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Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease
Journal of the Neurological Sciences, 104 (1991) 203-208 © 1991 Elsevier Science Publishers B.V. 0022-510X/91/$03.50
203
JNS 03581
Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease L . A . B i n d o f f ~, M . A . B i r c h - M a c h i n 1, N . E . F . C a r t l i d g e 1, W . D . P a r k e r , Jr. 2 a n d D . M . T u r n b u l l 1 Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, NE2 4HH (U.K.), and 2Departrnents of Pediatrics and Neurology, University of Colorado, Denver, CO (U.S.A.) (Received 20 August, 1990) (Revised, received 22 March, 1991) (Accepted 2 April, 1991)
Key words: Mitochondria; Parkinson's disease; Respiratory chain; Skeletal muscle
Summary Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and l-methyl-4-phenylpyridine (MPP ÷ ), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.
Introduction Parkinson's disease is a common neurodegenerative disorder characterised by the loss of pigmented cell nuclei from the brain. It has been estimated that between 2 and 3 ~o of the population will develop the disease (Kurland 1958) which, together with the long natural history and progressive physical and mental disability, make the disorder of major social and economic importance. The cause of Parkinson's disease is unknown; theories on the aetiology have included hereditary factors (Barbeau and Pourcher 1982), accelerated ageing (Calne and Langston 1983), viral infection (Poskanzer and Schwab 1963) and environmental toxins (Langston et al. 1983; Barbeau 1986; Snyder and D'Amato 1986). Recently, much of the research into the aetiology of Parkinson's disease has concentrated on the toxic effects of a class of compounds that cause parkinsonism in man and some animals. Davis et al. (1979) described a young man who developed parkinsonism after injecting himself with a self-synthesised drug he supposed was an analogue of Correspondence to: Professor D.M. Turnbull, Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
meperidine. It has been shown that the cause of this, and similar cases which followed, was a contaminant 1-methyl4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Langston et al. 1983). Further investigation showed that M P T P is oxidised to 1-methyl-4-phenylpyridine (MPP ÷ ) by monoamine oxidase type B (Chiba et al. 1984), probably in glial cells, and that this compound is actively taken up into striatal neurones by the dopamine uptake system (Javitch et al. 1985). Nicklas et al. (1985) demonstrated that MPP ÷ inhibited mitochondrial N A D H oxidation and subsequent studies have shown that this compound is actively accumulated by mitochondria (Ramsay and Singer, 1986) where it is a concentration dependent inhibitor of complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain (Ramsay et al. 1986). The mitochondrial respiratory chain is a series of 5 multi-subunit enzyme complexes located within the inner mitochondrial membrane comprising complex I (NADH : ubiquinone oxidoreductase), complex II (succinate : ubiquinone oxidoreductase), complex lII (ubiquinol-cytochrome c oxidoreductase), complex IV (cytochrome c oxidase) and complex V (ATP synthase). The function of this sequence of enzymes is the oxidation of reduced cofactors (NADH and FADH2) coupled to the synthesis of ATP. Disruption of this process would have numerous deleteri-
204 ous effects on cellular metabolism, including a decrease in ATP concentration (Di Monte et al. 1986) and generation of reactive oxygen species (Krishnamoorthy and Hinkle 1988). The observations on MPTP toxicity raise the possibility that a similar respiratory chain defect might be present in patients with idiopathic Parkinson's disease. Several groups have pursued this possibility: Schapira et al. (1989) showed low activity and Mizuno et al. (1990) low amount of complex I in striatum from patients with Parkinson's disease. Interestingly, more recently, Mizuno et al. (1990) have reported that complex III activity and not complex I, is lowered in the striata of patients with the disease. Parker et al. (1989) looked at platelets and found low complex I activity compared with controls. The present study was performed to establish whether respiratory chain defect(s) could be found in skeletal muscle since this tissue, like brain, is heavily dependent on oxidative metabolism for its energy supply. Preliminary results have been published in Bindoff et al. (1989).
