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Oxidative metabolites of 5-S-cysteinyldopamine inhibit the α-ketoglutarate dehydrogenase complex: Possible relevance to the pathogenesis of Parkinson's disease
Abstract
A characteristic change in the substantia nigra of Parkinson's disease patients is an apparent accelerated rate of dopamine oxidation as evidenced by an increased 5-S-cysteinyldopamine (5-S-CyS-DA) to dopamine ratio. However, 5-S-CyS-DA is more easily oxidized than dopamine to give 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). Previous studies have demonstrated that DHBT-1 can be accumulated by intact rat brain mitochondria and inhibits complex I but not complex II respiration. In this study, it is shown that DHBT-1 also inhibits the alpha-ketoglutarate dehydrogenase complex (alpha-KGDH) but not cytochrome c oxidase (complex IV). The inhibition of alpha-KGDH is dependent on the oxidation of DHBT-1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an electrophilic o-quinone imine that covalently modifies active site sulfhydryl residues. The latter conclusion is based on the ability of > or = equimolar glutathione to block the inhibition of alpha-KGDH by DHBT-1, without altering its rate of mitochondrial membrane-catalyzed oxidation, by scavenging the electrophilic o-quinone intermediate forming glutathionyl conjugates which have been isolated and spectroscopically characterized. Activities of mitochondrial alpha-KGDH and complex I, but not other respiratory complexes, are decreased in the parkinsonian substantia nigra. Such changes together with evidence for accelerated dopamine oxidation, increased formation of 5-S-CyS-DA and the ease of oxidation of this conjugate to DHBT-1 which inhibits alpha-KGDH and complex I, without affecting other respiratory enzyme complexes, suggests that the latter putative metabolite might be an endotoxin that contributes to the alpha-KGDH and complex I defects in Parkinson's disease.
... Indeed, 5-S-CyS-DA, normally a minor metabolite of DA, becomes a major metabolite in the parkinsonian SN c as indicated by a large increase in the 5-S-CyS-DA/DA concentration ratio (37). However, 5-S-CyS-DA is more easily oxidized than DA (31) to dihydrobenzothiazines (DHBTs) and benzothiazines (BTs) (38) that include irreversible inhibitors of mitochondrial complex I (39,40) and R-KGDH (41). This raises the possibility that DHBTs and BTs, oxidative metabolites of 5-S-CyS-DA, might be endogenously generated toxicants that contribute to the mitochondrial complex I (42) and R-KGDH (43) deficiencies and dopaminergic SN c cell death in PD. ...
... Freshly collected solutions of 8 and 9, prepared using preparative HPLC method IV, were bright yellow and exhibited identical UV/vis spectra (λ max ) 354 and 252 nm at pH 2.15) and ESI-MS data (m/z 576, MH + , 100%), indicating that they are isomers. Because of their decomposition during the final freeze-drying step, it was not possible to isolate 8 and 9. Nevertheless, the UV/vis spectra of 8 and 9 were identical to those of epimers of 2-S-glutathionyl-7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (ESI-MS, m/z 558) that differ only in the absence of the side chain -OH residue (41), suggesting that 8 and 9 are epimers of the 2-S-glutathionyl conjugate of BT-NE-1. Compounds 10 and 11, formed by the NaBH 4 reduction of 8 and 9, respectively, could be separated by preparative HPLC method IV. ...
... The UV spectra of 10 and 11, however, were different from those of 5 [λ max ) 318, 282 (sh), and 248 nm at pH 2.15] and 6 (λ max ) 318 and 248 nm at pH 2.15) as were their t R values in analytical (and preparative) HPLC ( Figure 6C). This indicated that 10 and 11 are 2-S-glutathionyl conjugates of DHBT-NE-1 and, indeed, that the UV spectra of these compounds are virtually identical to those of the diastereomeric 2-S-glutathionyl conjugates of 7-(2-aminoethyl)-5-hydroxy-2H-1,4-benzothiazine-3carboxylic acid (41). It was not possible to isolate sufficient quantities of 10 and 11 to permit their complete structure elucidation by spectroscopic methods ( 1 H NMR or FAB-MS). ...
The major initial product of the oxidation of norepinephrine (NE) in the presence of L-cysteine is 5-S-cysteinylnorepinephrine which is then further easily oxidized to the dihydrobenzothiazine (DHBT) 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1, 4-benzothiazine-3-carboxylic acid (DHBT-NE-1). When incubated with intact rat brain mitochondria, DHBT-NE-1 evokes rapid inhibition of complex I respiration without affecting complex II respiration. DHBT-NE-1 also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q(1) (CoQ(1)) reductase, the pyruvate dehydrogenase complex (PDHC), and alpha-ketoglutarate dehydrogenase (alpha-KGDH) when incubated with frozen and thawed rat brain mitochondria (mitochondrial membranes). The time dependence of the inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by DHBT-NE-1 appears to be related to its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane, to electrophilic intermediates which bind covalently to active site cysteinyl residues of these enzyme complexes. The latter conclusion is based on the ability of glutathione to block inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membrane-catalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates, several of which have been isolated and spectroscopically identified. The possible implications of these results to the degeneration of neuromelanin-pigmented noradrenergic neurons in the locus ceruleus in Parkinson's disease are discussed.
... DHBT-1 can be accumulated by intact rat brain mitochondria in vitro and inhibits complex I (pyruvate-and malate-supported) but not complex II (succinate-supported) respiration (296). Furthermore, when incubated with rat brain mitochondrial membranes, DHBT-1 evokes a time-dependent irreversible inhibition of NADH-CoQ 1 reductase (297) and R-KGDH but not cytochrome c oxidase (complex IV) (298). The inhibition of NADH-CoQ 1 reductase and R-KGDH by DHBT-1 is unaffected by SOD or catalase and, hence, does not involve reactive oxygen species. ...
... The inhibition of NADH-CoQ 1 reductase and R-KGDH by DHBT-1 is unaffected by SOD or catalase and, hence, does not involve reactive oxygen species. The time dependence of the inhibition of NADH-CoQ 1 reductase and R-KGDH appears to be related to its oxidation catalyzed by a presently unknown constituent of the inner mitochondrial membrane (297,298). In this reaction, DHBT-1 is catalytically oxidized by mitochondrial membranes to o-quinone imine 3 that rapidly rearranges to BT-1 and BT-2 ( Figure 11). ...
Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).
... Let us summarise some main structural and functional features of DA neurons (see Fig. 4). Only DA neurons of the SNc contain significant amount of DA, hence a possible explanation for the selective neuron loss in PD is that the cytosol DA oxidative adducts are toxic (Shen et al. 2000;Lee and Liu 2008). It is important noting that Ventral tegmental Area DA neurons have increased vesicular monoamine transporter expression compared with SNc neurons, so the DA cytosol levels are lower. ...
... On the basis of the above-mentioned data the scheme of Fig. 5 has been proposed, which maintains that Parkinsonism is caused by environmental and/or genetic factors acting on a very sensitive aspect of the SNc neurons: their high, constant, energy requirements. We put forward the hypothesis that a mitochondria dysfunction results in a sort of protein conformational disease and in the assemblage of abnormal PMs to which the action of cytosolic DA oxidative adducts contributes (Shen et al. 2000). As it will be discussed in ''Interconnections between mitochondrial overburden, energy failure and abnormal PMs assemblage Fig. 4 Schematic representation of some compartments and some signals characterising the structure and function of a DA neuron of Substantia Nigra pars compacta (SNc). ...
On the basis of the previously proposed hierarchic organisation of the central nervous system (CNS) and of its syntropic behaviour, a view of neurodegenerative diseases focusing on the assemblage of abnormal multimeric proteins (pathologic protein mosaics (PMs)) is proposed. Thus, the main focus of the present paper is on Parkinson's disease (PD) as a neurodegenerative disease, which has as crucial feature protein conformational alterations and formation of pathological PMs. Two interconnected cellular dysfunctions are discussed as main pathogenic factors of PD syndromes, namely mitochondrial deficits (i.e. energy failure, especially critical for Substantia Nigra DA neurons) and conformational protein alterations (due to genetic or environmental causes). Conformational protein alterations can trigger pathological phenomena via the loss and/or the gain of new functions. In particular, altered proteins can lead to the formation of abnormal PMs, which can, inter alia, cause distortion of cellular structures, toxic functions and/or formation of improper membrane ion channels. In view of the fact that disordered proteins can easily acquire unwanted conformation, the "disorder index" (DI) for proteins involved in PD has been evaluated. It has been found that both alpha-synuclein and tau-protein have high DI. This datum is in agreement with the observation that these two proteins synergistically promote polymerisation of each other into amyloid fibrils, favouring the formation of Lewy bodies.
... Besides, dopamine autoxidation can also form reactive quinones (LaVoie and Hastings 1999). The importance of DA-quinones in producing mitochondrial dysfunction is supported by findings showing inhibition of creatine kinase, α-ketoglutarate dehydrogenase and complex I by DA-quinones (Asanuma et al. 2003;Shen et al. 2000). Thus, it is tempting to speculate that high concentrations of tyrosine can increase dopamine levels, and this could be, at least in part, responsible for the impairment of energy metabolism found in the present study. ...
Tyrosinemia type II is a rare autosomal recessive disease caused by deficiency of hepatic tyrosine aminotransferase and is associated with neurologic and development difficulties in numerous patients. Considering that the mechanisms underlying the neurological dysfunction in hypertyrosinemic patients are poorly known and that high concentrations of tyrosine provoke mitochondrial dysfunction and oxidative stress, in the present study we investigated the in vivo influence of antioxidants (N-acetylcysteine, NAC; and deferoxamine, DFX) administration on the inhibitory effects on parameters of energy metabolism in cerebral cortex, hippocampus and striatum of rats, provoked by chronic administration of L.-tyrosine. Our results showed that chronic administration of L.-tyrosine results in a marked decrease in the activity of citrate synthase in all the analyzed structures and succinate dehydrogenase activities in hippocampus and striatum, and that antioxidants administration can prevent this inhibition in hippocampus and striatum. Moreover, chronic administration of L.-tyrosine inhibited the activity of complex I, II-III and IV in the striatum, which can be prevented by antioxidant treatment. However, the co-administration of NAC plus DFX could not prevent the inhibition of creatine kinase activity in the striatum. In conclusion, the present study demonstrates that the administration of antioxidants NAC and DFX attenuates the L.-tyrosine effects on enzymes of the Krebs cycle and the mitochondrial respiratory chain, suggesting that impairment of energy metabolism can be involved with oxidative stress. These results also indicate a possible neuroprotective role for NAC and DFX as a potential adjuvant therapy to the patients with Tyrosinemia type II.
... This suggests that phosphorylation mechanisms of thiamine in damaged organisms are deficient. Thus, neurodegenerative diseases frequently include abnormalities in glucose metabolism (Ibanez et al., 1998), with low levels of PDHC, KGDHC, CAT and glutathione system (Mizuno et al., 1994;Heroux et al., 1996) and supplementation therapy decreases and restores the damage (Shen et al., 2000). Moreover, the processes of 792 TPP in atrophic testicular changes insulin resistance and/or the central hypoinsulinemia with inflammation, overproduction of ROS and lipotoxicity, among others (Owen et al. 1996;Kidd, 2000;Orth and Schapira, 2001;Doherty, 2011;Schuh et al., 2011) have been implicated in various neurodegenerative disorders, and remain the centerpiece in the physiopathological development of most pathognomonic lesions found in Alzheimer 's and Parkinson's diseases, even in asymptomatic patients (Cholerton and Craft, 2011;de la Monte, 2012;Umegaki, 2012). ...
