mean ⫾ SEM. 3. Results 3.1. Hydrolysis of ATP and cytochrome c oxidase activity in AD and control brains No statistically significant difference was found between the AD and the control groups for gender, age, or postmor-
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Table 1 Cytochrome c oxidase and mitochondrial ATPase activities in mitochondria isolated from motor cortex and hippocampus from AD and control brains
COX OS-ATPase
AD CON AD CON
Motor cortex
Hippocampus
198.7 ⫾ 14.2 220.5 ⫾ 4.93 105.8 ⫾ 4.34 113.7 ⫾ 16.3
132.4 ⫾ 14.0*,† 208.1 ⫾ 22.1 116 ⫾ 15.5 111.4 ⫾ 21.6
Data are expressed as nmol/min/mg mitochondrial protein and are means ⫾ SEM of 6 different brains per group. * P ⬍ 0.05 vs AD motor cortex; † P ⬍ 0.05 vs control. CON ⫽ control; OS-ATPase ⫽ Oligomycin-sensitive ATPase.
tem interval with regard to motor cortex specimens. The mean activity of citrate synthase per gram of tissue, a mitochondrial matrix enzyme insensitive to abnormalities of the respiratory chain, did not differ between the AD and control groups (data not shown), indicating that the amount of mitochondria was not affected by AD pathology. Table 1 shows mean COX and OS-ATPase activities in mitochondria isolated from the motor cortex of AD patients and controls. A lack of statistical significance between the means is consistent with the minimal pathology usually observed in the AD motor cortex (unaffected region). In the AD hippocampus on the other hand, which normally demonstrates marked neuropathology, COX activity was decreased by 35– 40% compared to control (132.4 ⫾ 14.0 vs 208.1 ⫾ 22.1 nmol/min/mg, P ⬍ 0.05) and to activity in the AD motor cortex (198.7 ⫾ 14.2 nmol/min/mg, P ⬍ 0.05), confirming previous reports relative to the parietal cortex [37]. There was no significant difference in brain mitochondrial ATPase activity between AD and control groups. 3.2. Hydrolysis and synthesis of ATP, and cytochrome c oxidase activity in AD and control platelets Different methods used to prepare platelets from AD patients have produced discrepant data in different studies of respiratory chain enzyme activities [39,40,54]. Therefore, we took great care in preparing platelets and mitochondria. Our procedure [3] was rapid, gentle, and avoided exposing the platelets and mitochondria to sonic oscillations, which could impair their structure and functions of biologic systems. Moreover, our mitochondrial fractions contained only very small contaminants, as detected by measuring 5⬘-nucleotidase activity below the assay sensitivity (specific activity ⬍ 1 nmol/min/mg of protein). Citrate synthase activity ranged from 0.21– 0.25 mol/min/mg in mitochondrial preparations from AD and control subject. No significant difference in mean succinate-sustained ATP synthesis was found between AD and control platelet mitochondria (0.32 ⫾ 0.05 vs 0.30 ⫾ 0.05 nmol/min/mg protein). Mean ATP hydrolysis rates also did not differ significantly between the two groups (46.5 ⫾ 4 vs 48.5 ⫾ 6
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Fig. 1. Cytochrome c oxidase activity in platelet mitochondria from AD patients and controls. The reaction mixture consisted of 50 mM KCl, 10 mM Tris, 1 mM EDTA, pH 7.4, 40 M ferrocytochrome c and the reaction was started by addition of 10 g mitochondrial protein. Activity was assayed spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm. Results are mean ⫾ SEM (n ⫽ 20 in each group). **P ⬍ 0.01 vs controls.
nmol/min/mg). In contrast, COX activity was decreased by 30% (35 ⫾ 3.5 vs 48.5 ⫾ 2.5 nmol/min/mg, P ⬍ 0.01) in AD compared with control platelets (Fig. 1), as reported elsewhere [29,40]. No significant difference in the COX/ citrate synthase ratio was observed (data not shown) between the groups.