Case histories Case 1
A 36-year-old man who had a 2-year progressive history of stiffness then resting tremor affecting his right arm. He has increased tone in both arm and leg, but is functionally independent. He takes Sinemet 110 7 times daily. Case 2
A 38-year-old man who had a 3-year history of Parkinson's disease consisting of both resting tremor and stiffness affecting mainly left arm and leg. He takes Sinemet 275 4 times daily, Orphenadrine 50 thrice daily and Ibuprofen. Case 3
A 50-year-old man who had recently been diagnosed as having Parkinson's disease. His symptoms, which included resting tremor, rigidity and gait instability, had been present for around 12 months and were progressive. He was not on treatment at the time of study and was reasonably mobile. Subsequently he has been treated with an L-dopa preparation with considerable improvement. Case 4
A 62-year-old man who was diagnosed as having Parkinson's disease in 1973 when he presented with bilateral upper limb resting tremor. He subsequently became progressively bradykinetic and, despite treatment, is functionally limited requiring help with dressing, feeding and toileting. His current medications include Madopar 125 8 times daily, bromocriptine 2 mg twice daily and diazepam. Case 5
A 62-year-old man diagnosed as having Parkinson's disease 8 years ago. He has asymmetrical involvement (left worse than right) but remains independent. Current medications are Sinemet 110 8 times daily, Benzhexol 2 mg thrice daily and triazolam 0.125 mg daily.
There was no family history of extrapyramidal disease or any unusual feature in any of these cases to suggest a diagnosis other than idiopathic Parkinson's disease. All cases had two or more of the classical features of Parkinson's disease (resting tremor, rigidity, bradykinesia, gait instability) and all responded to treatment with L-dopa. There was no evidence of muscle weakness, nor symptoms compatible with muscle abnormalities in any of these patients.
Methods
Muscle samples (1-2g) were removed from vastus lateralis under local anaesthesia after informed consent. A portion of muscle was frozen for histopathology and histochemistry (Johnson 1983). Control values were obtained from subjects evaluated for neuromuscular disease in whom no abnormality was found. Mitochondrial fractions were prepared according to Watmough et al. (1988). Overall mitochondrial substrate oxidation was measured spectrophotometrically at 420 nm (reference 475) using hexacyanoferrate (III) which accepts electrons at the level of cytochrome c (Turnbull et al. 1982). NAD +-linked substrate oxidation (pyruvate and malate, glutamate and malate, and oxoglutarate) requires electron flux through complexes I and III whereas succinate oxidation requires flux through complexes II and III. The activities of complex I (NADH : ubiquinone oxidoreductase), and complex IV (cytochrome c oxidase) were measured as described by Birch-Machin et al. (1989). Complex II activity was measured at 30 °C in the presence of 65/~M ubiquinone-1 and 50 #M dichlorophenolindophenol (DCPIP), after preincubation of the enzyme with 20 mM succinate (Ackrell et al. 1978). Citrate synthase, a matrix enzyme used as a marker for mitochondria, was measured by the method of Shepherd and Garland (1969) and protein was estimated by a modified Lowry method (Pederson 1977). Low temperature reduced minus oxidized cytochrome spectra were recorded using an Hitachi 557 spectrophotometer after reduction of the sample cuvette with dithionite. Cytochrome concentrations were calculated using the extinction coefficients and simultaneous equations given by Tervoort et al. (1981) and the enhancement factors quoted by Wilson (1967). Mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis (Laemmli 1970) using a 5~o stacking gel and at 15 % separating gel. All samples contained 3 mM p-aminobenzamidine to minimise proteolysis. Proteins were transferred to nitrocellulose (0.45/~m pore size) (Towbin et al. 1979) with the addition of 0.1 ~o SDS to the transfer buffer. Antisera to holocomplex I was raised in rabbits against purified beef heart complex I. Immunoreactive peptides were detected by the immunoperoxidase method with 4-chloro-l-naphthol as substrate (Domin et al. 1984). The results were analysed using a Student t-test.
205 TABLE 1 SUBSTRATE OXIDATIONS BY SKELETAL MUSCLE MITOCHONDRIA Results are expressed as nmol of ferricyanidereduced per min per mg protein. The number of patients and controls is shown in parentheses and results are shown as mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test.