Aging is a multifactorial universal process and constitutes the most important risk factor for chronic-degenerative diseases. Although it is a natural process, pathological aging arises when these changes occur quickly and the body is not able to adapt. This is often associated with the generation of reactive oxygen species (ROS), inflammation, and a decrease in the endogenous antioxidant systems, constituting a physiopathological state commonly found in chronic-degenerative diseases. At the testicular level, aging is associated with tissue atrophy, decreased steroidogenesis and spermatogenesis, and sexual behavior disorders. This situation, in addition to the elevated generation of ROS in the testicular steroidogenesis, provides a critical cellular environment causing oxidative damage at diverse cellular levels. To assess the effects of a reduction in the levels of ROS, thiamine pyrophosphate (TPP) was chronically administered in senile Wistar rats. TPP causes an activation of intermediate metabolism routes, enhancing cellular respiration and decreasing the generation of ROS. Our results show an overall decrease of atrophic histological changes linked to aging, with higher levels of serum testosterone, sexual activity, and an increase in the levels of endogenous antioxidant enzymes in TPP-treated animals. These results suggest that TPP chronic administration decreases the progression of age-related atrophic changes by improving the intermediate metabolism, and by increasing the levels of antioxidant enzymes.
... It is well known that CysDA is an easily oxidizable molecule that may generate further oxidized molecules, one of which, 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid, the dihydrobenzothiazine-1, has been demonstrated to inhibit the pyruvate dehydrogenase and the a-ketoglutarate dehydrogenase complexes and is able to inhibit irreversibly complex I of the mitochondrial respiratory chain, altering the redox status of the cell Dryhurst, 1997, 2001;Shen et al., 2000). This evidence suggests that more toxic compounds may arise from Cys-DA autooxidation that could be the effective endogenous neurotoxins, which may contribute to oxidative stress, complex I damage, energy deficit, and neurodegeneration in SN of PD patients. ...
Parkinson's disease (PD) is a progressive neurodegenerative disorder whose etiology is still unclear in spite of extensive investigations. It has been hypothesized that 5-S-cysteinyldopamine (CysDA), a catechol-thioether metabolite of dopamine (DA), could be an endogenous parkinsonian neurotoxin. To gain further insight into its role in the neurodegenerative process, both CD1 mice and SH-SY5Y neuroblastoma cells were treated with CysDA, and the data were compared with those obtained by the use of 6-hydroxydopamine, a well-known parkinsonian mimetic. Intrastriatal injection of CysDA in CD1 mice caused a long-lasting depletion of DA, providing evidence of in vivo neurotoxicity of CysDA. Both in mice and in SH-SY5Y cells, CysDA treatment induced extensive oxidative stress, as evidenced by protein carbonylation and glutathione depletion, and affected the expression of two proteins, α-synuclein (α-Syn) and ERp57, whose levels are modulated by oxidative insult. Real-time PCR experiments support these findings, indicating an upregulation of both ERp57 and α-Syn expression. α-Syn aggregation was also found to be modulated by CysDA treatment. The present work provides a solid background sustaining the hypothesis that CysDA is involved in parkinsonian neurodegeneration by inducing extensive oxidative stress and protein aggregation. © 2013 Wiley Periodicals, Inc.
... Exposure of Chinese hampster ovary cells to hyperoxia results in cell death and complete inactivation of KGDHC, whereas KGDHC activity is preserved in cells that are resistant to hyperoxia (Schoonen et al. 1990Schoonen et al. , 1991). KGDHC is also inhibited by an oxidized derivative of dopamine (Shen et al. 2000) and other oxidants including hydroxynonenal, H 2 O 2 and peroxynitrite (Chinopoulos et al. 1999; Park et al. 1999; Gibson and Zhang 2002; Kumar et al. 2003). The inactivation of KGDHC by peroxynitrite is particularly interesting since peroxynitrite mediated neurotoxicity is strongly implicated in MPTP neurotoxicity (Schulz et al. 1995a; Przedborski et al. 1996 ). ...
The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.
... The inhibition requires oxidation of DHBT-1 by an unknown constituent of the inner mitochondrial membrane. The ability of glutathione to block the inhibition of KGDHC and PDHC by DHBT-1, without altering mitochondrial membrane-catalyzed oxidation of DHBT-1, suggests the oxidation product modifies the active site sulfhydryl residues (Fig. 4) (Shen et al., 2000). Norepinephrine neurons in the locus-ceruleus also die in PD. ...
Thiamine-dependent processes are diminished in brains of patients with several neurodegenerative diseases. The decline in thiamine-dependent enzymes can be readily linked to the symptoms and pathology of the disorders. Why the reductions in thiamine linked processes occur is an important experimental and clinical question. Oxidative stress (i.e. abnormal metabolism of free radicals) accompanies neurodegeneration and causes abnormalities in thiamine-dependent processes. The vulnerability of thiamine homeostasis to oxidative stress may explain deficits in thiamine homeostasis in numerous neurological disorders. The interactions of thiamine with oxidative processes may be part of a spiral of events that lead to neurodegeneration, because reductions in thiamine and thiamine-dependent processes promote neurodegeneration and cause oxidative stress. The reversal of the effects of thiamine deficiency by antioxidants, and amelioration of other forms of oxidative stress by thiamine, suggest that thiamine may act as a site-directed antioxidant. The data indicate that the interactions of thiamine-dependent processes with oxidative stress are critical in neurodegenerative processes.
... Thiocatechol adducts derived from quinoidal DA, DOPA, and DOPAC, elevated in PD brain [81], have received increased attention; they and their derived benzothiazines and mercapturates are neurotoxic [63,75]. MAO-mediated deamination of DA generates the aldehyde intermediate, dopaldehyde, which has had a modest history in PD research. ...
Idiopathic Parkinson's disease (PD), one of the most common neurodegenerative disorders associated with aging, is characterized neurochemically by abnormal and profound loss of nigrostriatal dopamine (DA) neurons. A prominent current view is that the excessive degeneration of the dopaminergic system is the outcome of extended insults by environmental neurotoxins or endogenous neurotoxic factors in genetically vulnerable or susceptible individuals. Recent insights into the identities and mechanisms of potential neurotoxic species, which span pesticides, environmental contaminants including heterocyclic amines with beta-carboline (betaC) and isoquinoline (IQ) structures, endogenous DA metabolites or intermediates, neuromelanin, metals, and infectious agents, are presented.
... Reactive oxygen species (ROS) produced during this biosynthetic pathway are known to contribute to tissue damage resulting from brain injury or ischemia (Sairanen et al., 1998). The production of ROS in this pathway may also contribute to dopamine oxidation, which has been implicated in the development of PD (Shen, Li, & Dryhurst, 2000). ROS may not be the only COX-2 products that are neurotoxic. ...
Neurodegenerative disorders have been reported to be associated with accumulation of ubiquitinated proteins in neuronal inclusions and also with signs of inflammation. In these disorders, the abnormal protein aggregates may, themselves, trigger the expression of inflammatory mediators, such as, cyclooxygenase 2 (COX-2). Impairment of the ubiquitin/proteasome pathway may contribute to this neurodegenerative process. Accordingly, proteasome inhibitors and oxidative stressors such as cadmium, were found to decrease survival, induce the accumulation of ubiquitinated proteins and elicit up-regulation of cyclooxygenase 2 in neuronal cell cultures. Products of cyclooxygenase 2, such as prostaglandin J2, can, in turn, increase the levels of ubiquitinated proteins and also cause cyclooxygenase 2 up-regulation, creating a "self-destructive" feedback mechanism. In neurodegenerative disorders characterized by neuronal inclusions containing ubiquitinated proteins, a disruption of the ubiquitin/proteasome pathway may, therefore, act in conjunction with cyclooxygenase 2 up-regulation to exacerbate the neurodegenerative process. Cyclooxygenase 2 inhibitors and agents that prevent protein aggregation could be of therapeutic value to these forms of neurodegeneration.
... Under these conditions, inhibition may arise from generation of ROS or through formation of DA-quinones, and 5-cysteinyldopamine metabolites. The potential importance of DA-quinones, 5-Scysteinyldopamine and the associated oxidized 5-S-cysteinyldopamine adduct, 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) in producing mitochondrial dysfunction is supported by findings showing inhibition of mitochondrial creatine kinase by DA-quinones (Van Laar et al. 2003) and a-ketoglutarate dehydrogenase and complex I of the electron transport chain by DHBT-1 (Shen et al. 2000). ...
A structure-potency study examining the ability of dopamine (DA), its major metabolites and related amine and acetate congeners to inhibit NADH-linked mitochondrial O(2) consumption was carried out to elucidate mechanisms by which DA could induce mitochondrial dysfunction. In the amine studies, DA was the most potent inhibitor of respiration (IC(50) 7.0 mm) compared with 3-methoxytryramine (3-MT, IC(50) 19.6 mm), 3,4-dimethoxyphenylethylamine (IC(50) 28.6 mm), tyramine (IC(50) 40.3 mm) and phenylethylamine (IC(50) 58.7 mm). Addition of monoamine oxidase (MAO) inhibitors afforded nearly complete protection against inhibition by phenylethylamine, tyramine and 3,4-dimethoxyphenylethylamine, indicating that inhibition arose from MAO-mediated pathways. In contrast, the inhibitory effects of DA and 3-MT were only partially prevented by MAO blockade, suggesting that inhibition might also arise from two-electron catechol oxidation and quinone formation by DA and one-electron oxidation of the 4-hydroxyphenyl group of 3-MT. In the phenylacetate studies, 3,4-dihydroxyphenylacetic acid (DOPAC) was equipotent with DA in inhibiting respiration (IC(50) 7.4 mm), further implicating the catechol reaction as the cause of inhibition. All other carboxylate congeners; phenylacetic acid (IC(50) 13.0 mm), 4-hydroxyphenylacetic acid (IC(50) 12.1 mm), 4-hydroxy-3-methoxyphenylacetic acid (HVA, IC(50) 12.0 mm) and 3,4-dimethoxyphenylacetic acid (IC(50) 10.2 mm), were equipotent respiratory inhibitors and two- to fourfold more potent than their corresponding amine. These latter findings suggest that the phenylacetate ion can also contribute independently to mitochondrial inhibition. In summary, mitochondrial respiration can be inhibited by DA and its metabolites by four distinct MAO-dependent and independent mechanisms.
... Peroxynitrite may also be involved in PD as shown by increases in 3-nitrotyrosine staining of Lewy bodies in the substantia nigra of patients with PD (Good et al. 1998;Giasson et al. 2000). KDGHC is also inhibited by oxidized derivatives of dopamine (Shen et al. 2000). Exposure of Chinese hamster ovary cells to hyperoxia results in cell death and complete inactivation of KDGHC, whereas KDGHC activity is preserved in cells that are resistant to hyperoxia (Schooner et al. 1990(Schooner et al. , 1991. ...
Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes alpha-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/-) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/- mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/- mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/- mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.
