J Neural Transm (1990) [Suppl] 29: 241-249 C£; by Springer-Verlag 1990
Oxidative stress: a role in the pathogenesis of Parkinson's disease M. E. Gotz, A. Freyberger, and P. Riederer Klinische Neurochemie, UniversiUits-Nervenklinik Wiirzburg, Wiirzburg, Federal Republic of Germany
Summary. The degeneration of nigro-striatal dopaminergic neurons is considered to be a predominant pathogenetic factor of Parkinson's disease (PD). However, the etiology of this degeneration is not known. Hypotheses assume accumulation of endogenous and/or exogenous toxins as trigger of the disease. An increase in the concentration of free radicals has been suggested to be toxic to cells, especially when combined with certain metals like free iron or copper. The role of melanin in the degenerative process is not clear, but auto xi dative reactions such as the oxidation of dopamine (DA) to melanin generating radicals and toxic metabolites seem to enhance the vulnerability of neurons in the substantia nigra (SN). Disappearance of melanin in the SN, increase of total iron and ferric iron, extreme decrease of glutathione (OSH) levels, reduced activity of enzymes involved in the detoxification of hydrogen peroxide, hydroxyl and superoxide radicals (peroxidases, catalase, glutathione peroxidase), an increase of monoamine oxidase B (MAO B) activity and the substantial increase of malondialdehyde, a marker of lipid peroxidation, in the SN seem to indicate a role of an oxidative stress syndrome in the SN causing or aggravating PD. Introduction It is generally agreed that Parkinson's disease (PD) is a progressive neurode-
generative disease characterized by a degeneration of nigrostriatal dopaminergic neurons including the loss of cell bodies in the pars compacta of the substantia nigra (SN). Thus, typical biochemical changes, such as the decrease of tyrosine hydroxylase (TH) activity and reduced levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HV A) are found (Birkmayer and Riederer, 1985). Moreover, there is general consensus that this biochemical pathology correlates with motor symptomes of PD. The beneficial effects on mainly akinesia and rigidity of direct (L-DOPA with or without decarboxylase and/or MAO inhibitors) or indirect (dopaminergic agonists, anticholinergic drugs) substitution of DA impressingly emphazises this correlation. Whilst morphological, clinical and functional aspects of PD have been in ten-
242
M. E. Gotz et al.
Table 1. Biochemical parameters that may contribute to increased vulnerability of substantia nigra neurons to degeneration in PD 1. 2. 3. 4. 5. 6. 7. 8.
High activity of Fe 2 + -dependent tyrosine hydroxylase. High concentration of reactive (free) iron. Reduced enzymatic capacity to detoxify free radicals (- OH, . O 2 - ) and H 20 2 . Low or reduced concentration of antioxidants (ascorbic acid, vitamin E, glutathione). High turnover of monoamines. Intraneuronal activity of MAO and aldehyde dehydrogenase. High affinity of neurotoxins for uptake mechanism. High rate of autoxidation and melaninization.