10 mM succinate 10 mM pyruvate + 1 mM malate 10 mM glutamate + 1 mM malate 10 mM oxoglutarate
Patients (5)
Controls (7)
Significance(P)
224.6 + 46.6 147.0 + 40.4 65.9 + 31.4 74.9 + 30.5
266.6 + 40.5 202.0 + 64.0 126.3 + 52.6 126.0 _+29.6
ns ns <0.03 <0.006
Results
Activities of complexes I, II and IV were all significantly lower in muscle mitochondria from patients with Parkin-
There were m i n o r morphological abnormalities seen in two patients on N A D H - t e t r a z o l i u m reductase staining. These consisted of " m o t h - e a t e n " and "floccular" fibres, changes that reflect an abnormality in the distribution of mitochondria, but which are not specific for mitochondrial disorders (M.A. J o h n s o n , personal communication). The m e a n age range of patients with P a r k i n s o n ' s disease (49.6 + 12.5; m e a n + SD) and controls (48.8 + 12.6) is similar. Citrate synthase activity did n o t differ significantly between the controls (0.938 + 0.18 # m o l . m i n - 1 . m g protein (mean + SD)) a n d patients with P a r k i n s o n ' s disease (0.847 + 0.16 # m o l . min - 1. m g - 1 protein) suggesting the n u m b e r of mitochondria is similar in the two groups. M e a s u r e m e n t of flux through the respiratory chain showed that m i t o c h o n d r i a from patients with P a r k i n s o n ' s disease oxidised N A D ÷-linked substrates more slowly than controis (Table 1). This is significant for oxoglutarate a n d glutamate oxidation, but the same trend was found for pyruvate oxidation. Succinate oxidation was only slightly slower in patients with P a r k i n s o n ' s disease.
son's disease than controls (Table 2). These abnormalities are apparent when the activity of the complexes is referenced to mitochondrial protein (Table 2) or to citrate synthase activity, although in the latter case, the significance diminished (to the 5~o level for complexes I and II and between 5 and 10% for complex IV). The percentage decrease in activity was very similar whether the result was expressed per protein or citrate synthase a n d this was also true for substrate oxidation m e a s u r e m e n t s (results not shown). The low temperature cytochrome spectra showed no significant difference in the concentrations of cytochromes in patients with P a r k i n s o n ' s disease a n d controls (Table 3). Western blot analysis showed that mitochondrial fractions from several patients contained less immunoreactive material than controls (Fig. 1). N o particular pattern was seen and in general all detectable subunits were decreased. However, since some subunits were relatively more affected in one patient compared to another these changes are not thought simply to represent the loading of less mitochondrial protein (Fig. 1).
TABLE 2 ACTIVITIES OF RESPIRATORY CHAIN COMPLEXES IN SKELETAL MUSCLE MITOCHONDRIA Results are expressed as nmol of NADH oxidized per min per mg protein (complex I), nmol of DCPIP reduced per min per mg protein (complex II) or apparent first order rate constant (s 1. mg protein - ~) (complex IV). The number of patients and controls is shown in parentheses and results are mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test. Only in 4 of the controls shown in Table 1 had we sufficient mitochondria to perform these assays.
Complex I Complex II Complex IV
Patients (5)
Controls (4)
Significance (P)
132.4 + 39.9 180.5 + 33.3 1.23 + 0.31
220.0 + 29.5 352.5 + 69.2 2.05 +_ 0.23
<0.004 <0.001 <0.002
TABLE 3 CONCENTRATION OF CYTOCHROMES MUSCLE MITOCHONDRIA
IN
SKELETAL
Results are expressed as nmol per mg protein. The number of patients and controls is shown in parentheses and results are shown as mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test. As in Table 2, only 4 of the controls were used due to insufficientmaterial.
Cytochrome a a Cytochrome b Cytochrome c
3
Patients (4)
Controls (4)
Significance(P)
0.184 + 0.062 0.149 + 0.042 0.280 + 0.074
0.213+ 0.030 0.149_+0.020 0.316_+0.075
ns ns ns
206 1
2
3
4
5
6
7
8
Mr
68 57 43 40 30
110 75 51 49 42 39 3O 24 2O 18 15 13 10
Fig. 1. Western blot analysis of complex I subunits in patient and control mitochondrial fractions. Lanes 1 and 8 purified bovine complex I (7.25 gg); lanes 2 (patient 2), 4 (patient 4) and 6 (patient 1) different patients with Parkinson's disease (100 #g each); lanes 3, 5 and 7 controls (100 #g each). Molecular weight marker positions are shown on the right and the subunit assignation (in kDa) shown on the left. The patients in lanes 2 and 4 have generally lower amounts of all immunodetectable subunits compared with both controls and the patient in lane 6. This also appears true for the transhydrogenase (110 kDa) except that this is highly variable even between controls (compare lanes 5 and 7 with patient in lane 6). In addition, the 75, 51 and 49 kDa bands in lane 2 are present in visibly greater amount than lane 4 whereas the converse is true for 20, 13 and 10 kDa bands suggesting that the differences are not simply due to differences in overall protein loading.