... Both proteins exhibited a strong decrease in fluorescence intensity following exposure of mitochondria to DAQ ( Figure 1D-E), with MtCK exhibiting the largest changes in relative fluorescence in Cys-CyDye 2-D DIGE gels. Other identified proteins whose relative fluorescence intensities were significantly reduced included several proteins of the Krebs cycle, including pyruvate carboxylase (−36±6%), succinate-CoA ligase (−43±4%), and oxoglutarate dehydrogenase (−49±5%), a protein previously shown to be inhibited by reactive metabolites of 5-cysteinyl-DA (Shen et al., 2000). ...
Oxidative stress and mitochondrial dysfunction have been linked to dopaminergic neuron degeneration in Parkinson disease. We have previously shown that dopamine oxidation leads to selective dopaminergic terminal degeneration in vivo and alters mitochondrial function in vitro. In this study, we utilized 2-D difference in-gel electrophoresis to assess changes in the mitochondrial proteome following in vitro exposure to reactive dopamine quinone. A subset of proteins exhibit decreased fluorescence labeling following dopamine oxidation, suggesting a rapid loss of specific proteins. Amongst these proteins are mitochondrial creatine kinase, mitofilin, mortalin, the 75 kDa subunit of NADH dehydrogenase, and superoxide dismutase 2. Western blot analyses for mitochondrial creatine kinase and mitofilin confirmed significant losses in isolated brain mitochondria exposed to dopamine quinone and PC12 cells exposed to dopamine. These results suggest that specific mitochondrial proteins are uniquely susceptible to changes in abundance following dopamine oxidation, and carry implications for mitochondrial stability in Parkinson disease neurodegeneration.
... 5-S-L-Cysteinyldopamine readily undergoes conversion in vitro to additional cysteine S-conjugates, two benzothiazines, and benzothiazine cysteine S-conjugates. Many of these metabolites are neu-rotoxic by interfering with brain metabolism [53][54][55][56][57][58][59][60][61]. Strong evidence suggests that 5-S-L-cysteinyldopamine is an important precursor of the pheomelanin component of the pigment neuromelanin in the substantia nigra [54,[62][63][64]. ...
Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate beta-lyase. The beta-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate beta-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and beta-lyase substrate of rhGTK. Moderate aminotransferase and beta-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The alpha-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed beta-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and beta-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.
In Drosophila, the same set of genes that are used for cuticle pigmentation and sclerotization are present in the nervous system and are responsible for neurotransmitter recycling. In this study, we carried out biochemical analysis to determine if insects have the enzymatic machinery to make melanic component of neuromelanin. We focused our attention on two key enzymes of melanogenesis, namely phenoloxidase and dopachrome decarboxylase/tautomerase. Activity staining of the proteins isolated from the Drosophila larval brain tissue, separated by native polyacrylamide gel electrophoresis indicated the presence of these two enzymes. Mass spectral sequence analysis of the band also supported this finding. To best of our knowledge, this is the first report on the presence of the enzymatic machinery to make melanin part of neuromelanin in any insect brain.
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Many factors appear to be involved in the neurotoxic mechanism that underlies the degeneration of neuromelanin-pigmented dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson’s disease (PD). These include: (1) a massive loss of glutathione (GSH) without corresponding increases of glutathione disulfide, a very early change in the parkinsonian SNpc; (2) increased intraneuronal superoxide (O
2-·) generation; (3) mobilization of low molecular weight iron from storage proteins, part of which is scavenged by neuromelanin; (4) increased activity of γ-glutamyl transpeptidase; (5) an accelerated rate of dopamine (DA) oxidation that does not lead to increased neuromelanin deposition but rather increased formation of 5-S-cysteinyldopamine (5-S-CyS-DA); and (6) decreased activities of mitochondrial complex I and α-ketoglutarate dehydrogenase (α-KGDH). Based on these changes and information derived from studies of the dopaminergic neurotoxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and methamphetamine, a new pathological mechanism is proposed that might contribute to an understanding of SNpc cell death in PD. This neurotoxic mechanism is suggested to be initiated by a transient dopaminergic neuron energy impairment that triggers a cascade of processes which ultimately result in the intraneuronal oxidation of DA by O
2-· in the presence of cysteine to form 5-S-CyS-DA. Further O
2-·mediated oxidation of 5-S-CyS-DA is proposed to lead to aberrant dihydrobenzothiazine and benzothiazine metabolites responsible for complex I and α-KGDH inhibition.
The past several decades have seen an outburst of research on dopaminergic neurotoxins that might underlie Parkinson’s disease (PD). These are summarized as endogenous, primarily dopamine (DA)-derived, and exogenous (environmental) agents. In some cases, important metabolic steps for neurotoxin activation, brain accumulation and selective uptake, i.e., oxidation (nitrogen or sulfur), N-methylation (quaternization) and/or conjugation routes, are necessitated.
Many potentially toxic electrophiles react with glutathione to form glutathione S-conjugates in reactions catalyzed or enhanced by glutathione S-transferases. The glutathione S-conjugate is sequentially converted to the cysteinylglycine-, cysteine- and N-acetyl-cysteine S-conjugate (mercapturate). The mercapturate is generally more polar and water soluble than the parent electrophile and is readily excreted. Excretion of the mercapturate represents a detoxication mechanism. Some endogenous compounds, such as leukotrienes, prostaglandin (PG) A2, 15-deoxy-?12,14-PGJ2, and hydroxynonenal can also be metabolized to mercapturates and excreted. On occasion, however, formation of glutathione S- and cysteine S-conjugates are bioactivation events as the metabolites are mutagenic and/or cytotoxic. When the cysteine S-conjugate contains a strong electron-withdrawing group attached at the sulfur, it may be converted by cysteine S-conjugate ?-lyases to pyruvate, ammonium and the original electrophile modified to contain an -SH group. If this modified electrophile is highly reactive then the enzymes of the mercapturate pathway together with the cysteine S-conjugate ?-lyases constitute a bioactivation pathway. Some endogenous halogenated environmental contaminants and drugs are bioactivated by this mechanism. Recent studies suggest that coupling of enzymes of the mercapturate pathway to cysteine S-conjugate ?-lyases may be more common in nature and more widespread in the metabolism of electrophilic xenobiotics than previously realized.
Many potentially toxic electrophilic xenobiotics and some endogenous compounds are detoxified by conversion to the corresponding glutathione S-conjugate, which is metabolized to the N-acetylcysteine S-conjugate (mercapturate) and excreted. Some mercapturate pathway components, however, are toxic. Bioactivation may occur when the glutathione S-conjugate (or mercapturate) is converted to a cysteine S-conjugate that undergoes a β-lyase reaction. If the sulfhydryl-containing fragment produced in this reaction is reactive, toxicity may ensue. Some drugs and halogenated environmental contaminants are bioactivated by this mechanism. On the other hand, cysteine S-conjugate β-lyases occur in nature as a means of generating biologically useful sulfhydryl-containing compounds.
Dopamine (DA) is the prevalent catecholaminergic neurotransmitter in the brain and dopaminergic neurons are mainly located
in the substantia nigra (SN), the ventral tegmental area (VTA), and arcuate nuclei. They constitute at least three important
dopaminergic pathways: the nigro-striatal, the mesocortical-mesolimbic, and the tubero-infundibular system, which are responsible
for locomotor movement, motivation and reward, and secretion of pituitary gland hormones, respectively (1,2). The dysfunction of these systems can lead to neurological, psychological and endocrinological diseases like, e.g., Parkinson’s
disease (PD), attention deficit hyperactivity disorder (ADHD), and prolactinoma (3). There is increasing evidence that DA, besides acting as a neurotransmitter, can become neurotoxic at high concentrations
or in an oxidative environment. Especially in idiopathic PD, even though the SN is not the only affected region in the brain,
the main pathological hallmark is the progressive degeneration of the neuromelanin-containing dopaminergic neurons in the
SN pars compacta (SNpc) causing the cardinal symptoms of tremor, rigidity, and bradykinesia. It is believed that the interplay
between the high DA content and a local pro-oxidative environment in the SN mutually promote the increase of oxidative stress
and thus the degeneration of dopaminergic neurons.
This review summarizes current knowledge on glutathione (GSH) associated cellular processes that play a central role in defense against oxidative stress. GSH itself is a critical factor in maintaining the cellular redox balance and has been demonstrated to be involved in regulation of cell signalling and repair pathways. Enhanced expression of various enzymes involved in GSH metabolism, including glutathione peroxidases, γ-glutamyl cysteinyl synthetase (γ-GCS), glutathione S-transferases (GST) and membrane proteins belonging to the ATP-binding cassette family, such as the multidrug resistance associated protein, have all been demonstrated to play a prominent role in cellular resistance towards oxidative stress. This review stresses the fact that aco-ordinateinterplay between these systems is essential for efficient protection against oxidative stress.
Parkinson's disease (PD) is the second most common form of neurodegeneration in the elderly population. PD is clinically characterized by tremors, rigidity, slowness of movement and postural imbalance. A significant association has been demonstrated between PD and low levels of thiamine in the serum, which suggests that elevated thiamine levels might provide protection against PD. Genetic studies have helped identify a number of factors that link thiamine to PD pathology, including the DJ-1 gene, excitatory amino acid transporters (EAATs), the α-ketoglutarate dehydrogenase complex (KGDHC), coenzyme Q10 (CoQ10 or ubiquinone), lipoamide dehydrogenase (LAD), chromosome 7, transcription factor p53, the renin-angiotensin system (RAS), heme oxygenase-1 (HO-1), and poly(ADP-ribose) polymerase-1 gene (PARP-1). Thiamine has also been implicated in PD through its effects on L-type voltage-sensitive calcium channels (L-VSCC), matrix metalloproteinases (MMPs), prostaglandins (PGs), cyclooxygenase-2 (COX-2), reactive oxygen species (ROS), and nitric oxide synthase (NOS). Recent studies highlight a possible relationship between thiamine and PD. Genetic studies provide opportunities to determine which proteins may link thiamine to PD pathology. Thiamine can also act through a number of non-genomic mechanisms that include protein expression, oxidative stress, inflammation, and cellular metabolism. Further studies are needed to determine the benefits of using thiamine as a treatment for PD.
Aberrant oxidative pathways of catecholamine neurotransmitters, i.e. dopamine and norepinephrine, are an important biochemical correlate of catecholaminergic neuron loss in some disabling neurodegenerative diseases of the elderly, notably Parkinson's disease. In an oxidative stress setting, under conditions of elevated lipid peroxidation, iron accumulation, impaired mitochondrial functioning and antioxidant depletion, catecholamines are oxidatively converted to the corresponding o-quinones, which may initiate a cascade of spontaneous reactions, including intramolecular cyclization, aminoethyl side chain fission and interaction with molecular targets. The overall outcome of the competing pathways may vary depending on contingent factors and the biochemical environment, and may include formation of nitrated derivatives, neuromelanin deposition, generation of chain fission products, conjugation with L-cysteine leading eventually to cytotoxic responses and altered cellular function. In addition, catecholamines may interact with products of lipid peroxidation and other species derived from oxidative breakdown of biomolecules, notably glyoxal and other aldehydes, leading e.g. to tetrahydroisoquinolines via Pictet-Spengler chemistry. After a brief introductory remark on oxidative stress biochemistry, the bulk of this review will deal with an overview of the basic chemical pathways of catecholamine oxidation, with special emphasis on the analogies and differences between the central neurotransmitters dopamine and norepinephrine. This chemistry will form the basis for a concise discussion of the latest advances in the mechanisms of catecholamine-associated neurotoxicity in neuronal degeneration.
Heidelberg, Univ., Diss., 2004 (Nicht für den Austausch).