4. Discussion This study demonstrates decreased COX activity in the hippocampus and platelets from AD patients compared to control subjects, but no difference in the motor cortex. Moreover, no significant group difference in ATP hydrolysis rate was observed in mitochondria from hippocampus, motor cortex or platelets. These data are in agreement with a reported 50%– 65% decrease in mRNA levels of the mitochondrialencoded COX subunits I and III in the middle temporal association neocortex, but not in the primary motor cortex from AD brains, as compared to control brains [13]. It is unlikely that the absence of a COX defect in AD motor cortex was due to our use of crude mitochondrial fractions. The effect likely reflects lesser neuropathology in motor cortex compared to hippocampus [2]. Furthermore, in previous reports changes in enzyme activities could be detected even using the whole brain homogenate preparations [29,33,37], even less purified than the ones we used. We also normalized our activities to the activity of citrate synthase, as a marker of mitochondrial number. Therefore, the decreased activity observed was due to a real effect directed to COX rather than to a mere loss of mitochondria. However, use of highly purified mitochondrial preparations could enhance the sensitivity of the assay and increase the chance of finding catalytic defects that are not otherwise seen. Taken together, these results support the idea that oxidative
phosphorylation enzyme impairment in the AD brain may be reflected in the periphery, particularly in platelets [11]. Reduced COX activity has been reported in parietal and temporal brain regions and in platelets from AD patients [29,37,39,40]. Whether the effect is primary or a consequence of AD pathology is unclear. The observed decrease in COX activity is unlikely to be merely a consequence of neuronal degeneration in AD, in which case other oxidative phosphorylation enzymes would be affected; however, we did not find any alteration in mitochondrial ATPase activity. It rather might be a consequence of lipid abnormalities or oxidative stress [37,38]. Indeed, A has been shown to favor the accumulation of hydrogen peroxide and lipid peroxides in cells [6]. Lipid peroxidation, which has been shown increased in AD [20, 30,31,36,41,42], can alter electrical potential, order, and ion permeability of biologic membranes [24]. Moreover, A can directly interact with membrane lipids, perturbing their fluidity and composition [25]. These adverse actions of both ROS and A could impair COX assembly and activity, which are particularly sensitive to the fluidity and composition of membrane lipids [38,53]. A second target of oxidative damage could be mitochondrial DNA, whose localization in the mitochondrial matrix makes it particularly vulnerable to ROS [21,24], and the catalytic subunits of the F1F0-complex are encoded by nuclear DNA while the catalytic subunits I-III of COX are encoded by mitochondrial DNA. Our results show, for the first time, that rates of succinate-sustained ATP synthesis (oxidative phosphorylation rate) of platelet mitochondria from AD patients and agedmatched controls do not differ significantly, despite a reduced COX activity in the AD mitochondria. In recent reports, the control coefficient of COX over oxidative phosphorylation was shown to be very low in several tissues including brain [44]. The relative capacity of COX in cells was found to be in excess of that required to support endogenous respiration [55,58]. Therefore, a 30 –35% reduction in COX activity in AD platelets might not be sufficient to cause a significant decrease of the overall ATP synthesis rate under physiological conditions. However, in the presence of reduced oxygen concentration, COX might be ratelimiting in determining the rate of oxidative phosphorylation, as found in some experimental models [58]. Indeed, positron emission tomography has demonstrated reduced cerebral blood flow in AD during functional activation, which might indicate reduced oxygen availability in the cells during activation [10,35].
Acknowledgments This research was supported by a grant (co-fin 1999) from Italian MURST, Roma.