From Riederer and Youdim (1987) with permission
sively investigated in the past, research work on the etiology of the disease was initiated only recently. A preliminary answer to the question about an increased vulnerability of the nigrostriatal system towards toxic species may be the finding that both the SN and the locus coeruleus (LC) have biochemical features different from those of other brain regions. Some of these are shown in Table 1 and they will be discussed in the following text. Autoxidation of dopamine
Dopamine (DA) in solution is unstable, even at neutral pH-values, and easily undergoes autoxidation. Molecular oxygen, free radicals, hydrogen peroxide (H 2 0 2), quinones, and other substances readily oxidize DA in good yields. Oxidation of DA to melanin by these cytotoxic products can be demonstrated in vitro and the reaction is accelerated in alkaline solution. Direct evidence for the toxic effects of such autoxidative reactions can be obtained from cell culture experiments. Two distinct mechanisms are probably involved in the cytotoxicity of DA: reaction of DA-quinone with cellular nuc1eophiles and the formation of reactive oxygen species during autoxidation of DA (Graham et a1., 1978). The neurodegenerative effect of amphetamine on dopaminergic striatal axons, observed under specific experimental conditions, is considered to be the result of increased DA liberation followed by its autoxidation (Sonsalla et a1., 1989). It is likely that ascorbic acid (AA) protects DA from autoxidation both in the cytosol and in the extracellular space. However, when oxidation products are formed, AA can also act as a pro-oxidant and can increase the formation of H 2 0 2 . The formation of both 5-S-cysteinyldopamine and 5-S-cysteinyldopa,
Oxidative stress in Parkinson's disease
243
which reflects a synthesis of DA-quinone and DOPA-quinone, has been demonstrated in brain tissues (Rosengren et aI., 1985). This finding may also indicate the formation of quinoid intermediates during the biosynthesis of melanin in the SN and in the LC. Recent in vitro investigations on DA-autoxidation and its interaction with AA underline a possible role of a DA induced toxicity in neuronal systems. Monoamine oxidase, H 2 0 2, and GSH/GSSG-Equilibrium Since the oxidative deamination of DA by MAO results in the production of H 2 0 2 , the actual DA turnover in axons by MAO is, at least theoretically, associated with oxidative stress (Fig. 1). Administration of L-DOPA should result in increased DA turnover and an increase in H 2 0 2 levels. Moreover, a rise in DA turnover could catalyze its own metabolism, since there is some evidence for a stimulation of MAO B by H 2 0 2 (Konradi et aI., 1986; Table 2). In incubations of striatal synaptosomes with L-DOPA and glucose in the presence or absence of 10 ~M reserpine, H 20 2 concentrations were estimated by measuring oxidized glutathione (GSSG) (Spina and Cohen, 1988). GSSG increased in a concentration-dependent manner (O.04-1.0mM L-DOPA). At the highest L-DOPA-concentration an increase ofGSSG by 38.0± 4.5% was found compared to controls. This experiment demonstrates that H 20 2 generated by MAO is detoxified by glutathione peroxidase with the concomitant formation of GSSG. Spina and Cohen (1988, 1989) also investigated the influence of the MAO inhibitors c10rgyline and pargyline. The increase of GSSG levels was inhibited by 88-92% and the synthesis of DOPAC was also reduced by these inhibitors. The measured rise of GSSG levels probably underestimates the real increase of this compound, since reducing equivalents provided by the glucose/ glucose oxidase system may regenerate reduced glutathione (GSH). However, the moderate increase of GSSG levels indicates an imbalance of the GSH/ GSSG-equilibrium and thus reflects the appearance of "oxidative stress" Table 2. Kinetics of MAO-A and MAO-B in the presence of hydrogen peroxide in homogenates of human parietal cortex
Controls H 2 0 2 10-3 M
2-Phenylethylamine (MAO-B)
5-Hydroxytryptamine (MAO-A)
1.62 12.5*
135 1,016*
0.71 2.6*
2
7.6*
Data from Konradi et al. (1989) with permission. n=2; each determination in duplicate; * p 80%) of the SN with an increase of DA turnover (relative rise of acidic metabolites) in remaining axons of the striatum (Hefti et aI., 1983), amplified DA-liberation and -reuptake, are all processes that may contribute to the progressive loss of DA-neurons in PD (Cohen, 1983). These data may support the conclusion that L-DOPA can compensate for the loss of DA in PD, but cannot stop the progression of the disease. Our own studies on PD treated with L-DOPA provide some evidence for an increase of the DA-Ioss in the advanced decompensatory phase ofPD (Riederer and Wuketich, 1976; Table 3). The hypothesis mentioned above is further substantiated by a positive effect of (-)-deprenyl (MoverganTM, IumexTM, EldeprylTM) on the life expectancy of PD patients (Birkmayer et aI., 1985). Deprenyl inhibits MAO activity and concomitantly reduces the generation of H 20 2 . Of further interest is the finding that in PD a significant increase of malondialdehyde levels (indicative of membrane lipid peroxidation) (Dexter et aI., 1989) is found which coincides with an extreme loss of GSH (Perry et Table 3. Time course of the loss of dopamine in human brain Patients
Age group years
45-55 56-65 Healthy controls 66-75 76-85 86-95
(4)
Parkinsonian patientsa Parkinsonian patientsb
(14) (13) (9) (3)
61-67 67-73 74-80 80-84
(7)
(8) (6) (3)
Loss of dopamine in caudate nucleus % 15.3 15.7 9.8 10.7 mean = 12.87 ± 3.05 ± s.d. per decade 28.55 ±9.00 46.55 ±6.85 28.30±4.16 38.60± 5.79
Data from Riederer and Wuketich (1976) with permission. a Beginning of the disease = 60 ± 1 years. b Beginning of the disease = 73 ± 1 years