Trichochromes, the peculiar pigments of red human hair, featuring the Delta(2,2)(')-bi(2H-1,4-benzothiazine) skeleton, are known to arise from cysteinyldopas, mainly the 5-S-isomer (5). However, the mode of formation and the direct precursors have remained largely undefined. To fill this gap, we investigated the oxidation of 5 in air or with chemical and enzymatic agents under biomimetic conditions. In the presence of zinc ions, which occur in epidermal tissues at significant concentrations, the reaction course is diverted toward the formation of a labile 3-carboxy-2H-1,4-benzothiazine intermediate (11), which was identified by direct NMR analysis. Structural formulation was supported by characterization of the analogous compound 13 isolated from oxidation of the model 5-methyl-3-S-cysteinylcatechol (12) after methylation. In the further stages of the oxidation, diastereomeric 2,2'-bi(2H-1,4-benzothiazine) 15 and 14 were obtained from 5 and 12, respectively, the reaction proceeding at a higher rate and to a greater extent in the presence of acids. The dimers were shown to readily convert to each other in the presence of acids. In the case of the methylated dimers 14, a 2,2'-bi(4H-1,4-benzothiazine) intermediate (16) was isolated and characterized. In acidic media, trichochrome C (1a), the most abundant in red human hair, was smoothly formed from aerial oxidation of 15, and under similar conditions, trichochrome-related products (17 and 18) were obtained from 14 prior to or after methylation. The presence of 1a and precursors 5 and 15 was investigated by HPLC analysis of red hair samples following mild proteolytic digestion. On the basis of these data, a likely biosynthetic route to trichochrome pigments of red human hair is depicted.
During the past two or three decades an immense amount of research has been carried out to understand the fundamental molecular mechanisms that underlie the neurotoxicity evoked by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), methamphetamine (MA) and related amphetamine drugs of abuse. The neurotoxicity of MPTP is of particular interest because it not only mimics the symptoms and major pathobiochemical changes that occur in Parkinson’s disease (PD) but also arguably provides the best model of PD in animals (Gerlach et al.,1991; Gerlach and Riederer, 1996; Royland and Langston, 1998). Interest in the neurotoxicity evoked by MA derives not only from the fact that this compound is a widely abused psychoactive drug but in animals it also mimics many of the major pathobiochemical changes that occur in PD (Gerlach and Riederer, 1996) although it is a less selective neurotoxin than MPTP. Transient cerebral ischemia, or ischemia-reperfusion (I-R), can also lead to neurodegeneration although this is even less selective than MA. Nevertheless, there are a rather striking number of similar factors that appear to be key steps in a complex cascade of processes that compromise the neurotoxic mechanisms evoked by MPTP, MA and I-R. In this communication similarities associated with the neurotoxicity evoked by MPTP, MA and I-R will be briefly reviewed. Subsequently, these will be integrated into a working hypothesis for the underlying neurotoxic mechanisms in which one or more of the neurotransmitters dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT; serotonin) are proposed to be the precursors of biologically reactive intermediates in the pathological processes.
The principal neuropathological feature of Parkinson's disease is the degeneration of melanized dopamine neurons in the substantia nigra pars compacta (SNc). Characteristic pathobiochemical changes in the parkinsonian SNc include a fall of both dopamine (DA) and glutathione levels (GSH), increased activity of gamma-glutamyl transpeptidase, a key enzyme involved in the degradation of GSH to L-cysteine (CySH), together with evidence for elevated intraneuronal superoxide (O2-*), nitric oxide (NO.) and thence peroxynitrite (ONOO-) generation, and accelerated DA oxidation as indicated by a large rise of the 5-S-cysteinyldopamine (5-S-CyS-DA)/DA concentration ratio. The latter effect is consistent with an increased rate of DA oxidation by O2-* and ONOO- forming DA-o-quinone which reacts with CySH forming 5-S-CyS-DA. However, 5-S-CyS-DA is readily further oxidized to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1). Previous studies have demonstrated that DHBT-1 is rapidly accumulated by isolated intact rat brain mitochondria and selectively inhibits complex I respiration and the alpha-ketoglutarate dehydrogenase (alpha-KGDH) complex. In this study it is demonstrated that DHBT-1 also inhibits the pyruvate dehydrogenase complex (PDHC). The mechanism underlying the inhibition of all of these enzyme complexes involves bioactivation of intramitochondrial DHBT-1 by oxidation to highly electrophilic metabolites that covalently bind to active site cysteine residues. Thus, oxidative metabolites of intraneuronal 5-S-CyS-DA may contribute to impaired mitochondrial complex I and alpha-KGDH activities known to occur in the parkinsonian SNc and suggest that impaired PDHC evoked by the same metabolites may also occur in PD.
Impairments of glucose and mitochondrial function are important causes of brain dysfunction and therefore of brain disease. Abnormalities have been found in association with disease of the nervous system in most of the components of glucose/mitochondrial metabolism. In many, molecular genetic abnormalities have been defined. Brain glucose oxidation is abnormal in common diseases of the nervous system, including Alzheimer disease and other dementias, Parkinson disease, delirium, probably schizophrenia and other psychoses, and of course cerebrovascular disease. Defects in a single component and even a single mutation can be associated with different clinical phenotypes. The same clinical phenotype can result from different genotypes. The complex relationship between biological abnormality in brain glucose utilization and clinical disorder is similar to that in other disorders that have been intensively studied at the genetic level. Genes for components of the pathways of brain glucose oxidation are good candidate genes for disease of the brain. Preliminary data support the proposal that treatments to normalize abnormalities in brain glucose oxidation may benefit many patients with common brain diseases.
Parkinson's disease (PD) is associated with mitochondrial dysfunction, specifically a deficiency of complex I of the electron transport chain. Most, although not all, studies indicate that this deficiency is limited to brain regions with neurodegeneration. The current studies tested for deficiencies in other mitochondrial components in PD brain in a neuropathologically unaffected region where the abnormality cannot be attributed to secondary effects of neurodegeneration. The activity of a key (and arguably rate-limiting) tricarboxylic acid cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), was measured in the cerebellum of patients with PD. Activity in 19 PD brains was 50.5% of that in 18 controls matched for age, sex, post-mortem interval, and method of preservation (P<0.0019). The protein subunits of KGDHC were present in normal amounts in PD brains, indicating a relatively discrete abnormality in the enzyme. The activities of another mitochondrial enzyme, glutamate dehydrogenase (GDH), were normal in PD brains. These results demonstrate that specific reductions in KGDHC occur even in pathologically unaffected areas in PD, where the decline is unlikely to be a non-specific result of neurodegeneration. Reductions in the activity of this enzyme, if widespread in the brain, may predispose vulnerable regions to further damage.
Oxidative stress contributes to the cascade leading to dopamine cell degeneration in Parkinson's disease (PD). However, oxidative stress is intimately linked to other components of the degenerative process, such as mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity and inflammation. It is therefore difficult to determine whether oxidative stress leads to, or is a consequence of, these events. Oxidative damage to lipids, proteins, and DNA occurs in PD, and toxic products of oxidative damage, such as 4-hydroxynonenal (HNE), can react with proteins to impair cell viability. There is convincing evidence for the involvement of nitric oxide that reacts with superoxide to produce peroxynitrite and ultimately hydroxyl radical production. Recently, altered ubiquitination and degradation of proteins have been implicated as key to dopaminergic cell death in PD. Oxidative stress can impair these processes directly, and products of oxidative damage, such as HNE, can damage the 26S proteasome. Furthermore, impairment of proteasomal function leads to free radical generation and oxidative stress. Oxidative stress occurs in idiopathic PD and products of oxidative damage interfere with cellular function, but these form only part of a cascade, and it is not possible to separate them from other events involved in dopaminergic cell death.
The antioxidant glutathione (GSH) is essential for the cellular detoxification of reactive oxygen species in brain cells. A compromised GSH system in the brain has been connected with the oxidative stress occuring in neurological diseases. Recent data demonstrate that besides intracellular functions GSH has also important extracellular functions in brain. In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons. Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multidrug resistance protein 1 participates in both the export of GSH and GSSG from astrocytes. This review focuses on recent results on the export of GSH and GSSG from brain cells as well as on the functions of extracellular GSH in the brain. In addition, implications of disturbed GSH pathways in brain for neurodegenerative diseases will be discussed.
The effects of increasing mitochondrial oxidative phosphorylation (OXPHOS), by enhancing electron transport chain components, were evaluated on 1-methyl-4-phenylpyridinium (MPP+) toxicity in brain neuroblastoma cells. Although glucose is a direct energy source, ultimately nicotinamide and flavin reducing equivalents fuel ATP produced through OXPHOS. The findings indicate that cell respiration/mitochondrial O(2) consumption (MOC) (in cells not treated with MPP+) is not controlled by the supply of glucose, coenzyme Q(10) (Co-Q(10)), NADH+, NAD or nicotinic acid. In contrast, MOC in whole cells is highly regulated by the supply of flavins: riboflavin, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), where cell respiration reached up to 410% of controls. In isolated mitochondria, FAD and FMN drastically increased complex I rate of reaction (1300%) and (450%), respectively, having no effects on complex II or III. MPP+ reduced MOC in whole cells in a dose-dependent manner. In isolated mitochondria, MPP+ exerted mild inhibition at complex I, negligible effects on complexes II-III, and extensive inhibition of complex IV. Kinetic analysis of complex I revealed that MPP+ was competitive with NADH, and partially reversible by FAD and FMN. Co-Q(10) potentiated complex II ( approximately 200%), but not complex I or III. Despite positive influence of flavins and Co-Q(10) on complexes I-II function, neither protected against MPP+ toxicity, indicating inhibition of complex IV as the predominant target. The nicotinamides and glucose prevented MPP+ toxicity by fueling anaerobic glycolysis, evident by accumulation of lactate in the absence of MOC. The data also define a clear anomaly of neuroblastoma, indicating a preference for anaerobic conditions, and an adverse response to aerobic. An increase in CO(2), CO(2)/O(2) ratio, mitochondrial inhibition or O(2) deprivation was not directly toxic, but activated metabolism through glycolysis prompting depletion of glucose and starvation. In conclusion, the results of this study indicate that the mechanism of action for MPP+, involves the inhibition of complex I and and more specifically complex IV, leading to impaired OXPHOS and MOC. Moreover, flavin dervatives control the rate of complex I/cellular respiration and Co-Q10 augments complex II [corrected].
In 0.1 M phosphate buffer, pH 7.4, dopamine reacts with glyoxal, a cytotoxic and genotoxic alpha-oxoaldehyde produced by oxidative degradation of carbohydrates, to give three main products, two of which could be isolated and identified as the isomeric tetrahydrobiisoquinolines 1 and 2 by extensive two-dimensional NMR and mass spectrometric analysis. Time course studies indicated that 1 is the first intermediate in the process and changes slowly to 2 via an unstable species that escaped all efforts at isolation and structural identification. Products 1 and 2 were detected also among the species formed by the interaction of dopamine with oxidized carbohydrates, such as glucose, ribose, and fructose. Mechanistic evidence suggests that the formation of 1 proceeds by an unusual reaction pathway involving intramolecular cyclization of a double Schiff base intermediate followed by glyoxal-induced oxidation of the resulting octahydrobiisoquinoline intermediate (4). Subsequent conversion of 1 to 2 would involve a complex redox mechanism depending on an initial oxidation step. Product 2 was only poorly toxic to PC12 cells, whereas its methylated derivative 3 was as toxic as salsolinol, an established neurotoxin. Overall, these results throw light on a novel pathway of dopamine modification of potential relevance to the mechanisms underlying neurodegenerative changes in Parkinson's disease and other disorders characterized by a prooxidant state.
Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP+ (1-methyl-4-phenylpyridinium) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like syndromes in humans and animals. MPTP/MPP+ treatment produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been elucidated. In order to gain insights into MPP+-induced neurotoxicity, a gene expression microarray study was performed using a midbrain-derived dopaminergic neuronal cell line, MN9D. Utilizing a two-color reference design, Agilent mouse oligonucleotide microarrays were used to examine relative gene expression changes in MN9D cells treated with 40microM MPP+ compared with controls. Bioinformatics tools were used for data evaluation. Briefly, raw data were imported into the NCTR ArrayTrack database, normalized using a Lowess method and data quality was assessed. The Student's t-test was used to determine significant changes in gene expression (set as p<0.05, fold change >1.5). Gene Ontology for Function Analysis (GOFFA) and Ingenuity Pathway Analysis were employed to analyze the functions and roles of significant genes in biological processes. Of the 51 significant genes identified, 44 were present in the GOFFA or Ingenuity database. These data indicate that multiple pathways are involved in the underlying mechanisms of MPP+-induced neurotoxicity, including apoptosis, oxidative stress, iron binding, cellular metabolism, and signal transduction. These data also indicate that MPP+-induced toxicity shares common molecular mechanisms with the pathogenesis of PD and further pathway analyses will be conducted to explore these mechanisms.
Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.
Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP+ (1-methyl-4-phenylpyridinium ion) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like symptoms in humans and animals. MPTP/MPP+ produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been fully elucidated. Recently, we reported in a microarray study using a midbrain-derived dopaminergic neuronal cell line, MN9D, that MPP+ induced significant changes in a number of genes known to be associated with the dopaminergic system. In this study, we investigated the expression time courses of six genes using real-time RT-PCR, and compared them with the progressive dopaminergic depletion caused by MPP+. Our data showed that dopamine content was significantly decreased after 0.5h of MPP+ (200 microM) exposure and was completely depleted after 40 h. The expression of Gpr37, which is closely related to the pathogenesis of autosomal recessive juvenile Parkinsonism, was up-regulated after 0.5h, and stayed up-regulated up to 48 h. Txnip, which is critical to the adjustment of cellular redox status, was down-regulated after 1h and stayed down-regulated up to 48 h. Ldh1 and Cdo1, which are also involved in oxidative stress, were down-regulated after 16 h and stayed down-regulated up to 48 h. Two pro-apoptotic genes, Egln3 and Bnip3, were down-regulated after 2 and 4h, and stayed down-regulated up to 48 h. These findings suggested that the time course of expression for multiple genes correlated with the dopaminergic depletion; and MPP+-induced neurotoxicity in MN9D cells could be used as a model to further explore the roles of these and other genes in the pathogenesis and possible treatment of PD.
Cysteinyldopas are naturally occurring conjugates of cysteine and dopa (3,4-dihydroxy-l-phenylalanine) that are precursors to red pheomelanin pigments. Metal ions are known to influence pheomelanogenesis in vitro and may be regulatory factors in vivo. Cydo (3-[(2-amino-ethyl)sulfanyl]-4,6-di-tert-butylbenzene-1,2-diol) and CarboxyCydo (2-amino-3-(4,6-di-tert-butyl-2,3-dihydroxyphenylsulfanyl)-propionic acid) are model compounds of cysteinyldopa that retain its metal-binding functionalities but cannot polymerize due to the presence of blocking tert-butyl groups. Cydo reacts readily with zinc(II) acetate or nickel(II) acetate to form a cyclized 1,4-benzothiazine (zine) intermediate that undergoes ring contraction to form benzothiazole (zole) unless it is stabilized by coordination to a metal ion. The crystal structure of [Ni(zine)2] is reported. The acetate counteranion is required for the zinc-promoted reactivity, as neither zinc(II) sulfate nor zinc(II) chloride alone promotes the transformation. The counterion is less important for redox-active copper and iron, which both readily promote the oxidation of Cydo to zine and zole species; Cu(II) complexes of both zine and zole have been characterized by X-ray crystallography. In the case of CarboxyCydo, a 3-carboxy-1,4-benzothiazine intermediate decarboxylates to form [Cu(zine)2] under basic conditions, but in the absence of base forms a mixture of products that includes the carboxylated dimer 2,2'-bibenzothiazine (bi-zine). These products are consistent with species implicated in the pheomelanogenesis biosynthetic pathway and emphasize how metal ions, their counteranions, and reaction conditions can alter pheomelanin product distribution.
The cause of Parkinson's disease (PD) is currently unknown. Although a genetic cause has been implicated in familial PD, the vast majority of cases are considered idiopathic. Environmental toxins have been implicated as a cause for PD by many investigators. Unfortunately, the magnitude of this exposure would likely need to be very high and as a result, would likely have been identified by the many epidemiological studies performed to date. Recently, we inadvertently realized that exposure to neurotoxins while still in utero may also represent a risk factor. Thus, exposure to the bacteriotoxin, lipopolysaccharide (LPS) during a critical developmental window in rats, leads to the birth of animals with fewer than normal dopamine (DA) neurons. This DA neuron loss is apparently permanent as it is still present in 16 months old animals (the longest period studied to date). Moreover, the loss of DA neurons seen in these animals increases with age thereby mimicking the progressive pattern of cell loss seen in human PD. The DA neuron loss is accompanied by reductions in striatal DA, increases in DA activity, and increased production of the pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). These are also characteristics of the PD brain. This model therefore shares many of the same characteristics with PD, and most importantly exhibits a slow, protracted loss of DA neurons - a characteristics of this animal model not found in other models. Interestingly, a common complication of pregnancy is a condition known as bacterial vaginosis (BV), which is known to produce increased levels of LPS and pro-inflammatory cytokines in the chorioamniotic environment of the fetus. This raises the interesting possibility that BV may be a risk factor for PD. The possibility that prenatal toxin exposure may contribute to the development of a neurodegenerative disease of the aged raises interesting new pathogenic questions and draws attention to the possibility that in utero exposure to neurotoxins may represent a here to fore unrecognized cause of PD.
We report in this paper that highly purified Escherichia coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase are inactivated by O2- with second order rate constants in the range of 10(6) to 10(7) M-1 s-1. Each of these enzymes belongs to the hydrolyase class and contains catalytically active [4Fe-4S] clusters. Simultaneous with inactivation by O2- is the release of iron from their clusters. Our working hypothesis is O2- inactivates these enzymes by oxidizing their clusterS to an unstable oxidation state, and cluster degradation follows. Consistent with this hypothesis is our observation that spinach dihydroxy-acid dehydratase, a member of the hydro-lyase class that has a catalytically active [2Fe-2S] cluster, is not inactivated and does not lose iron in the presence of O2-. Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is also not inactivated by O2-. We also report the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1. E. coli dihydroxy-acid dehydratase has been reported previously to be inactivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions. We report here that E. coli fumarase A, fumarase B, and aconitase are also inactivated in vivo by hyperbaric O2. Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated.
The substrate and cofactor requirements and some kinetic properties of the α-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for α-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg²⁺ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex > cerebellum ≧ midbrain > striatum = hippocampus > hypothalamus > pons and medulla > olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2–2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both β-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 μM, calcium slightly stimulated (by 10–15%) the brain mitochondrial KGDHC.
Permanent parkinsonism was observed in a man with chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate). Symptoms developed at 37 years of age, two years after exposure had ceased. To our knowledge, this is the second report on parkinsonism associated with exposure to maneb. Manganese is a well-known parkinsonigen toxin in humans. More recently, it has been shown that dithiocarbamates can also induce extrapyramidal syndromes. The biochemical effects of manganese and dithiocarbamates are reviewed and their possible neurotoxic mechanisms are discussed. Both of these components may have played a role in this case.
The mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I), which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD). The origin of this lesion and its role in the neurodegeneration of PD are unknown. To address these questions, we created an in vitro system in which the potential contributions of environmental toxins, complex I nuclear DNA mutations, and mitochondrial DNA mutations could be systematically analyzed. A clonal line of human neuroblastoma cells containing no mitochondrial DNA was repopulated with mitochondria derived from the platelets of PD or control subjects. After 5 to 6 weeks in culture, these cytoplasmic hybrid (cybrid) cell lines were assayed for electron transport chain activities, production of reactive oxygen species, and sensitivity to induction of apoptotic cell death by 1-methyl-4-phenyl pyridinium (MPP+). In PD cybrids we found a stable 20% decrement in complex I activity, increased oxygen radical production, and increased susceptibility to 1-methyl-4-phenyl pyridinium-induced programmed cell death. The complex I defect in PD appears to be genetic, arising from mitochondrial DNA, and may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins.
A change in drug clearance with age is considered an important factor in determining the high prevalence of adverse drug reactions associated with prescribing medications for the elderly. Despite this, no general principles have been available to guide drug administration in the elderly, although a substantial body of clearance and metabolism data has been generated in humans and experimental animals. A review of age-related change in drug clearances established that patterns of change are not simply explained in terms of hepatic blood flow, hepatic mass and protein binding changes. In particular, the maintained clearance of drugs subject to conjugation processes while oxygen-dependent metabolism declines, and all in vitro tests of enzyme function have been normal, requires new explanations.
Reduction in hepatic oxygen diffusion as part of a general change in hepatocyte surface membrane permeability and conformation does provide one explanation for the paradoxical patterns of drug metabolism, and increased hepatocyte volume would also modify oxygen diffusion path lengths (the ‘oxygen diffusion barrier’ hypothesis). The reduction in clearances of high extraction drugs does correlate with observed reduction in hepatic perfusion.
Dosage guidelines emerge from these considerations. The dosage of high clearance drugs should be reduced by approximately 40% in the elderly while the dosage of low clearance drugs should be reduced by approximately 30%, unless the compound is principally subject to conjugation mechanisms. If the hepatocyte diffusion barrier hypothesis is substantiated, this concept may lead to therapeutic (preventative and/or restorative) approaches to increased hepatocyte oxygenation in the elderly. This may lead to approaches for modification of the aging process in the liver.
In a combined biochemical and histochemical investigation the following quantitative data on a unilateral nigro-neostriatal dopamine neuron system in the rat have been obtained or calculated. — The number of neurons is 3,500 on the average. — There are about 120 pg dopamine per neuron (1 pg = 10-12 g). — The terminals contain 50 (or 150) times more dopamine than the cell bodies. —The terminal system of one neuron varies mainly between 55 and 77 cm in length and contains about 500,000 varicosities. — The neostriatum, with a volume of 33 mm3, contains 109 dopamine varicosities occupying 0.3 % of the volume, and 4,000,000 nerve cell bodies (not containing dopamine). — One average dopamine varicosity, with a diameter of 0.4 μ, contains 2.5 × 10-4 pg dopamine, and has a dopamine concentration around 8,000 μg/g. — One dopamine cell body of the substantia nigra, with a mean diameter of 30 μ, contains 2.5 (or 0.8) pg dopamine, and has a dopamine concentration around 200 (or 60) μg/g.