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Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer’s disease Francesca Bosettia,b, Francesca Brizzia, Silvia Barogia, Michelangelo Mancusoc, Gabriele Sicilianoc, Elisabetta A. Tendib, Luigi Murric, Stanley I. Rapoportb, Giancarlo Solainia,* a
Scuola Superiore di Studi Universitari e di Perfezionamento S. Anna, Via G. Carducci 40, 56127 Pisa, Italy b Brain Physiology and Metabolism Section, National Institute on Aging, NIH; Bethesda, MD 20892, USA c Dipartimento di Neuroscienze, Clinica Neurologica, University of Pisa, Italy Received 8 May 2001; received in revised form 24 August 2001; accepted 24 August 2001
Abstract Evidence suggests that mitochondrial dysfunction is prominent in Alzheimer’s disease (AD). A failure of one or more of the mitochondrial electron transport chain enzymes or of F1F0-ATPase (ATP synthase) could compromise brain energy stores, generate damaging reactive oxygen species (ROS), and lead to neuronal death. In the present study, cytochrome c oxidase (COX) and F1F0-ATPase activities of isolated mitochondria from platelets and postmortem motor cortex and hippocampus from AD patients and age-matched control subjects were assayed. Compared with controls, COX activity was decreased significantly in platelets (-30%, P ⬍ 0.01, n ⫽ 20) and hippocampus (-35 to -40%, P ⬍ 0.05, n ⫽ 6), but not in motor cortex from the AD patients. In contrast, in AD platelets and brain tissues, F1F0-ATP hydrolysis activity was not significantly changed. Moreover, the ATP synthesis rate was similar in mitochondria of platelets from AD patients and controls. These results demonstrate that COX but not F1F0-ATPase is a mitochondrial target in AD, in both a brain association area and in platelets. A reduced COX activity may make the tissue vulnerable to excitotoxicity or reduced oxygen availability. Abbreviations: A: amyloid beta; AD: Alzheimer’s disease; COX: cytochrome oxidase; OS-ATPase: oligomycin-sensitive ATPase; ROS: reactive oxygen species. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer; Mitochondria; F1F0-ATPase; Cytochrome c oxidase; Platelets; Brain
1. Introduction Alzheimer’s disease (AD) affects 5–15% of the population over the age of 65 years [28]. Causes involve multiple genetic and environmental factors. Recently, attention has been directed to the possible contribution of mitochondrial dysfunction and oxidative damage in late-onset neurodegenerative disorders, including the familial and sporadic forms of AD [5,46,56]. The pathology of AD is characterized by progressive accumulation of intraneuronal fibrillary tangles with abnormally phosphorylated tau protein, and by amyloidogenic peptide (A) condensed within extracellular neuritic plaques, often surrounded by proliferating activated micro* Corresponding author. Tel.: ⫹39-050-883-320; fax: ⫹39-050-883215. E-mail address: [email protected] (G. Solaini).
glia and astrocytes [48]. Abnormal proteolytic processing of amyloid precursor protein (APP) has been shown to contribute to A deposition in the cerebrovasculature of AD patients [1,16,18]. Incubation with aggregated A also has been shown to decrease the redox activity and consequently the viability of cultured primary neurons, astrocytes, HeLa and PC12 cells, suggesting that A impairs mitochondrial function and initiates a neurotoxic cascade [26,27,47]. However, the basis of A toxicity is poorly understood, but likely involves free radical damage [6]. It has been suggested that sporadic AD involves mutations in the mitochondrial genome of genes encoding complex IV (COX, cytochrome c oxidase) of the electron transport chain [39,52]. How this would translate into loss of specific neuronal populations, including cholinergic neurons in the forebrain, hippocampus, and neocortex is unclear. One possibility (oxidative stress hypothesis) is that as yet unknown factors cause an imbalance that favors the
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generation of reactive oxygen species (ROS), leading to oxidative damage of cell lipids, proteins and DNA [32]. Neuronal factors that may increase brain sensitivity to oxidative stress include a high oxygen consumption rate, an abundant polyunsaturated fatty acid content and a short half-life of mitochondrial DNA. Reduced activity of one or more of the mitochondrial electron transport chain enzymes, or of F1F0-ATPase (ATP synthase, complex V) could compromise brain ATP synthesis and induce damaging ROS production, and if severe could lead to neuronal death [7]. Deficient COX activity has been reported by several authors in different brain regions [29,33,37,50,59], as well as in platelets [39] and fibroblasts [17]. Moreover, COX activity and mRNA levels of COX subunits I and III have been shown to be decreased in association cortex from AD patients [15]. It is unknown whether reduced COX subunit mRNA is part of a pathological cascade or reflects physiological downregulation due to reduced energy need. The toxic A fragment 25–35 has been reported to selectively decrease COX activity in rat brain mitochondria, without affecting other components of the respiratory chain [12]. However, almost a normal amount of COX was found in mitochondria of the postmortem AD hippocampus [45]. Involvement of F1F0-ATPase in AD is even more controversial. Hydrolysis of ATP has been reported to be slightly increased in both frontal and occipital areas in AD [37]. These data do not correspond to the marked decrease (50 – 60%) of the mRNA level of ATP synthase subunit  found in temporal cortex of AD patients [14], or to the reduced amount of ATP synthase observed in hippocampal extracts from patients [45]. To our knowledge, the ATP synthesis rate of F1F0-ATPase of mitochondria from platelets of AD patients has not been reported. In the present study, we investigated rates of ATP synthesis and hydrolysis catalyzed by F1F0-ATPase, as well as COX activity, in mitochondria from platelets of AD patients and age-matched control subjects. We also assayed ATPase and COX enzyme activities in mitochondria from two regions of autopsied brain from pathologically-confirmed AD patients and age-matched controls. We chose the hippocampus because of its high densities of senile plaques and neurofibrillary tangles [2], and the motor cortex, which usually is little affected by AD pathology.
