The proto-oncogene Bcl-2 inhibits cellular toxicity of dopamine: possible implications for Parkinson's Disease

It is currently believed that excessive oxidant stress induced by metabolism of dopamine (DA), plays a major role in the pathogenesis of the selective nigrostriatal neuronal loss in Parkinson's disease. We recently showed that the neurotransmitter DA, in physiological concentrations, is capable of initiating apoptosis in cultured, post-mitotic sympathetic neurons. Bcl-2 is a proto-oncogene that blocks apoptosis. We now report that Bcl-2 is a powerful inhibitor of DA toxicity in PC-12 pheochromocytoma cells. We induced stable expression of Bcl-2 in PC-12 cells by transfection with recombinant pCMV5 expression vector, containing mouse bcl-2 (coding-sequence) cDNA. Cells expressing Bcl-2 manifested marked resistance to otherwise lethal (300 uM) in vitroconcentrations of DA. This protective effect was reflected in the trypan-blue test of cell survival, 3 H-thymidine incorporation and inhibition of the characteristic apoptotic morphologic alterations in scanning electron microscopic studies. Bcl-2 and associated control systems of apoptosis may have an important physiological role in restraining the apop-tosis-triggering potential of DA in nigrostriatal neurons. This novel field of research may yield insights into the pathogenesis of Parkinson's disease and lead to development of novel therapeutic approaches.     

Protein aggregation in Huntington's and Parkinson's disease: implications for therapy

The accumulation of highly insoluble intracellular protein aggregates in neuronal inclusions is a hallmark of Huntington's disease (HD) and Parkinson's disease (PD) as well as several other late-onset neurodegenerative disorders. The aggregates formed in vitro and in vivo generally have a fibrillar morphology, consist of individual beta-strands and are resistant to proteolytic degradation. Although the causal relationship between aggregate formation and disease remains to be proven, the gradual deposition of mutant protein in neurons is consistent with the late-onset and progressive nature of symptoms. Recently, circumstantial evidence from mouse and Drosophila model systems suggests that abnormal protein folding and aggregation play a key role in the pathogenesis of both HD and PD. Therefore, a detailed understanding of the molecular mechanisms of protein aggregation and its effects on neuronal cell death could open new opportunities for therapy.     

Inhibition of brain mitochondrial respiration by dopamine and its metabolites: implications for Parkinson's disease and catecholamine-associated diseases

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.     

Increased SOD1 enzymatic activity and gene modifications in orangutans: evolutionary implications

Summary Superoxide dismutase CuZn (SOD1) enzymatic activity was measured in five orangutans (Pongo pygmaeus, PPY) and compared to that of man, chimpanzee, and gorilla. It was found to be increased by a factor of two in one orangutan (Ralfina) and by a factor of 1.5 in the four others. In situ hybridization of the SOD1 cDNA human probe showed a heterozygous intrachromosomal rearrangement of pair PPY XXI, possibly an insertion, in Ralfina. Southern blotting showed that the SOD1 gene is modified in the three orangutans that were investigated and that a further modification of the 5-end of the gene had occurred in Ralfina. The evolutionary implications of these observations are discussed.     

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).     

Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson's disease

To investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia-rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 micro m DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide-dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mm DA established almost constant extracellular concentrations of H2O2 of 5 microm and 15 microm, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenaline and noradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brain's GSH metabolism in Parkinson's disease.     

Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease

Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone‐induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the ‘catecholaldehyde hypothesis,’ buildup of the autotoxic dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F‐labeled catechols were measured in PC12 cells incubated with rotenone (0–1000 nM, 180 min), without or with F‐dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F‐DOPAL, and 3,4‐dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD⁺ was supplied, whereas the direct‐acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone‐induced toxicity in dopaminergic neurons.

     

Dextran enhances calcium-induced aggregation of phosphatidylserine liposomes: possible implications for exocytosis

We have studied the calcium-induced aggregation of phosphatidylserine liposomes in the presence of various concentration of a high-molecular water-soluble polysaccharide dextran. It has been shown that threshold concentrations of calcium necessary to induce liposome aggregation in the presence of approximately 1 mM concentration of dextran is about one order lower than in the absence of dextran. Soluble intracellular polymers may thus play an important role in the process of exocytosis.     

Human carboxylesterase 2: Studies on the role of glycosylation for enzymatic activity

Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation.Partial or non-glycosylated forms of a secreted form of hCES2 have been obtained by three approaches: (i) enzymatic deglycosylation with peptide N-glycosidase F; (ii) incubation with the inhibitor tunicamycin; ii) site directed mutagenesis of each or both N-glycosylation sites.Deglycosylated protein did not show a detectable decrease in enzyme activity. On the other hand, tunicamycin led to decreased levels of secreted hCES2 but the enzyme was still active. In agreement, mutation of each and both N-glycosylation sites led to decreased levels of secreted active hCES2. However, the thermostability of the glycosylation mutants was decreased.The results indicated that glycans are involved, to some extent in protein folding in vivo, however, removal of glycans does not abrogate the activity of secreted hCES2.     