Discussion
All the patients we studied had a well established clinical diagnosis of idiopathic Parkinson's disease and all but one (case 3) had been on treatment for several years. Only one of our patients experienced prolonged immobility: the others were independent and, apparently, normally mobile. Although we have carefully age-matched our control subjects, our experience does not mirror that of Trounce et al. (1989), who found a decline in mitochondrial oxidative capacity with age. Our control data shows no significant age-related difference in the activity of respiratory chain complexes of 18 subjects aged 8 months to 69 years (results not shown). The morphological changes found in two patients suggest that there may be abnormal distribution of mitochondria in some fibres. Electron microscopy was not performed, but ultrastructural abnormalities in muscle have previously been described in patients with Parkinson's disease (Alhqvist et al. 1975).
This study was by necessity based on small numbers of patients. Nevertheless, our results demonstrate a difference in the activity of the skeletal muscle mitochondrial respiratory chain between patients with Parkinson's disease and control subjects. We found low activities of complex I, II and IV, together with slow substrate oxidation. Citrate synthase activity per g wet weight (controls 9.92 + 3.72 # m o l . g - 1 (mean _+ SD); patients 9.1 + 2.68 # m o l . g - 1) and per mg mitochondrial protein was similar in patients and controls suggesting that equivalent amounts of mitochondria were present in both groups. That the differences in respiratory chain activity were present, whether expressed per milligram of protein or citrate synthase activity, suggests that the findings reflect a true decrease. Although the relatively normal oxidation of succinate by intact mitochondria despite abnormal complex II activity appears paradoxical, complex II is not the rate limiting step in electron transport to cytochrome c (B.A.C. Ackrell, personal communication) and a partial defect of complex II will not necessarily, therefore, impair the oxidation of succinate. The changes we found in skeletal muscle mitochondria from patients with Parkinson's disease are different to those reported in brain homogenates (Schapira et al. 1989). These differences may reflect the different tissues studied or that Schapira et al. (1989) did not measure the individual complexes directly, rather they determined the flux through two complexes. Interestingly, these changes were not confirmed in a recent study which showed that complex III activity and not complex I was low in striata from patients with Parkinson's disease (Mizuno et al. 1990). Moreover, this group showed that complex I activity was the most labile of all the respiratory chain complexes, declining in activity in the interval between death and freezing the brain. Further studies on brain mitochondria should clarify the situation. Previous studies using platelet mitochondrial fractions (Parker et al, 1989) have also shown low complex I activity, but did not demonstrate deficient complex IV or succinate cytochrome c reductase activity. As we state above, the latter does not preclude lowered complex II activity. These differences may be due to the relatively quick turnover of platelets compared to skeletal muscle, thus allowing additional changes to develop in the skeletal muscle mitochondria. The mitochondrial abnormalities we have described are not severe, but do appear to be significant. The mechanism giving rise to these abnormalities, and whether they are primary or secondary, is unknown. A primary genetic defect involving a nuclear gene is unlikely because of the lack of positive family history in the majority of cases of Parkinson's disease. A defect in the mitochondrial genome could give rise to multiple respiratory chain abnormalities,
207 but would not affect complex II which does not have any subunits encoded by mitochondrial DNA (Bindoff et al. 1990). An alternative explanation is that, whether involved in the pathogenesis or not, the respiratory chain changes are secondary. Using the MPTP model, it is possible that an environmental toxin poisons the respiratory chain which subsequently results in tissue damage due to lowered ATP synthesis. Herbicides (Barbeau et al. 1985) and other compounds such as isoquinolones (Makino et al. 1988) have been suggested and the latter have also been shown to produce parkinsonism (Nagatsu and Yoshida 1988) and inhibit complex I of the respiratory chain (Suzuki et al. 1988). Alternatively, the respiratory chain may be damaged by another toxic process such as induced by free radicals. Indeed, free radicals are formed by the respiratory chain especially if electron flux is slowed (Turrens and Boveris 1980) and evidence of increased free radical damage has been found in Parkinson's disease (Dexter et al. 1989). Lastly, lowered respiratory chain activity may be secondary and unrelated to the pathogenesis of Parkinson's disease. Disease or drug treatment may affect the respiratory chain causing a decreased activity which, although statistically different, is not sufficient to affect oxidative metabolism. Acknowledgements We would like to thank Dr. H.S.A. Sherratt and Professor F.E. Frerman for their helpful comments and discussion, Dr. C. I. Ragan for the gift of complex I antibodies and Mrs. S. Lowe for help with preparing the manuscript. We are grateful for financial support from the Muscular Dystrophy Group of Great Britain, Newcastle University Research Committee, The Wolfson Foundation, and NIH grants 24872 and NS01047.