246
M. E. Gotz et al.
Table 4. Total glutathione and its dependence on the severity of Parkinson's disease (PD) Group Controls PD PD PD
Brain areas (n)
Grade a (0-4+ )
Glutathione a
23
0 1+ 2+ 3-4+
56.86 ± 12.95 29.66± 12.24 24.77 ± 8.44 6.29± 2.70b
II
4 7
(~g/g)
Data from Riederer et al. (1989) with permission. Values for total glutathione are means ± s.d.; n number of brain areas (from putamen < 3 >, globus paUidus < 4 >, substantia nigra < 3 >, nucleus basalis of Meynert < 4 >, thalamus < 3 >, amygdaloid nucleus < 3 >, and frontal cortex < 2 > ). GSH was measured by HPLC with electrochemical detection. a Regression analysis between grade of degeneration and GSH concentrations shows r = - 0.96 and p < 0.05. b P < 0.0 1 compared to controls
Table 5. Oxidized glutathione (GSSG) levels in control (C) and MPTP treated OF 1 mice Day Hour Item
Striatum n A.C.SN
GSSG-C 28± 16 GSSG-MPTP 35± 12
5
8
GSSG-C GSSG-MPTP -
8
GSSG-C 38 ± 18 GSSG-MPTP 44± 18
n
3 48± 12 5 5 139±26* 5 29± 13 55± 19
3 5
3 53± 15 3 3 155±1483
Data from Riederer et al. (1988) with permission. ~g/ g; n number of mice; A.C.SN. area containing substantia nigra; mean ± s.d.; Student's t test (C-MPTP): *p
Oxidative stress: a role in the pathogenesis of Parkinson's disease M. E. Gotz, A. Freyberger, and P. Riederer Klinische Neurochemie, UniversiUits-Nervenklinik Wiirzburg, Wiirzburg, Federal Republic of Germany
Summary. The degeneration of nigro-striatal dopaminergic neurons is considered to be a predominant pathogenetic factor of Parkinson's disease (PD). However, the etiology of this degeneration is not known. Hypotheses assume accumulation of endogenous and/or exogenous toxins as trigger of the disease. An increase in the concentration of free radicals has been suggested to be toxic to cells, especially when combined with certain metals like free iron or copper. The role of melanin in the degenerative process is not clear, but auto xi dative reactions such as the oxidation of dopamine (DA) to melanin generating radicals and toxic metabolites seem to enhance the vulnerability of neurons in the substantia nigra (SN). Disappearance of melanin in the SN, increase of total iron and ferric iron, extreme decrease of glutathione (OSH) levels, reduced activity of enzymes involved in the detoxification of hydrogen peroxide, hydroxyl and superoxide radicals (peroxidases, catalase, glutathione peroxidase), an increase of monoamine oxidase B (MAO B) activity and the substantial increase of malondialdehyde, a marker of lipid peroxidation, in the SN seem to indicate a role of an oxidative stress syndrome in the SN causing or aggravating PD. Introduction It is generally agreed that Parkinson's disease (PD) is a progressive neurode-
generative disease characterized by a degeneration of nigrostriatal dopaminergic neurons including the loss of cell bodies in the pars compacta of the substantia nigra (SN). Thus, typical biochemical changes, such as the decrease of tyrosine hydroxylase (TH) activity and reduced levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HV A) are found (Birkmayer and Riederer, 1985). Moreover, there is general consensus that this biochemical pathology correlates with motor symptomes of PD. The beneficial effects on mainly akinesia and rigidity of direct (L-DOPA with or without decarboxylase and/or MAO inhibitors) or indirect (dopaminergic agonists, anticholinergic drugs) substitution of DA impressingly emphazises this correlation. Whilst morphological, clinical and functional aspects of PD have been in ten-
242
M. E. Gotz et al.
Table 1. Biochemical parameters that may contribute to increased vulnerability of substantia nigra neurons to degeneration in PD 1. 2. 3. 4. 5. 6. 7. 8.