Sustained extracellular signal-regulated kinase activation by 6-hydroxydopamine: implications for Parkinson's disease

Although the toxin 6-hydroxydopamine (6-OHDA) is utilized extensively in animal models of Parkinson's disease, the underlying mechanism of its toxic effects on dopaminergic neurons is not completely understood. We examined the effects of 6-OHDA on the CNS-derived tyrosine hydroxylase expressing B65 cell line, with particular attention to the regulation of the extracellular signal-regulated protein kinases (ERK). 6-OHDA elicited a dose-dependent cytotoxicity in B65 cells. Toxic doses of 6-OHDA also elicited a biphasic pattern of ERK phosphorylation with a prominent sustained phase, a pattern that differed from that observed with hydrogen peroxide (H(2)O(2)) treatment. 6-OHDA-elicited ERK phosphorylation was blocked by PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK. PD98059 also conferred protection against 6-OHDA cytotoxicity, but did not affect H(2)O(2) toxicity in B65 cells. These results suggest that ERK activation plays a direct mechanistic role in 6-OHDA toxicity, rather than representing a protective compensatory response, and raise the possibility that abnormal patterns of ERK activation may contribute to dopaminergic neuronal cell death.     

Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.     

Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinson's disease

The research on central synaptic neurotransmission has greatly benefited from the use of the neurotoxin 2,4,5-trihydroxyphenylethylamine, or 6-hydroxydopamine (6-OHDA), that destroys catecholamine-containing neuronal cell bodies and nerve terminals. Refinements in the use of this neurotoxin led to the use of dopamine-denervated animals as models of human Parkinson's disease, in which the loss of dopaminergic neurons is a prominent feature. Here we review structural, pharmacological, and biochemical studies carried out in the adult and neonatal 6-OHDA lesioned animals. These models have become useful and interesting paradigms to examine alterations in the expression of receptors and in their sensitivity to agonist drugs; some of these modifications may underlie the altered responsiveness of the dopamine-lesioned animals to dopamine, but also to other compounds, including serotoninergic drugs. We have also reviewed studies of amino acids as well as of monoamine metabolism and of uptake mechanisms that may underlie some of the behavioural alterations in these models that have become relevant for our understanding of the sprouting and plastic properties of spared neurons, and of the alternate neuronal projections that replace lesioned terminals, enabling compensatory adaptations. Although 6-OHDA-lesioned animals, that display some biochemical characteristics of Parkinson's disease in humans, do not express all of the neurological features exhibited by patients, the increasing knowledge that can be obtained from studies in simplified experimental models will undoubtedly lead to the development of innovative drugs and other replacement therapies for degenerative brain diseases.     

Human serum dopamine beta-hydroxylase: correlation of enzymatic activity with immunoreactive protein in genetically defined samples.

An antibody against human adrenal dopamine beta-hydroxylase (DBH) was used to quantitate immunoreactive DBH protein in human serum by an immunoprecipitation technique. A significant correlation was found between DBH enzyme activity and immunoreactive DBH protein in randomly selected serum samples (r = 0.94; N = 38; p less than .001). Studies of sera from obligate heterozygotes and individuals homozygous for the allele responsible for very low serum DBH enzymatic activity were compatible with a genetically mediated decrease in the quantity of circulating DBH protein in these subjects.     

Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson's disease

Degenerating neurons of Parkinson's disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wildtype (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (MEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.     

Destabilization of DJ-1 by familial substitution and oxidative modifications: implications for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by oxidative stress and protein aggregation. Both toxic phenomena are mitigated by DJ-1, a homodimeric protein with proposed antioxidant and chaperone activities. The neuroprotective function of DJ-1 is modulated by oxidation of cysteine 106, a residue that may act as an oxidative stress sensor. Loss-of-function mutations in the DJ-1 gene have been linked to early onset PD, and age-dependent over-oxidation of DJ-1 is thought to contribute to sporadic PD. The familial mutant L166P fails to dimerize and is rapidly degraded, suggesting that protein destabilization accounts for the dysfunction of this mutant. In this study, we investigated how the structure and stability of DJ-1 are impacted by two other pathogenic substitutions (M26I and E64D) and by over-oxidation with H2O2. Whereas the recombinant wild-type protein and E64D both adopted a stable dimeric structure, M26I showed an increased propensity to aggregate and decreased secondary structure. Similar to M26I, over-oxidized wild-type DJ-1 exhibited reduced secondary structure, and this property correlated with destabilization of the dimer. The engineered mutant C106A had a greater thermodynamic stability and was more resistant to oxidation-induced destabilization than the wild-type protein. These results suggest that (i) the M26I substitution and over-oxidation destabilize dimeric DJ-1, and (ii) the oxidation of cysteine 106 contributes to DJ-1 destabilization. Our findings provide a structural basis for DJ-1 dysfunction in familial and sporadic PD, and they suggest that dimer stabilization is a reasonable therapeutic strategy to treat both forms of this disorder.     