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203
JNS 03581
Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease L . A . B i n d o f f ~, M . A . B i r c h - M a c h i n 1, N . E . F . C a r t l i d g e 1, W . D . P a r k e r , Jr. 2 a n d D . M . T u r n b u l l 1 Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, NE2 4HH (U.K.), and 2Departrnents of Pediatrics and Neurology, University of Colorado, Denver, CO (U.S.A.) (Received 20 August, 1990) (Revised, received 22 March, 1991) (Accepted 2 April, 1991)
Key words: Mitochondria; Parkinson's disease; Respiratory chain; Skeletal muscle
Summary Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and l-methyl-4-phenylpyridine (MPP ÷ ), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.
Introduction Parkinson's disease is a common neurodegenerative disorder characterised by the loss of pigmented cell nuclei from the brain. It has been estimated that between 2 and 3 ~o of the population will develop the disease (Kurland 1958) which, together with the long natural history and progressive physical and mental disability, make the disorder of major social and economic importance. The cause of Parkinson's disease is unknown; theories on the aetiology have included hereditary factors (Barbeau and Pourcher 1982), accelerated ageing (Calne and Langston 1983), viral infection (Poskanzer and Schwab 1963) and environmental toxins (Langston et al. 1983; Barbeau 1986; Snyder and D'Amato 1986). Recently, much of the research into the aetiology of Parkinson's disease has concentrated on the toxic effects of a class of compounds that cause parkinsonism in man and some animals. Davis et al. (1979) described a young man who developed parkinsonism after injecting himself with a self-synthesised drug he supposed was an analogue of Correspondence to: Professor D.M. Turnbull, Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
meperidine. It has been shown that the cause of this, and similar cases which followed, was a contaminant 1-methyl4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Langston et al. 1983). Further investigation showed that M P T P is oxidised to 1-methyl-4-phenylpyridine (MPP ÷ ) by monoamine oxidase type B (Chiba et al. 1984), probably in glial cells, and that this compound is actively taken up into striatal neurones by the dopamine uptake system (Javitch et al. 1985). Nicklas et al. (1985) demonstrated that MPP ÷ inhibited mitochondrial N A D H oxidation and subsequent studies have shown that this compound is actively accumulated by mitochondria (Ramsay and Singer, 1986) where it is a concentration dependent inhibitor of complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain (Ramsay et al. 1986). The mitochondrial respiratory chain is a series of 5 multi-subunit enzyme complexes located within the inner mitochondrial membrane comprising complex I (NADH : ubiquinone oxidoreductase), complex II (succinate : ubiquinone oxidoreductase), complex lII (ubiquinol-cytochrome c oxidoreductase), complex IV (cytochrome c oxidase) and complex V (ATP synthase). The function of this sequence of enzymes is the oxidation of reduced cofactors (NADH and FADH2) coupled to the synthesis of ATP. Disruption of this process would have numerous deleteri-
204 ous effects on cellular metabolism, including a decrease in ATP concentration (Di Monte et al. 1986) and generation of reactive oxygen species (Krishnamoorthy and Hinkle 1988). The observations on MPTP toxicity raise the possibility that a similar respiratory chain defect might be present in patients with idiopathic Parkinson's disease. Several groups have pursued this possibility: Schapira et al. (1989) showed low activity and Mizuno et al. (1990) low amount of complex I in striatum from patients with Parkinson's disease. Interestingly, more recently, Mizuno et al. (1990) have reported that complex III activity and not complex I, is lowered in the striata of patients with the disease. Parker et al. (1989) looked at platelets and found low complex I activity compared with controls. The present study was performed to establish whether respiratory chain defect(s) could be found in skeletal muscle since this tissue, like brain, is heavily dependent on oxidative metabolism for its energy supply. Preliminary results have been published in Bindoff et al. (1989).