: The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on ATP levels in different areas of mouse brain were studied after rapid fixation of cerebral tissue in situ by microwave irradiation. ATP levels in the striatum, ventral mesencephalon, and cerebellum of untreated C57BL/6 mice killed by microwave irradiation were 2-3 times greater than values measured in the brains of animals killed by cervical dislocation. In microwaved mice, administration of MPTP (40 mg/kg s.c.) caused a 10-20% decrease in ATP concentrations as compared to control animals injected with saline. This decrease was relatively rapid and selective because it occurred in both the striatum and ventral mesencephalon, but not in the cerebellar and frontal cortex, at 30, 60, 120, and 240 min after MPTP exposure. Furthermore, ATP loss in the striatum was prevented by mazindol, a catecholamine uptake blocker, indicating a rather selective effect of MPTP on the ATP content of dopaminergic terminals. Results of this study are consistent with mitochondrial damage in the MPTP model of parkinsonism and provide the first direct experimental evidence in vivo that a decrease in ATP may play a role in MPTP-induced neurotoxicity.
: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between l-3,4-dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.
Dihydrolipoamide succinyltransferase (E2, EC 2.3.1.61, chromosome 14q24.2-3) is a specific subunit of human alpha-ketoglutarate dehydrogenase complex (KGDHC). A biallelic intragenic polymorphism was identified in E2 gene of KGDHC. It was a single nucleotide substitution between G (in allele 1) and A (in allele 2) at the position that does not change amino acid code. Using this intragenic polymorphism as a marker, we investigated the association between this gene and Parkinson's disease. Frequencies of the genotypes that carry allele 2 were significantly higher in the Parkinson's disease group than in the control group. The results indicated that a genetic variant of the E2 gene itself or in close proximity to the gene constitutes one of the genetic risk factors for Parkinson's disease.
We report an immunohistochemical study of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) in the substantia nigra in Parkinson's disease. The KGDHC, the three enzyme complex catalyzing the oxidation of alpha-ketoglutarate to succinate through succinic semialdehyde, is the rate-regulating enzyme of the TCA cycle. The mitochondrial toxin, MPP+, inhibits not only complex I but also the KGDHC. Therefore, we investigated this enzyme complex in Parkinson's disease. In the control patients (n = 6), the immunostaining of the melanized nigral neurons was generally uniform; most of the melanized neurons showed good immunostaining with some neurons showing somewhat reduced staining. In Parkinson's disease (n = 9), many melanized neurons showed reduced immunostaining for the KGDHC, and those neurons were more frequently seen in the lateral one-third of substantia nigra. The decrease in the immunostaining for the KGDHC correlated roughly with the severity of degeneration. The KGDHC is more vulnerable to degeneration than complex II, III, and IV as noted in our previous immunohistochemical study. Even if secondary, the loss may play a role in the progression of the disease.
To include weak electron spin rovibronic and nuclear interaction other than electrostatic interaction we invoke the coupling of the spin rovibronic states of the molecular fragments as they form Van der Waals complexes. It is shown how concerted and cooperative rotation/vibration, etc. will preserve the relative geometry and potential for the complex. Stateto-state symmetry correlations, including the Fermion symmetry of protons are performed for the formation of a hypothetical dimer of C2h symmetry from a monomer ABH3 of C3v symmetry, and for the formation of a hypothetical complex of C3v symmetry from ABH3 coupled with C6H6 Cor C6F6) of D6h symmetry.
We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-d-aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and l-glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm3), as did coinjection of malonate with S-AMPA (11.0 ± 1.0 mm3) or glutamate (12.8 ± 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm3). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 ± 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by ∼40% (7.5 ± 0.9 mm3). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm3, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 ± 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.
Oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l-DOPA, DA, and other catecholamines to a system generating O2•− radical led to O2•−-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l-DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l-DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l-DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l-DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l-DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.
Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured in various brain areas (substantia nigra, putamen, caudate nucleus, globus pallidus, and cerebral cortex) from patients dying with Parkinson's disease, progressive supranuclear palsy, multiple-system atrophy, and Huntington's disease and from control subjects with no neuropathological changes in substantia nigra. GSH levels were reduced in substantia nigra in Parkinson's disease patients (40% compared to control subjects) and GSSG levels were marginally (29%) but insignificantly elevated; there were no changes in other brain areas. The only significant change in multiple-system atrophy was an increase of GSH (196%) coupled with a reduction of GSSG (60%) in the globus pallidus. The only change in progressive supranuclear palsy was a reduced level of GSH in the caudate nucleus (51%). The only change in Huntington's disease was a reduction of GSSG in the caudate nucleus (50%). Despite profound nigral cell loss in the substantia nigra in Parkinson's disease, multiple-system atrophy, and progressive supranuclear palsy, the level of GSH in the substantia nigra was significantly reduced only in Parkinson's disease. This suggests that the change in GSH in Parkinson's disease is not solely due to nigral cell death, or entirely explained by drug therapy, for multiple-system atrophy patients were also treated with levodopa. The altered GSH/GSSG ratio in the substantia nigra in Parkinson's disease is consistent with the concept of oxidative stress as a major component in the pathogenesis of nigral cell death in Parkinson's disease.
Recent studies suggest that elevated rates of autoxidation of dopamine (DA) in the cytoplasm of neuromelanin-pigmented dopaminergic cell bodies in the substantia nigra (SN) and/or reactions of its proximate oxidation product, DA-o-quinone, with glutathione (GSH) or L-cysteine (CySH) might yield endotoxins that play roles in the pathogenesis of idiopathic Parkinson′s Disease (PD). In this study the reactions between DA-o-quinone and CySH and some cysteine derivatives have been studied. The reactions between DA-o-quinone and CySH or cysteine methyl ester are rapid and give the corresponding 5-S-cysteinyl conjugates of DA as the predominant products. By contrast, the reaction between the sterically hindered D-penicillamine methyl ester (PME) and DA-o-quinone is much slower to give, initially, a mixture of the 2-S-, 5-S-, and 6-S-PME conjugates of DA. These conjugates are then further oxidized by unreacted DA-o-quinone to give a complex mixture of products which include 7-(2-aminoethyl)-5-hydroxy-2,2-dimethyl-1,4-benzothiazine-3-carboxylic acid methyl ester (13). Large increases in the 5-S-cysteinyldopamine (5-S-CyS-DA)/DA concentration ratio have been measured in the Parkinsonian SN and have been interpreted to reflect elevated rates of DA autoxidation in this structure that degenerates in PD. However, the present study reveals that at physiological pH 5-S-CyS-DA is not only more easily oxidized than DA but a bicyclic o-quinone imine (5) intermediate is formed that can chemically oxidize the former conjugate in a reaction that leads to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (6). Compound 6 is lethal when administered into the brains of mice. However, this putative dihydrobenzothiazine endotoxin is even more easily oxidized than 5-S-CyS-DA in a rather complex reaction that ultimately forms 7-(2-aminoethyl)-5-hydroxy-1,4-benzothiazine-3-carboxylic acid (15). Although this benzothiazine could not be isolated it was identified by comparison of its electrochemical and spectroscopic properties in solution with those of 13. The results of this study suggest that benzothiazine 15 might serve as a better analytical marker molecule than 5-S-CyS-DA for either elevated rates of DA autoxidation and/or for roles of GSH and CySH in the neurodegenerative mechanisms in the SN that contribute to PD.
Dieldrin can be retained for decades in lipid-rich tissue and has been measured in some postmortem PD brains. Dieldrin has been reported to deplete brain monoamines in several species and has been shown to inhibit mitochondrial respiration. To further investigate the possibility that it may be involved in the pathogenesis of parkinsonism, its toxicity for dopaminergic (DA) neurons was assessed in a mesencephalic cell culture model. Primary neuronal cultures of mesencephalic neurons were prepared from fetal rats or fetal mice, grown for 1 week and incubated with Dieldrin (0.01-100 microM) for 24 or 48 h. Toxicity for DA neurons was determined by measuring density of surviving tyrosine hydroxylase immunoreactive (TH-ir) cells. Toxicity for gamma-aminobutyric acid (GABA)-ergic neurons was determined by measuring survival of glutamate decarboxylase (GAD)-ir neurons. General, nonselective cytotoxicity was determined by counting cells visualized by phase contrast microscopy or by DAPI-stained cells with fluorescence microscopy. Dieldrin exposure for 24 h resulted in a dose-dependent decrease in survival of TH-IR cells (DA neurons) with a 50% decrease (EC50) produced by 12 microM in rat mesencephalic cultures. Dieldrin also produced a dose- and time-dependent decrease in mouse DA-ergic and GABA-ergic neurons in mouse mesencephalic cultures. GABA-ergic neurons were less sensitive to the toxin compared to DA-ergic neurons. Cellular uptake of 3H-DA was also affected by lower concentrations of Dieldrin (EC50 = 7.98 microM) than uptake of 3H-GABA (EC50 = 43 microM). Thus, Dieldrin appears to be a relatively selective DA-ergic neurotoxin in mesencephalic cultures. Dieldrin, which may be ubiquitous in the environment, is proposed as an agent which can initiate and promote dopaminergic neurodegeneration in susceptible individuals.
Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromobimane by reversed-phase HPLC. Only glutathione and cysteine were detected. The Ca(2+)-dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o-phthalaldehyde reagent solution, and analysis with reversed-phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons-medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.
alpha-Tocopherol (vitamin E) levels in normal brain were lower in the cerebellum than in the cerebral cortex or basal ganglia. There was no difference in alpha-tocopherol levels in the cerebellum, basal ganglia, or cerebral cortex between control subjects and patients with Parkinson's disease.
The contribution of neuromelanin (NM) to the pathogenesis of Parkinson's disease (PD) has long been suspected. In particular, a correlation has been reported between the estimated cell loss in the mesencephalic dopaminergic cell groups and the percentage of NM-pigmented neurons in these cell groups. To test whether the amount of pigment per cell is a critical factor or whether the presence of NM within a neuron is sufficient to account for the degeneration of dopaminergic neurons, the NM content was measured in each neuron from representative sections throughout the ventral mesencephalon of four controls subjects and four patients with PD. Intraneuronal NM was quantified by a densitometric method, using known amounts of synthetic melanin as standards. In control brains, the distribution of melanized neurons in the nigral complex showed a high proportion of lightly melanized neurons in the ventral tegmental area and the pars alpha and gamma of the substantia nigra (SN), whereas heavily melanized neurons were mostly located in the pars beta and lateralis of the SN. An inverse relationship was observed between the percentage of surviving neurons in PD compared with controls and the amount of NM they contain, suggesting that the vulnerability of the dopaminergic neurons is related to their NM content. Factors other than NM may be involved in the differential vulnerability of catecholaminergic neurons in PD. In particular, the constant topography of the cell loss suggests that cell position within the nigral complex is a key factor.
Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mM cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mM fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.
The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with idiopathic Parkinson's disease has provided new understanding into the possible mechanisms that may underlie this neurodegenerative disorder. The biochemical defect is identical to that induced in humans, primates and mice exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We have studied mitochondrial respiratory chain function in various brain regions, in skeletal muscle and in blood platelets from patients with idiopathic Parkinson's disease and from matched controls. We provide evidence suggesting that the complex I deficiency in Parkinson's disease is limited to the brain and that this defect is specific for the substantia nigra. The tissue specificity of the complex I deficiency in Parkinson's disease and its localization to the substantia nigra support the proposition that complex I deficiency may be directly involved in the cause of dopaminergic cell death in Parkinson's disease. An understanding of the molecular basis of this biochemical defect will provide valuable insight into the cause of Parkinson's disease. Our findings of normal mitochondrial function in platelet homogenates suggests that this tissue cannot be used to develop a 'diagnostic test' for Parkinson's disease.