Synaptic and non-synaptic striatal dopamine D2 receptors: possible implications in normal and pathological behaviour

Various levels of organisation in the central nervous system can be distinguished, ranging from the molecular, the cellular, the multicellular and the neuronal system level. The relationship between receptor function and behaviour is focussed to the dopamine D2 type receptor of the striatal complex in relation to extrapyramidal and limbic systems. In the striatal complex a striosomal and a matrix compartment can be distinguished. The matrix compartment can be considered as a part of the extrapyramidal system and is innervated by the motor cortex and by the dopaminergic neurons of the ventral tegmental, the dorsal substantia nigra and the retrorubral area. This compartment has a relatively high density of D2 receptors. The striosomes are innervated by e.g. the prelimbic cortex and dopamine neurones of the ventral part of the substantia nigra; here the density of D2 receptors are lower. Under normal conditions most of the D2 receptors are occupied by endogenous dopamine, and postsynaptic (e.g. cholinergic) function is therefore sensitive to antagonists; e.g. antipsychotics. Exposure to drugs such as amphetamine produces a substantial overflow of dopamine from nerve terminals leading to the activation of remote dopamine receptors, that may belong to the system that normally is not influenced by these nerve terminals (defined here as extra synaptic receptor activation). A loss of the normal spatial-temporal relationships may also occur during L-DOPA therapy in Parkinson's disease. In this illness, due to degeneration of dopaminergic innervation, several dopamine receptors have become non-synaptic. In these states of intoxication the normal spatial/temporal organization is lost and such a loss may contribute to behavioural impairments.     

Enhancement of dopaminergic neurotoxicity by the mercapturate of dopamine: relevance to Parkinson's disease

The mechanisms that underlie dopaminergic neurodegeneration in Parkinson's disease (PD) are not known but have been proposed to involve oxidation of dopamine and related catechols. In other organ systems, cytotoxicity from catechol oxidation is profoundly influenced by mercapturate metabolism. Here we have tested the hypothesis that catechol thioethers produced in the mercapturic acid pathway may act as dopaminergic neurotoxins. A rat mesencephalic/neuroblastoma hybrid (MES) cell line was exposed to dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), or eight different catechol thioethers for up to 24 h, and the extent of apoptosis was quantified by a microculture kinetic assay. Apoptosis also was confirmed morphologically with Giemsa-stained cultures and by demonstration of internucleosomal DNA fragmentation. The results showed that dopamine at 5-50 microM produced concentration-dependent increases in the percentage of apoptotic MES cells. At 25 and 50 microM dopamine, the maximal proportions of apoptotic cells were detected at approximately 19 (20.7 +/- 2.0%) and 14 h (30.3 +/- 3.5%), respectively. None of the catechol thioethers (up to 5 microM) alone induced significant apoptosis in MES cells. However, when MES cells were incubated with dopamine (25 microM) and catechol thioethers (5 microM) to mimic pathological conditions, 5-S-N-acetylcysteinyldopamine, 5-S-homocysteinyldopamine, and 5-S-homocysteinyl-DOPAC significantly increased the percentage of apoptotic cells compared with dopamine alone. These results suggest that mercapturate metabolism of endogenous catechols may yield products that facilitate dopaminergic neurodegeneration.     

Beta-synuclein regulates Akt activity in neuronal cells. A possible mechanism for neuroprotection in Parkinson's disease

Recent studies have shown that the neurodegenerative process in disorders with Lewy body formation, such as Parkinson's disease and dementia with Lewy bodies, is associated with alpha-synuclein accumulation and that beta-synuclein might protect the central nervous system from the neurotoxic effects of alpha-synuclein. However, the mechanisms are unclear. The main objective of the present study was to investigate the potential involvement of the serine threonine kinase Akt (also known as protein kinase B) signaling pathway in the mechanisms of beta-synuclein neuroprotection. For this purpose, Akt activity and cell survival were analyzed in synuclein-transfected B103 neuroblastoma cells and primary cortical neurons. Beta-synuclein transfection resulted in increased Akt activity and conferred protection from the neurotoxic effects of rotenone. Down-regulation of Akt expression resulted in an increased susceptibility to rotenone toxicity, whereas transfection with a lentiviral vector encoding for beta-synuclein was protective. The effects of beta-synuclein on the Akt pathway appear to be by direct interaction between these molecules and were independent of upstream signaling molecules. Taken together, these results indicate that the mechanisms of beta-synuclein neuroprotection might involve direct interactions between beta-synuclein and Akt and suggest that this signaling pathway could be a potential therapeutic target for neurological conditions associated with parkinsonism and alpha-synuclein aggregation.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400μM) for up to 24h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.