Case histories Case 1
A 36-year-old man who had a 2-year progressive history of stiffness then resting tremor affecting his right arm. He has increased tone in both arm and leg, but is functionally independent. He takes Sinemet 110 7 times daily. Case 2
A 38-year-old man who had a 3-year history of Parkinson's disease consisting of both resting tremor and stiffness affecting mainly left arm and leg. He takes Sinemet 275 4 times daily, Orphenadrine 50 thrice daily and Ibuprofen. Case 3
A 50-year-old man who had recently been diagnosed as having Parkinson's disease. His symptoms, which included resting tremor, rigidity and gait instability, had been present for around 12 months and were progressive. He was not on treatment at the time of study and was reasonably mobile. Subsequently he has been treated with an L-dopa preparation with considerable improvement. Case 4
A 62-year-old man who was diagnosed as having Parkinson's disease in 1973 when he presented with bilateral upper limb resting tremor. He subsequently became progressively bradykinetic and, despite treatment, is functionally limited requiring help with dressing, feeding and toileting. His current medications include Madopar 125 8 times daily, bromocriptine 2 mg twice daily and diazepam. Case 5
A 62-year-old man diagnosed as having Parkinson's disease 8 years ago. He has asymmetrical involvement (left worse than right) but remains independent. Current medications are Sinemet 110 8 times daily, Benzhexol 2 mg thrice daily and triazolam 0.125 mg daily.
There was no family history of extrapyramidal disease or any unusual feature in any of these cases to suggest a diagnosis other than idiopathic Parkinson's disease. All cases had two or more of the classical features of Parkinson's disease (resting tremor, rigidity, bradykinesia, gait instability) and all responded to treatment with L-dopa. There was no evidence of muscle weakness, nor symptoms compatible with muscle abnormalities in any of these patients.
Methods
Muscle samples (1-2g) were removed from vastus lateralis under local anaesthesia after informed consent. A portion of muscle was frozen for histopathology and histochemistry (Johnson 1983). Control values were obtained from subjects evaluated for neuromuscular disease in whom no abnormality was found. Mitochondrial fractions were prepared according to Watmough et al. (1988). Overall mitochondrial substrate oxidation was measured spectrophotometrically at 420 nm (reference 475) using hexacyanoferrate (III) which accepts electrons at the level of cytochrome c (Turnbull et al. 1982). NAD +-linked substrate oxidation (pyruvate and malate, glutamate and malate, and oxoglutarate) requires electron flux through complexes I and III whereas succinate oxidation requires flux through complexes II and III. The activities of complex I (NADH : ubiquinone oxidoreductase), and complex IV (cytochrome c oxidase) were measured as described by Birch-Machin et al. (1989). Complex II activity was measured at 30 °C in the presence of 65/~M ubiquinone-1 and 50 #M dichlorophenolindophenol (DCPIP), after preincubation of the enzyme with 20 mM succinate (Ackrell et al. 1978). Citrate synthase, a matrix enzyme used as a marker for mitochondria, was measured by the method of Shepherd and Garland (1969) and protein was estimated by a modified Lowry method (Pederson 1977). Low temperature reduced minus oxidized cytochrome spectra were recorded using an Hitachi 557 spectrophotometer after reduction of the sample cuvette with dithionite. Cytochrome concentrations were calculated using the extinction coefficients and simultaneous equations given by Tervoort et al. (1981) and the enhancement factors quoted by Wilson (1967). Mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis (Laemmli 1970) using a 5~o stacking gel and at 15 % separating gel. All samples contained 3 mM p-aminobenzamidine to minimise proteolysis. Proteins were transferred to nitrocellulose (0.45/~m pore size) (Towbin et al. 1979) with the addition of 0.1 ~o SDS to the transfer buffer. Antisera to holocomplex I was raised in rabbits against purified beef heart complex I. Immunoreactive peptides were detected by the immunoperoxidase method with 4-chloro-l-naphthol as substrate (Domin et al. 1984). The results were analysed using a Student t-test.
205 TABLE 1 SUBSTRATE OXIDATIONS BY SKELETAL MUSCLE MITOCHONDRIA Results are expressed as nmol of ferricyanidereduced per min per mg protein. The number of patients and controls is shown in parentheses and results are shown as mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test.
10 mM succinate 10 mM pyruvate + 1 mM malate 10 mM glutamate + 1 mM malate 10 mM oxoglutarate
Patients (5)
Controls (7)
Significance(P)
224.6 + 46.6 147.0 + 40.4 65.9 + 31.4 74.9 + 30.5
266.6 + 40.5 202.0 + 64.0 126.3 + 52.6 126.0 _+29.6
ns ns <0.03 <0.006
Results
Activities of complexes I, II and IV were all significantly lower in muscle mitochondria from patients with Parkin-
There were m i n o r morphological abnormalities seen in two patients on N A D H - t e t r a z o l i u m reductase staining. These consisted of " m o t h - e a t e n " and "floccular" fibres, changes that reflect an abnormality in the distribution of mitochondria, but which are not specific for mitochondrial disorders (M.A. J o h n s o n , personal communication). The m e a n age range of patients with P a r k i n s o n ' s disease (49.6 + 12.5; m e a n + SD) and controls (48.8 + 12.6) is similar. Citrate synthase activity did n o t differ significantly between the controls (0.938 + 0.18 # m o l . m i n - 1 . m g protein (mean + SD)) a n d patients with P a r k i n s o n ' s disease (0.847 + 0.16 # m o l . min - 1. m g - 1 protein) suggesting the n u m b e r of mitochondria is similar in the two groups. M e a s u r e m e n t of flux through the respiratory chain showed that m i t o c h o n d r i a from patients with P a r k i n s o n ' s disease oxidised N A D ÷-linked substrates more slowly than controis (Table 1). This is significant for oxoglutarate a n d glutamate oxidation, but the same trend was found for pyruvate oxidation. Succinate oxidation was only slightly slower in patients with P a r k i n s o n ' s disease.