The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.
The micro-architecture of the substantia nigra was studied in control cases of varying age and patients with parkinsonism. A single 7 mu section stained with haematoxylin and eosin was examined at a specific level within the caudal nigra using strict criteria. The pars compacta was divided into a ventral and a dorsal tier, and each tier was further subdivided into 3 regions. In 36 control cases there was a linear fallout of pigmented neurons with advancing age in the pars compacta of the caudal substantia nigra at a rate of 4.7% per decade. Regionally, the lateral ventral tier was relatively spared (2.1% loss per decade) compared with the medial ventral tier (5.4%) and the dorsal tier (6.9%). In 20 Parkinson's disease (PD) cases of varying disease duration there was an exponential loss of pigmented neurons with a 45% loss in the first decade. Regionally, the pattern was opposite to ageing. Loss was greatest in the lateral ventral tier (average loss 91%) followed by the medial ventral tier (71%) and the dorsal tier (56%). The presymptomatic phase of PD from the onset of neuronal loss was estimated to be about 5 yrs. This phase is represented by incidental Lewy body cases: individuals who die without clinical signs of PD or dementia, but who are found to have Lewy bodies at post-mortem. In 7 cases cell loss was confined to the lateral ventral tier (average loss 52%) congruent with the lateral ventral selectivity of symptomatic PD. It was calculated that at the onset of symptoms there was a 68% cell loss in the lateral ventral tier and a 48% loss in the caudal nigra as a whole. The regional selectivity of PD is relatively specific. In 15 cases of striatonigral degeneration the distribution of cell loss was similar, but the loss in the dorsal tier was greater than PD by 21%. In 14 cases of Steele-Richardson-Olszewski syndrome (SRO) there was no predilection for the lateral ventral tier, but a tendency to involve the medial nigra and spare the lateral. These findings suggest that age-related attrition of pigmented nigral cells is not an important factor in the pathogenesis of PD.
The concentrations of dopamine (DA) and of the excitatory amino acids (EAAs) glutamate (Glu) and aspartate (Asp) were measured in dialysates from the striatum of awake rats in order to study the link between the release of DA and of EAAs induced by the infusion of 1-methyl-4-phenylpyridinium ion (MPP+). DA and EAAs were detected simultaneously by HPLC-EC. The infusion of MPP+ at the concentration of 1 mM elevated DA levels in the perfusates, but did not affect EAA release. However, MPP+ at 10 mM maximally stimulated Glu and Asp release to 230- and 68-fold of baseline, respectively. In this condition, pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (5 mg/kg, i.p.) prevented the MPP(+)-induced EAA release. In contrast, MK-801 had no effect on DA release induced either by 1 or 10 mM MPP+. These results suggest that MPP(+)-induced DA and EAA release are independently regulated processes. In addition, the finding that MK-801 inhibits MPP(+)-induced EAA release suggests that EAAs may act on NMDA receptors to stimulate their own release through a positive-feedback mechanism.
The relationships between mitochondrial transmembrane potential, ATP concentration, and cytotoxicity were evaluated after exposure of isolated rat hepatocytes to different mitochondrial poisons. Both the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its fully oxidized metabolite, the 1-methyl-4-phenylpyridinium (MPP+) ion, caused a concentration- and time-dependent depolarization of mitochondrial membranes which followed ATP depletion and preceded cytotoxicity. The effect of MPTP, but not that of MPP+, was prevented by deprenyl, an inhibitor of MPTP conversion to MPP+ via monoamine oxidase type B. Addition of fructose to the hepatocyte incubations treated with either MPTP or MPP+ counteracted the loss of mitochondrial transmembrane potential. Fructose was also effective in protecting against the mitochondrial membrane depolarization as well as ATP depletion and cytotoxicity induced by antimycin. A, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and valinomycin. Data confirm the key role played by MPP(+)-induced mitochondrial damage in MPTP toxicity and indicate that (i) ATP produced via the glycolytic pathway can be utilized by hepatocytes to maintain mitochondrial electrochemical gradient, and (ii) a loss of mitochondrial membrane potential may occur only when supplies of ATP are depleted.
The influence of reducing the KGD non-cooperative form by DTT on the KG binding by the enzyme was investigated. The chemical modification of KGD by DEP has revealed that reduction of KGD cysteine residues results in the appearance of the interaction of the dimer active sites upon the enzyme-substrate complex formation. The reduction of 2 SH-groups per KGD subunit: the most reactive one and a buried one--was established to be sufficient for the appearance of KGD cooperative properties in substrate binding as well as for the change in the enzyme activity plots versus substrate concentration. It is suggested that KGD can be regulated by thiol-disulfide exchange in the cell.
Uptake of the catecholamines (CA), dopamine (DA) and norepinephrine (NE) into synaptosomes prepared from rat and bovine brains was potentiated by ATP (from 0.1 to 5.0 mM) in a dose-dependent manner. Other nucleotides, particularly the nonhydrolyzable ATP analogs beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP) and beta,gamma-methyladenosine-5'-triphosphate (AMP-PCP) also potentiated [3H]DA and [3H]NE uptake. Several endogenous 5'-nucleotide triphosphates (e.g. GTP, UTP and CTP) potentiated [3H]CA uptake, but were less effective than ATP. Among the ATP metabolites, only ADP potentiated uptake whereas AMP and adenosine did not. [3H]Dopamine uptake measured in Krebs bicarbonate buffer had a Km of 2.1 microM and a Vmax of 163.9 pmol/mg prot./min. In presence of ATP, [3H]DA uptake had much higher affinity (Km = 0.56 microM) and larger capacity (Vmax = 333 pmol/mg prot./min) than uptake in absence of added ATP. Furthermore, [3H]DA uptake in presence of ATP had faster rate of uptake, and was independent of temperature while in absence of added ATP it was temperature-dependent. This ATP-dependent [3H]DA uptake was retained by synaptosomal ghosts that were obtained after lysing the striatal synaptosomes and removing their contents of synaptic vesicles and mitochondria. It is proposed that, in addition to the carrier-mediated (neuronal) uptake of CA, there is neuronal uptake that is regulated by ATP and inhibited by cocaine, which may be more relevant for terminating the synaptic action of CA because of its faster rate of uptake and larger capacity.
Perfusion of rat striatum with 10 mM 1-methyl-4-phenylpyridinium (MPP+) during 1 min caused a rapid reversible increase in dopamine (DA) efflux (maximal after 4 min) that was similar to the effects of 1 or 15 min 10 mM amphetamine. A massive, prolonged, and irreversible release of DA occurred after 15 min 10 mM MPP+, showing a biphasic trend: the rapid amphetamine-like increase was followed by a slower and larger DA efflux, reaching its maximum after 25 min. One minute perfusions with 1-100 mM MPP+ caused a gradual increase in the overflow of striatal lactate, reaching a maximum effect after 20-30 min. The similarity between the time courses of the MPP+-induced efflux of DA and lactate suggests that both effects are probably consequences of inhibition of aerobic glycolysis by MPP+. A comparison with the courses of DA and lactate efflux after death of control rats showed a delay of several minutes before the toxic effects of MPP+ become apparent. These in vivo data are consistent with the hypothesis that MPP+ accumulates and then kills dopaminergic cells, possibly by the irreversible inhibition of mitochondrial respiration.
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor appears to play a pivotal role in enabling glutamate to express its neurotoxic potential in a variety of neurological disorders. Our results show that the transition of glutamate from neurotransmitter to neurotoxin is facilitated when cellular energy is limited in cultured cerebellar neurons. Omission of glucose, exclusion of oxygen, or inclusion of inhibitors of oxidative phosphorylation or of the sodium/potassium pump, enables the excitatory amino acids glutamate or NMDA to express their neurotoxic potential. We interpret these results as demonstrating that glucose metabolism, ATP production, and functioning Na+,K+-ATPases are necessary to generate a resting potential sufficient to maintain the voltage-dependent Mg2+ block of the NMDA receptor channel; relief of the Mg2+ block enables the excitatory amino acids to act persistently at the NMDA receptor, resulting in the opening of ion channels and subsequent neuronal damage. These findings are discussed in the context of perturbations or abnormalities which lead to decreased availability or utilization of glucose and oxygen in the brain which may trigger endogenous excitatory amino acids to become neurotoxic by this mechanism.
Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinson's disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinson's disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinson's disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinson's disease.
The concentrations of the 5-S-cysteinyl adducts of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylalanine (DOPA) and the levels of noradrenaline (NA), DA, DOPAC and DOPA were determined in the putamen (PUT), caudate nucleus (CN) and substantia nigra (SN) of human post mortem brains with or without depigmentation and degeneration of the SN. The levels of DA, DOPAC and DOPA decreased with the degree of depigmentation and degeneration in the three brain regions while NA levels only decreased in SN and PUT. In general, the concentrations of the 5-S-cysteinyl adducts did not differ, but the ratios of 5-S-cysteinyl-DA/DA, 5-S-cysteinyl-DOPAC/DOPAC and 5-S-cysteinyl-DOPA/DOPA were higher in patients with a more depigmentated and degenerated SN, except for the 5-S-cysteinyl-DA/DA ratio in the PUT. Higher ratios were also found in the cell body areas compared to the neuron terminal areas.
Thus depigmentation and degeneration of dopaminergic SN neurons, seem to be correlated to enhanced rates of autoxidation, possibly due to an impaired antioxidant capacity.
The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinson's disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)-binding, protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.
Autografting of dopamine-producing adrenal medullary tissue to the striatal region of the brain is now being attempted in patients with Parkinson's disease. Since the success of this neurosurgical approach to dopamine-replacement therapy may depend on the selection of the most appropriate subregion of the striatum for implantation, we examined the pattern and degree of dopamine loss in striatum obtained at autopsy from eight patients with idiopathic Parkinson's disease. We found that in the putamen there was a nearly complete depletion of dopamine in all subdivisions, with the greatest reduction in the caudal portions (less than 1 percent of the dopamine remaining). In the caudate nucleus, the only subdivision with severe dopamine reduction was the most dorsal rostral part (4 percent of the dopamine remaining); the other subdivisions still had substantial levels of dopamine (up to approximately 40 percent of control levels). We propose that the motor deficits that are a constant and characteristic feature of idiopathic Parkinson's disease are for the most part a consequence of dopamine loss in the putamen, and that the dopamine-related caudate deficits (in "higher" cognitive functions) are, if present, less marked or restricted to discrete functions only. We conclude that the putamen--particularly its caudal portions--may be the most appropriate site for intrastriatal application of dopamine-producing autografts in patients with idiopathic Parkinson's disease.
C57 black mice showed significantly decreased glutathione (GSH) content in the brainstem 24 h after a single s.c. injection of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 40 mg/kg. This loss of GSH could be prevented by pretreating animals with large amounts of alpha-tocopherol or beta-carotene. Increasing dopamine turnover of nigrostriatal neurons in mice by dietary administration of L-dihydroxy-phenylalanine and carbidopa did not accentuate the neurotoxic effects of MPTP. It seems likely that MPTP metabolites directly damage dopaminergic nigrostriatal neurons and that GSH is consumed in attempts to detoxify these metabolites in the substantia nigra.