2. Methods Patients with a clinical diagnosis of senile dementia of the Alzheimer’s type (n ⫽ 20, 9 men and 11 women, 65.2 ⫾ 8.5 (SD) years, range 53–77 years) and age-matched controls (n ⫽ 20, 10 men and 10 women, 63.4 ⫾ 9.1 (SD) years, range 50 – 81 years) without evidence of neuropsychiatric diseases were recruited. The diagnosis of AD was based on DSM IV [19] and NINCDS-ADRDA criteria [34]. All AD patients underwent a battery of neuropsychological
tests, including Mini Mental State Evaluation [22] (mean values 17.7 ⫾ 4.8, range 11–27), and Global Deterioration Scale (mean values 4.4 ⫾ 1.1, range 2– 6) [43]. Patients scored 4 or less on the Hachinski scale [23], indicating that they likely did not include subjects with vascular dementia. No participant in the study had a history of any other neurological, psychiatric or drug abuse disorder, or was taking antioxidant therapy or medication that might interfere with cognitive function. The protocol was approved by our Institution’s Committee on Human Experimentation of the University of Pisa. Informed consent was obtained from each subject or from a relative, after the purposes and procedures of the study had been explained. 2.1. Brain tissue samples Post-mortem samples from the hippocampus and primary motor cortex were obtained from 6 clinically diagnosed and pathologically confirmed AD patients as well as from 6 neurologically and cognitively normal controls matched for age, gender, and post-mortem interval. Brains were obtained from the Brain Physiology and Metabolism Section of the National Institute on Aging, National Institutes of Health, Bethesda, MD. Autopsies were performed within 24 h of death. The brain was frozen at – 80°C immediately following autopsy. A clinical diagnosis of AD was confirmed neuropathologically according to the multifocal distribution of senile plaques and neurofibrillary tangles [60]. The motor cortex and the hippocampus were dissected from these brains for examination. 2.2. Isolation of brain mitochondria All chemicals, of reagent grade, were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified and were used without further purification. The brain sections were put in an ice-cold homogenization buffer (0.32 M sucrose, 1 mM K⫹EDTA, and 10 mM Tris-Cl, pH 7.4), containing 2.5 mg/ml of fatty acid-free bovine serum albumin (BSA) and 0.3 mM phenylmethylsulfonyl fluoride (PMSF, a protease inhibitor). The sections were minced and homogenized with 2 ml of homogenization buffer per gram of tissue, using a Teflon-glass homogenizer. The homogenate was centrifuged at 500 ⫻ g at 4°C for 5 min. The pellet was washed with the homogenization buffer and centrifuged at 500 ⫻ g for 5 min (4°C). The supernatant was centrifuged at 10,000 ⫻ g at 4°C for 15 min. The mitochondrial pellet was washed once and then resuspended in 500 l of homogenization buffer. Samples were stored on ice until enzyme activity was measured. Protein concentration in the mitochondrial suspension was determined by the Bradford method using BSA Fraction V as standard [9].