son's disease than controls (Table 2). These abnormalities are apparent when the activity of the complexes is referenced to mitochondrial protein (Table 2) or to citrate synthase activity, although in the latter case, the significance diminished (to the 5~o level for complexes I and II and between 5 and 10% for complex IV). The percentage decrease in activity was very similar whether the result was expressed per protein or citrate synthase a n d this was also true for substrate oxidation m e a s u r e m e n t s (results not shown). The low temperature cytochrome spectra showed no significant difference in the concentrations of cytochromes in patients with P a r k i n s o n ' s disease a n d controls (Table 3). Western blot analysis showed that mitochondrial fractions from several patients contained less immunoreactive material than controls (Fig. 1). N o particular pattern was seen and in general all detectable subunits were decreased. However, since some subunits were relatively more affected in one patient compared to another these changes are not thought simply to represent the loading of less mitochondrial protein (Fig. 1).
TABLE 2 ACTIVITIES OF RESPIRATORY CHAIN COMPLEXES IN SKELETAL MUSCLE MITOCHONDRIA Results are expressed as nmol of NADH oxidized per min per mg protein (complex I), nmol of DCPIP reduced per min per mg protein (complex II) or apparent first order rate constant (s 1. mg protein - ~) (complex IV). The number of patients and controls is shown in parentheses and results are mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test. Only in 4 of the controls shown in Table 1 had we sufficient mitochondria to perform these assays.
Complex I Complex II Complex IV
Patients (5)
Controls (4)
Significance (P)
132.4 + 39.9 180.5 + 33.3 1.23 + 0.31
220.0 + 29.5 352.5 + 69.2 2.05 +_ 0.23
<0.004 <0.001 <0.002
TABLE 3 CONCENTRATION OF CYTOCHROMES MUSCLE MITOCHONDRIA
IN
SKELETAL
Results are expressed as nmol per mg protein. The number of patients and controls is shown in parentheses and results are shown as mean + SD. The significance(controls vs. patients) was calculated using an unpaired t-test. As in Table 2, only 4 of the controls were used due to insufficientmaterial.
Cytochrome a a Cytochrome b Cytochrome c
3
Patients (4)
Controls (4)
Significance(P)
0.184 + 0.062 0.149 + 0.042 0.280 + 0.074
0.213+ 0.030 0.149_+0.020 0.316_+0.075
ns ns ns
206 1
2
3
4
5
6
7
8
Mr
68 57 43 40 30
110 75 51 49 42 39 3O 24 2O 18 15 13 10
Fig. 1. Western blot analysis of complex I subunits in patient and control mitochondrial fractions. Lanes 1 and 8 purified bovine complex I (7.25 gg); lanes 2 (patient 2), 4 (patient 4) and 6 (patient 1) different patients with Parkinson's disease (100 #g each); lanes 3, 5 and 7 controls (100 #g each). Molecular weight marker positions are shown on the right and the subunit assignation (in kDa) shown on the left. The patients in lanes 2 and 4 have generally lower amounts of all immunodetectable subunits compared with both controls and the patient in lane 6. This also appears true for the transhydrogenase (110 kDa) except that this is highly variable even between controls (compare lanes 5 and 7 with patient in lane 6). In addition, the 75, 51 and 49 kDa bands in lane 2 are present in visibly greater amount than lane 4 whereas the converse is true for 20, 13 and 10 kDa bands suggesting that the differences are not simply due to differences in overall protein loading.