High activity of Fe 2 + -dependent tyrosine hydroxylase. High concentration of reactive (free) iron. Reduced enzymatic capacity to detoxify free radicals (- OH, . O 2 - ) and H 20 2 . Low or reduced concentration of antioxidants (ascorbic acid, vitamin E, glutathione). High turnover of monoamines. Intraneuronal activity of MAO and aldehyde dehydrogenase. High affinity of neurotoxins for uptake mechanism. High rate of autoxidation and melaninization.
From Riederer and Youdim (1987) with permission
sively investigated in the past, research work on the etiology of the disease was initiated only recently. A preliminary answer to the question about an increased vulnerability of the nigrostriatal system towards toxic species may be the finding that both the SN and the locus coeruleus (LC) have biochemical features different from those of other brain regions. Some of these are shown in Table 1 and they will be discussed in the following text. Autoxidation of dopamine
Dopamine (DA) in solution is unstable, even at neutral pH-values, and easily undergoes autoxidation. Molecular oxygen, free radicals, hydrogen peroxide (H 2 0 2), quinones, and other substances readily oxidize DA in good yields. Oxidation of DA to melanin by these cytotoxic products can be demonstrated in vitro and the reaction is accelerated in alkaline solution. Direct evidence for the toxic effects of such autoxidative reactions can be obtained from cell culture experiments. Two distinct mechanisms are probably involved in the cytotoxicity of DA: reaction of DA-quinone with cellular nuc1eophiles and the formation of reactive oxygen species during autoxidation of DA (Graham et a1., 1978). The neurodegenerative effect of amphetamine on dopaminergic striatal axons, observed under specific experimental conditions, is considered to be the result of increased DA liberation followed by its autoxidation (Sonsalla et a1., 1989). It is likely that ascorbic acid (AA) protects DA from autoxidation both in the cytosol and in the extracellular space. However, when oxidation products are formed, AA can also act as a pro-oxidant and can increase the formation of H 2 0 2 . The formation of both 5-S-cysteinyldopamine and 5-S-cysteinyldopa,
Oxidative stress in Parkinson's disease
243
which reflects a synthesis of DA-quinone and DOPA-quinone, has been demonstrated in brain tissues (Rosengren et aI., 1985). This finding may also indicate the formation of quinoid intermediates during the biosynthesis of melanin in the SN and in the LC. Recent in vitro investigations on DA-autoxidation and its interaction with AA underline a possible role of a DA induced toxicity in neuronal systems. Monoamine oxidase, H 2 0 2, and GSH/GSSG-Equilibrium Since the oxidative deamination of DA by MAO results in the production of H 2 0 2 , the actual DA turnover in axons by MAO is, at least theoretically, associated with oxidative stress (Fig. 1). Administration of L-DOPA should result in increased DA turnover and an increase in H 2 0 2 levels. Moreover, a rise in DA turnover could catalyze its own metabolism, since there is some evidence for a stimulation of MAO B by H 2 0 2 (Konradi et aI., 1986; Table 2). In incubations of striatal synaptosomes with L-DOPA and glucose in the presence or absence of 10 ~M reserpine, H 20 2 concentrations were estimated by measuring oxidized glutathione (GSSG) (Spina and Cohen, 1988). GSSG increased in a concentration-dependent manner (O.04-1.0mM L-DOPA). At the highest L-DOPA-concentration an increase ofGSSG by 38.0± 4.5% was found compared to controls. This experiment demonstrates that H 20 2 generated by MAO is detoxified by glutathione peroxidase with the concomitant formation of GSSG. Spina