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2.3. Isolation of platelet mitochondria Human platelets were isolated and purified from 40 ml of venous blood, as previously reported [3]. Mitochondria were resuspended at a concentration of 10 mg/ml in 0.25 M sucrose and 2 mM EDTA, pH 8.0. The protein concentration of the mitochondrial suspension was determined by the Bradford method [9]. 2.4. Enzymatic activities Enzyme activity was determined as nmol/min/mg. In mitochondria from both brain and platelets, COX activity was determined spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm [57] using an extinction coefficient ⌬⑀red-ox ⫽ 19.1 mM⫺1 cm⫺1. Citrate synthase activity, a marker of the total number of mitochondria, was determined as previously reported [49]. 5⬘-Nucleotidase activity, a marker of mitochondrial contaminants, was assayed spectrophotometrically as previously described [4]. In mitochondrial preparations isolated from brain, ATPase activity was determined at 30°C with an ATP-regenerating system by following the decrease of NADH absorption at 340 nm in a Perkin Elmer spectrophotometer, as previously reported [8]. The time-dependent ATPase activity was determined both in the presence and absence of 1 g/ml oligomycin, a specific inhibitor, in order to exclude other enzymatic and non-enzymatic oxidation of NADH and to measure the oligomycin sensitive-ATPase (OS-ATPase). At least three assays of enzyme activity were carried out for each mitochondrial preparation. Intrasample variability was less than 5%. 2.5. Chemiluminescent methods for monitoring ATP synthesis and hydrolysis In mitochondria isolated from platelets, ATP synthesis and hydrolysis rates were assayed as previously described [3]. ATP was determined by the luciferin/luciferase chemiluminescent method [51]. All the assays were carried out at least in triplicate. 2.6. Statistical analysis Mean values were compared by an unpaired t test analysis (two-tailed). P ⬍ 0.05 was considered statistically significant. The data were expressed as the mean ⫾ SEM. 3. Results 3.1. Hydrolysis of ATP and cytochrome c oxidase activity in AD and control brains No statistically significant difference was found between the AD and the control groups for gender, age, or postmor-
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Table 1 Cytochrome c oxidase and mitochondrial ATPase activities in mitochondria isolated from motor cortex and hippocampus from AD and control brains
COX OS-ATPase
AD CON AD CON
Motor cortex
Hippocampus
198.7 ⫾ 14.2 220.5 ⫾ 4.93 105.8 ⫾ 4.34 113.7 ⫾ 16.3
132.4 ⫾ 14.0*,† 208.1 ⫾ 22.1 116 ⫾ 15.5 111.4 ⫾ 21.6
Data are expressed as nmol/min/mg mitochondrial protein and are means ⫾ SEM of 6 different brains per group. * P ⬍ 0.05 vs AD motor cortex; † P ⬍ 0.05 vs control. CON ⫽ control; OS-ATPase ⫽ Oligomycin-sensitive ATPase.
tem interval with regard to motor cortex specimens. The mean activity of citrate synthase per gram of tissue, a mitochondrial matrix enzyme insensitive to abnormalities of the respiratory chain, did not differ between the AD and control groups (data not shown), indicating that the amount of mitochondria was not affected by AD pathology. Table 1 shows mean COX and OS-ATPase activities in mitochondria isolated from the motor cortex of AD patients and controls. A lack of statistical significance between the means is consistent with the minimal pathology usually observed in the AD motor cortex (unaffected region). In the AD hippocampus on the other hand, which normally demonstrates marked neuropathology, COX activity was decreased by 35– 40% compared to control (132.4 ⫾ 14.0 vs 208.1 ⫾ 22.1 nmol/min/mg, P ⬍ 0.05) and to activity in the AD motor cortex (198.7 ⫾ 14.2 nmol/min/mg, P ⬍ 0.05), confirming previous reports relative to the parietal cortex [37]. There was no significant difference in brain mitochondrial ATPase activity between AD and control groups. 3.2. Hydrolysis and synthesis of ATP, and cytochrome c oxidase activity in AD and control platelets Different methods used to prepare platelets from AD patients have produced discrepant data in different studies of respiratory chain enzyme activities [39,40,54]. Therefore, we took great care in preparing platelets and mitochondria. Our procedure [3] was rapid, gentle, and avoided exposing the platelets and mitochondria to sonic oscillations, which could impair their structure and functions of biologic systems. Moreover, our mitochondrial fractions contained only very small contaminants, as detected by measuring 5⬘-nucleotidase activity below the assay sensitivity (specific activity ⬍ 1 nmol/min/mg of protein). Citrate synthase activity ranged from 0.21– 0.25 mol/min/mg in mitochondrial preparations from AD and control subject. No significant difference in mean succinate-sustained ATP synthesis was found between AD and control platelet mitochondria (0.32 ⫾ 0.05 vs 0.30 ⫾ 0.05 nmol/min/mg protein). Mean ATP hydrolysis rates also did not differ significantly between the two groups (46.5 ⫾ 4 vs 48.5 ⫾ 6
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Fig. 1. Cytochrome c oxidase activity in platelet mitochondria from AD patients and controls. The reaction mixture consisted of 50 mM KCl, 10 mM Tris, 1 mM EDTA, pH 7.4, 40 M ferrocytochrome c and the reaction was started by addition of 10 g mitochondrial protein. Activity was assayed spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm. Results are mean ⫾ SEM (n ⫽ 20 in each group). **P ⬍ 0.01 vs controls.