The oxidation of NAD+-linked substrates by rat brain mitochondria is completely inhibited by pre-incubation with 0.5 mM N-methyl-4-phenylpyridine (MPP+). The effect is dependent on the integrity of the mitochondria because far higher concentrations of MPP+ are required to inhibit NADH oxidation in inverted mitochondria or isolated inner membrane preparations. The reason for this difference in behavior has been traced to a novel system for the uptake of MPP+ into mitochondria against a concentration gradient. The uptake system is energized by the transmembrane potential, as shown by the fact that valinomycin plus K+, which collapses this gradient, abolishes MPP+ uptake, while agents which collapse the proton gradient have no effect on the process. If an uncoupler is added to mitochondria preloaded with MPP+, efflux of the latter occurs with the concentration gradient. The uptake system has been studied in liver, whole brain, cortex, and midbrain preparations from rats. It may be readily distinguished from the synaptic dopamine reuptake system, since the former is blocked by uncouplers and respiratory inhibitors, but not by dopamine or mazindol, whereas the synaptic system is blocked by mazindol and competitively inhibited by dopamine but is not affected by respiratory inhibitors or uncouplers. Energy-driven uptake of MPP+ by brain mitochondria may be a crucial step in the complex sequence of events leading to the neurotoxic actions of its precursor, MPTP.
Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration.
The numbers of nerve cells in the substantia nigra and locus caeruleus were counted, and the volume of their nucleolus and the amount of neuromelanin pigment they contain measured, in patients with Parkinson's disease and in age-matched and elderly individuals free from neurological illness. The number of pigmented nerve cells of both types is reduced by about 20% in the elderly when compared with age-matched controls. The melanin content of the remaining cells in the elderly is lessened (by 11% in substantia nigra and 21% in locus caeruleus) in such a way as to indicate that this age depletion in cell number results from a preferential loss of those nerve cells which contain the greatest amounts of melanin pigment. In Parkinson's disease there is a greater overall reduction in the amount of melanin within remaining cells (15% in substantia nigra, 25% in locus caeruleus) because of a more severe (80%) loss of the heavier pigmented cells. The basis for Parkinson's disease may therefore lie with an aggravation, possibly by secondary factors, of changes that occur within neurones of substantia nigra and locus caeruleus as part of their "normal" process of ageing.
Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.
Neuronal injury resulting from acute brain insults and some neurodegenerative diseases implicates N-methyl-D-aspartate (NMDA) glutamate receptors. The fact that antioxidants reduce some types of brain damage suggests that oxygen radicals may have a role. It has been shown that mutations in Cu/Zn-superoxide dismutase (SOD), an enzyme catalysing superoxide (O2.-) detoxification in the cell, are linked to a familial form of amyotrophic lateral sclerosis (ALS). Here we report that O2.- is produced upon NMDA receptor stimulation in cultured cerebellar granule cells. Electron paramagnetic resonance was used to assess O2.- production that was due in part to the release of arachidonic acid. Activation of kainic acid receptors, or voltage-sensitive Ca2+ channels, did not produce detectable O2.-. We also find that the nitrone DMPO (5,5-dimethyl pyrroline 1-oxide), used as a spin trap, is more efficient than the nitric oxide synthase inhibitor, L-NG-nitro-arginine, in reducing NMDA-induced neuronal death in these cultures.
Striatal levels of dopamine and its metabolites 3-methoxy-4-hydroxy-phenylacetic acid (DOPAC) and homovanillic acid (HVA) decreased 7 days after subcutaneous injection of MPTP (20 mg/kg) to the mouse. Striatal GSH contents decreased and GSSG/GSH ratios increased one hour after subcutaneous administration of MPTP. Pretreatments of both cysteamine (200 mg/kg, s.c.) and dimercaprol (20 mg/kg, i.m.) reduced the MPTP-induced decreases in striatal dopamine, DOPAC and HVA, and also prevented the MPTP-induced decreases in GSH levels and increases in GSSG/GSH ratios. On the other hand, injection of cysteamine did not modify the MPTP-induced decreases in striatal levels of dopamine and its metabolites when it was done 2 hours after MPTP administration. Moreover, pretreatment of cysteamine did not affect striatal concentrations of MPP+ in MPTP-treated mice. These results suggest that sulfhydryl drugs such as cysteamine and dimercaprol may reduce neurotoxicity of MPTP probably via changes in redox cycle of glutathione in the brain.
Glutathione levels in neurons and glial cells were investigated in a neuronal-glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neuronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate-limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cystine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cysteine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.
We have examined the biochemical and histological effects of high concentrations of dopamine (0.05-1.0 micromol) injected into the rat striatum. Twenty-four hours after such injections, the oxidation products of dopamine and dihydroxyphenylacetic acid were detected as both free and protein-bound cysteinyl dopamine and cysteinyl dihydroxyphenylacetic acid. Protein-bound cysteinyl catechols were increased 7- to 20-fold above control tissue levels. By 7 days postinjection, the protein-bound cysteinyl catechols were still detectable, although reduced in concentration, whereas the free forms could no longer be measured. Histological examination of striatum at 7 days revealed a central core of nonspecific damage including neuronal loss and gliosis. This core was surrounded by a region containing a marked reduction in tyrosine hydroxylase immunoreactivity but no apparent loss of serotonin or synaptophysin immunoreactivity. When dopamine was injected with an equimolar concentration of either ascorbic acid or glutathione, the formation of protein-bound cysteinyl catechols was greatly reduced. Moreover, the specific loss of tyrosine hydroxylase immunoreactivity associated with dopamine injections was no longer detectable, although the nonspecific changes in cytoarchitecture were still apparent. Thus, following its oxidation, dopamine in high concentrations binds to protein in the striatum, an event that is correlated with the specific loss of dopaminergic terminals. We suggest that the selective degeneration of dopamine neurons in Parkinson's disease may be caused by an imbalance between the oxidation of dopamine and the availability of antioxidant defenses.
The initial step in the genesis of neuromelanin, a black polymeric pigment normally found in the cytoplasm of dopaminergic cell bodies in the substantia nigra (SN), is the autoxidation of dopamine (DA) to DA-o-quinone (1). In this investigation, it is demonstrated that in the presence of L-cysteine (CySH) o-quinone 1 is scavenged to give 5-S-cysteinyldopamine (5-S-Cys-DA, major product) and 2-S-cysteinyldopamine (2-S-CyS-DA, minor product). These cysteinyl conjugates are more easily oxidized than DA. The relative yields of the resulting products are dependent on the concentration of free CySH. These products include 2,5-bi-S-cysteinyldopamine (2,5-bi-S-CyS-DA) and 2,5,6-tri-S-cysteinyldopamine (2,5,6-tri-S-CyS-DA), 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1), 8-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-5), and a number of cysteinyl conjugates of these dihydrobenzothiazines (DHBTs). 2,5-Bi-S-CyS-DA, DHBT-1, the 6-S-cysteinyl conjugate of DHBT-1, DHBT-5, and the 6-S-cysteinyl conjugate of DHBT-5 were lethal when administered into the brains of laboratory mice and evoke a very characteristic hyperactivity syndrome and episodes of severe tremor. These and related results provide support for the hypothesis that the massive, irreversible loss of glutathione (GSH), increased 5-S-CyS-DA/DA concentration ratio, and depigmentation of dopaminergic neurons in the SN that all occur in Parkinson's disease (PD) might be caused by the gamma-glutamyl transpeptidase-mediated translocation of CySH (and/or GSH) into these cells. Furthermore, the resulting cysteinyldopamines and DHBTs might include endotoxic metabolites responsible for the selective degeneration of nigrostriatal dopaminergic neurons and PD.
Animal models are an important aid in experimental medical science because they enable one to study the pathogenetic mechanisms and the therapeutic principles of treating the functional disturbances (symptoms) of human diseases. Once the causative mechanism is understood, animal models are also helpful in the development of therapeutic approaches exploiting this understanding. On the basis of experimental and clinical findings. Parkinson's disease (PD) became the first neurological disease to be treated palliatively by neurotransmitter replacement therapy. The pathological hallmark of PD is a specific degeneration of nigral and other pigmented brainstem nuclei, with a characteristic inclusion, the Lewy body, in remaining nerve cells. There is now a lot of evidence that degeneration of the dopaminergic nigral neurones and the resulting striatal dopamine-deficiency syndrome are responsible for its classic motor symptoms akinesia and bradykinesia. PD is one of many human diseases which do not appear to have spontaneously arisen in animals. The characteristic features of the disease can however be more or less faithfully imitated in animals through the administration of various neurotoxic agents and drugs disturbing the dopaminergic neurotransmission. The cause of chronic nigral cell death in PD and the underlying mechanisms remain elusive. The partial elucidation of the processes underlie the selective action of neurotoxic substances such as 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has however revealed possible molecular mechanisms that give rise to neuronal death. Accordingly, hypotheses concerning the mechanisms of these neurotoxines have been related to the pathogenesis of nigral cell death in PD. The present contribution starts out by describing some of the clinical, pathological and neurochemical phenomena of PD. The currently most important animal models (e.g. the reserpine model, neuroleptic-induced catalepsy, tremor models, experimentally-induced degeneration of nigrostriatal dopaminergic neurons with 6-OHDA, methamphetamine, MPTP, MPP+, tetrahydroisoquinolines, beta-carbolines, and iron) critically reviewed next, and are compared with the characteristic features of the disease in man.
The role of oxidative stress in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurotoxicity is as yet unclear and the evidence for generation of oxygen free radicals as a primary event in the neurotoxicity is yet to be demonstrated. The present study was undertaken to ascertain the potential role of oxidative damage, and the protective role, if any, of the antioxidant, glutathione (GSH), in MPTP-induced neurotoxicity. Exposure of sagittal slices of mouse brain to MPTP resulted in significant increases of reactive oxygen species (ROS) and malondialdehyde (MDA, the product of lipid peroxidation) and decreases in GSH content. Pretreatment of mouse brain slices, in vitro, with GSH or GSH isopropyl ester attenuated MPTP toxicity as assessed by the tissue activity of the mitochondrial enzyme, NADH-dehydrogenase (NADH-DH), and by leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), from the slice into the medium. In vivo administration of MPTP (30 mg/kg body weight, s.c.), to mice resulted in significant lowering of GSH in the striatum and midbrain, 2 h after dosage; ROS levels in the striatum and midbrain increased after 4 and 8 h, respectively. In the striatum significant inhibition of rotenone-sensitive NADH ubiquinone-1 oxido-reductase (Complex 1) was observed transiently 1 h after MPTP administration. The enzyme activity recovered thereafter; significant inhibition of mitochondrial Complex I was observed in the striatum only 18 h after MPTP dose. In the midbrain, mitochondrial Complex I was inhibited only 18 h after MPTP dose; no change was observed at the early time points examined. Thus, the depletion of GSH and increased ROS formation preceded the inhibition of the mitochondrial enzyme in the midbrain. Evidence presented herein from both in vitro and in vivo studies support that MPTP exposure generates ROS resulting in oxidative stress.
The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme gamma-glutamyl transpeptidase was inhibited by acivicin. The activity of gamma-glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 +/- 0.3 nmol/(min x mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of gamma-glutamyl transpeptidase activity was found at concentrations of about 5 microM and 50 microM, respectively. In the presence of acivicin at a concentration above 10 microM the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 +/- 0.3 nmol/(h x mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 microM and 50 microM acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme gamma-glutamyl transpeptidase of these cells.