and Cohen (1988, 1989) also investigated the influence of the MAO inhibitors c10rgyline and pargyline. The increase of GSSG levels was inhibited by 88-92% and the synthesis of DOPAC was also reduced by these inhibitors. The measured rise of GSSG levels probably underestimates the real increase of this compound, since reducing equivalents provided by the glucose/ glucose oxidase system may regenerate reduced glutathione (GSH). However, the moderate increase of GSSG levels indicates an imbalance of the GSH/ GSSG-equilibrium and thus reflects the appearance of "oxidative stress" Table 2. Kinetics of MAO-A and MAO-B in the presence of hydrogen peroxide in homogenates of human parietal cortex
Controls H 2 0 2 10-3 M
2-Phenylethylamine (MAO-B)
5-Hydroxytryptamine (MAO-A)
1.62 12.5*
135 1,016*
0.71 2.6*
2
7.6*
Data from Konradi et al. (1989) with permission. n=2; each determination in duplicate; * p 80%) of the SN with an increase of DA turnover (relative rise of acidic metabolites) in remaining axons of the striatum (Hefti et aI., 1983), amplified DA-liberation and -reuptake, are all processes that may contribute to the progressive loss of DA-neurons in PD (Cohen, 1983). These data may support the conclusion that L-DOPA can compensate for the loss of DA in PD, but cannot stop the progression of the disease. Our own studies on PD treated with L-DOPA provide some evidence for an increase of the DA-Ioss in the advanced decompensatory phase ofPD (Riederer and Wuketich, 1976; Table 3). The hypothesis mentioned above is further substantiated by a positive effect of (-)-deprenyl (MoverganTM, IumexTM, EldeprylTM) on the life expectancy of PD patients (Birkmayer et aI., 1985). Deprenyl inhibits MAO activity and concomitantly reduces the generation of H 20 2 . Of further interest is the finding that in PD a significant increase of malondialdehyde levels (indicative of membrane lipid peroxidation) (Dexter et aI., 1989) is found which coincides with an extreme loss of GSH (Perry et Table 3. Time course of the loss of dopamine in human brain Patients
Age group years
45-55 56-65 Healthy controls 66-75 76-85 86-95
(4)
Parkinsonian patientsa Parkinsonian patientsb
(14) (13) (9) (3)
61-67 67-73 74-80 80-84
(7)
(8) (6) (3)
Loss of dopamine in caudate nucleus % 15.3 15.7 9.8 10.7 mean = 12.87 ± 3.05 ± s.d. per decade 28.55 ±9.00 46.55 ±6.85 28.30±4.16 38.60± 5.79
Data from Riederer and Wuketich (1976) with permission. a Beginning of the disease = 60 ± 1 years. b Beginning of the disease = 73 ± 1 years
246
M. E. Gotz et al.
Table 4. Total glutathione and its dependence on the severity of Parkinson's disease (PD) Group Controls PD PD PD
Brain areas (n)
Grade a (0-4+ )
Glutathione a
23
0 1+ 2+ 3-4+
56.86 ± 12.95 29.66± 12.24 24.77 ± 8.44 6.29± 2.70b
II
4 7
(~g/g)
Data from Riederer et al. (1989) with permission. Values for total glutathione are means ± s.d.; n number of brain areas (from putamen < 3 >, globus paUidus < 4 >, substantia nigra < 3 >, nucleus basalis of Meynert < 4 >, thalamus < 3 >, amygdaloid nucleus < 3 >, and frontal cortex < 2 > ). GSH was measured by HPLC with electrochemical detection. a Regression analysis between grade of degeneration and GSH concentrations shows r = - 0.96 and p < 0.05. b P < 0.0 1 compared to controls
Table 5. Oxidized glutathione (GSSG) levels in control (C) and MPTP treated OF 1 mice Day Hour Item
Striatum n A.C.SN
GSSG-C 28± 16 GSSG-MPTP 35± 12
5
8
GSSG-C GSSG-MPTP -
8
GSSG-C 38 ± 18 GSSG-MPTP 44± 18
n
3 48± 12 5 5 139±26* 5 29± 13 55± 19
3 5
3 53± 15 3 3 155±1483
Data from Riederer et al. (1988) with permission. ~g/ g; n number of mice; A.C.SN. area containing substantia nigra; mean ± s.d.; Student's t test (C-MPTP): *p