nmol/min/mg). In contrast, COX activity was decreased by 30% (35 ⫾ 3.5 vs 48.5 ⫾ 2.5 nmol/min/mg, P ⬍ 0.01) in AD compared with control platelets (Fig. 1), as reported elsewhere [29,40]. No significant difference in the COX/ citrate synthase ratio was observed (data not shown) between the groups.
4. Discussion This study demonstrates decreased COX activity in the hippocampus and platelets from AD patients compared to control subjects, but no difference in the motor cortex. Moreover, no significant group difference in ATP hydrolysis rate was observed in mitochondria from hippocampus, motor cortex or platelets. These data are in agreement with a reported 50%– 65% decrease in mRNA levels of the mitochondrialencoded COX subunits I and III in the middle temporal association neocortex, but not in the primary motor cortex from AD brains, as compared to control brains [13]. It is unlikely that the absence of a COX defect in AD motor cortex was due to our use of crude mitochondrial fractions. The effect likely reflects lesser neuropathology in motor cortex compared to hippocampus [2]. Furthermore, in previous reports changes in enzyme activities could be detected even using the whole brain homogenate preparations [29,33,37], even less purified than the ones we used. We also normalized our activities to the activity of citrate synthase, as a marker of mitochondrial number. Therefore, the decreased activity observed was due to a real effect directed to COX rather than to a mere loss of mitochondria. However, use of highly purified mitochondrial preparations could enhance the sensitivity of the assay and increase the chance of finding catalytic defects that are not otherwise seen. Taken together, these results support the idea that oxidative
phosphorylation enzyme impairment in the AD brain may be reflected in the periphery, particularly in platelets [11]. Reduced COX activity has been reported in parietal and temporal brain regions and in platelets from AD patients [29,37,39,40]. Whether the effect is primary or a consequence of AD pathology is unclear. The observed decrease in COX activity is unlikely to be merely a consequence of neuronal degeneration in AD, in which case other oxidative phosphorylation enzymes would be affected; however, we did not find any alteration in mitochondrial ATPase activity. It rather might be a consequence of lipid abnormalities or oxidative stress [37,38]. Indeed, A has been shown to favor the accumulation of hydrogen peroxide and lipid peroxides in cells [6]. Lipid peroxidation, which has been shown increased in AD [20, 30,31,36,41,42], can alter electrical potential, order, and ion permeability of biologic membranes [24]. Moreover, A can directly interact with membrane lipids, perturbing their fluidity and composition [25]. These adverse actions of both ROS and A could impair COX assembly and activity, which are particularly sensitive to the fluidity and composition of membrane lipids [38,53]. A second target of oxidative damage could be mitochondrial DNA, whose localization in the mitochondrial matrix makes it particularly vulnerable to ROS [21,24], and the catalytic subunits of the F1F0-complex are encoded by nuclear DNA while the catalytic subunits I-III of COX are encoded by mitochondrial DNA. Our results show, for the first time, that rates of succinate-sustained ATP synthesis (oxidative phosphorylation rate) of platelet mitochondria from AD patients and agedmatched controls do not differ significantly, despite a reduced COX activity in the AD mitochondria. In recent reports, the control coefficient of COX over oxidative phosphorylation was shown to be very low in several tissues including brain [44]. The relative capacity of COX in cells was found to be in excess of that required to support endogenous respiration [55,58]. Therefore, a 30 –35% reduction in COX activity in AD platelets might not be sufficient to cause a significant decrease of the overall ATP synthesis rate under physiological conditions. However, in the presence of reduced oxygen concentration, COX might be ratelimiting in determining the rate of oxidative phosphorylation, as found in some experimental models [58]. Indeed, positron emission tomography has demonstrated reduced cerebral blood flow in AD during functional activation, which might indicate reduced oxygen availability in the cells during activation [10,35].
Acknowledgments This research was supported by a grant (co-fin 1999) from Italian MURST, Roma.
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