Discussion
All the patients we studied had a well established clinical diagnosis of idiopathic Parkinson's disease and all but one (case 3) had been on treatment for several years. Only one of our patients experienced prolonged immobility: the others were independent and, apparently, normally mobile. Although we have carefully age-matched our control subjects, our experience does not mirror that of Trounce et al. (1989), who found a decline in mitochondrial oxidative capacity with age. Our control data shows no significant age-related difference in the activity of respiratory chain complexes of 18 subjects aged 8 months to 69 years (results not shown). The morphological changes found in two patients suggest that there may be abnormal distribution of mitochondria in some fibres. Electron microscopy was not performed, but ultrastructural abnormalities in muscle have previously been described in patients with Parkinson's disease (Alhqvist et al. 1975).
This study was by necessity based on small numbers of patients. Nevertheless, our results demonstrate a difference in the activity of the skeletal muscle mitochondrial respiratory chain between patients with Parkinson's disease and control subjects. We found low activities of complex I, II and IV, together with slow substrate oxidation. Citrate synthase activity per g wet weight (controls 9.92 + 3.72 # m o l . g - 1 (mean _+ SD); patients 9.1 + 2.68 # m o l . g - 1) and per mg mitochondrial protein was similar in patients and controls suggesting that equivalent amounts of mitochondria were present in both groups. That the differences in respiratory chain activity were present, whether expressed per milligram of protein or citrate synthase activity, suggests that the findings reflect a true decrease. Although the relatively normal oxidation of succinate by intact mitochondria despite abnormal complex II activity appears paradoxical, complex II is not the rate limiting step in electron transport to cytochrome c (B.A.C. Ackrell, personal communication) and a partial defect of complex II will not necessarily, therefore, impair the oxidation of succinate. The changes we found in skeletal muscle mitochondria from patients with Parkinson's disease are different to those reported in brain homogenates (Schapira et al. 1989). These differences may reflect the different tissues studied or that Schapira et al. (1989) did not measure the individual complexes directly, rather they determined the flux through two complexes. Interestingly, these changes were not confirmed in a recent study which showed that complex III activity and not complex I was low in striata from patients with Parkinson's disease (Mizuno et al. 1990). Moreover, this group showed that complex I activity was the most labile of all the respiratory chain complexes, declining in activity in the interval between death and freezing the brain. Further studies on brain mitochondria should clarify the situation. Previous studies using platelet mitochondrial fractions (Parker et al, 1989) have also shown low complex I activity, but did not demonstrate deficient complex IV or succinate cytochrome c reductase activity. As we state above, the latter does not preclude lowered complex II activity. These differences may be due to the relatively quick turnover of platelets compared to skeletal muscle, thus allowing additional changes to develop in the skeletal muscle mitochondria. The mitochondrial abnormalities we have described are not severe, but do appear to be significant. The mechanism giving rise to these abnormalities, and whether they are primary or secondary, is unknown. A primary genetic defect involving a nuclear gene is unlikely because of the lack of positive family history in the majority of cases of Parkinson's disease. A defect in the mitochondrial genome could give rise to multiple respiratory chain abnormalities,
207 but would not affect complex II which does not have any subunits encoded by mitochondrial DNA (Bindoff et al. 1990). An alternative explanation is that, whether involved in the pathogenesis or not, the respiratory chain changes are secondary. Using the MPTP model, it is possible that an environmental toxin poisons the respiratory chain which subsequently results in tissue damage due to lowered ATP synthesis. Herbicides (Barbeau et al. 1985) and other compounds such as isoquinolones (Makino et al. 1988) have been suggested and the latter have also been shown to produce parkinsonism (Nagatsu and Yoshida 1988) and inhibit complex I of the respiratory chain (Suzuki et al. 1988). Alternatively, the respiratory chain may be damaged by another toxic process such as induced by free radicals. Indeed, free radicals are formed by the respiratory chain especially if electron flux is slowed (Turrens and Boveris 1980) and evidence of increased free radical damage has been found in Parkinson's disease (Dexter et al. 1989). Lastly, lowered respiratory chain activity may be secondary and unrelated to the pathogenesis of Parkinson's disease. Disease or drug treatment may affect the respiratory chain causing a decreased activity which, although statistically different, is not sufficient to affect oxidative metabolism. Acknowledgements We would like to thank Dr. H.S.A. Sherratt and Professor F.E. Frerman for their helpful comments and discussion, Dr. C. I. Ragan for the gift of complex I antibodies and Mrs. S. Lowe for help with preparing the manuscript. We are grateful for financial support from the Muscular Dystrophy Group of Great Britain, Newcastle University Research Committee, The Wolfson Foundation, and NIH grants 24872 and NS01047.
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