Dopamine Neuronal Damage Research Articles

Pogostemon Cabin prevents the MPTP-induced loss of dopaminergic neurons by inhibiting CD4+ T cell infiltration in the brain

Parkinson’s disease (PD) involves the neurodegeneration of dopaminergic neurons, which is accompanied by neuroinflammation in the substantia nigra (SN). Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibit microglial activation-induced inflammation as well as DA neuronal damage. In this study, treatment with Pogostemon Cabin (PC) prevented the degeneration of the Dopamine (DA) neurons in the substantia nigra (SN). This neuro-protective effect of PC was associated with microglial deactivation and a reduction in CD4+ T cell infiltration. Therefore, the results of this study suggest that PC exerts its neuroprotective function via its anti-inflammatory properties and that PC could be employed as a novel therapeutic agent for PD associated with neuroinflammation and CD4+ T cell infiltration into central nerve system.

Damage of neuroblastoma cell SH-SY5Y mediated by MPP+ inhibits proliferation of T-cell leukemia Jurkat by co-culture system.

The adaptive immune system has implications in pathology of Parkinson’s disease (PD). Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+) for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM) arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2) and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM) induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

LPS-induced oxidative stress and inflammatory reaction in the rat striatum

Inflammation-induced microglia activation and increased oxidative stress have been observed in neurodegenerative disorders, such as Parkinson's disease. The aim of our study was to determine the appropriate dose and route of LPS administration to study hydroxyl radical generation and extracellular level of dopamine (DA), glutamate (GLU) and adenosine (ADN) in the rat striatum as markers of DA neuron damage and glial cell activation. The effect of LPS administration on DA, DOPAC, HVA and hydroxyl radical tissue level was also examined. LPS was given to rats in a single dose of 10 mg/kg ip, repeatedly for 5 days in a dose of 5 mg/kg ip and intrastriatally at doses 5, 20 and 40 μg/4 μl. The extracellular level of DA, hydroxyl radical, ADN and GLU were assayed in striatal dialysates using HPLC with electrochemical, fluorescence and VIS detection, respectively. A single ip LPS (10 mg/kg) administration increased hydroxyl radical production but did not affect extracellular DA, GLU and ADN level. Repeated ip LPS (5 × 5 mg/kg) treatment decreased extracellular level of DA, GLU, ADN and production of hydroxyl radical. LPS (5 and 10 μg) given intrastriatally increased hydroxyl radical production, extracellular GLU and ADN level from 0 to 180 min after administration, but did not influence DA level. LPS (5, 20 and 40 μg) decreased striatal DA and DOPAC content, but increased HVA and hydroxyl radical level 72 h after intrastriatal administration. Our data indicate that local intrastriatal LPS administration activates glial cells and increases production of free radicals and secretion of GLU and ADN in early phase of inflammation. The damage of DA neurons is observed 72 h after local LPS administration.

Effects of adenosine receptor antagonists on the in vivo LPS-induced inflammation model of Parkinson's disease.

The study shows effects of the nonselective adenosine A1/A2A receptor antagonist caffeine and the selective A2A receptor antagonist KW6002 on LPS-induced changes in the extracellular levels of dopamine (DA), glutamate, adenosine, hydroxyl radical, and A2A receptor density in the rat striatum. Intrastriatal LPS (10 μg) injection decreased extracellular level of DA and increased the level of adenosine, glutamate, and hydroxyl radical on the ipsilateral side 24 h after LPS administration. Caffeine (10 and 20 mg/kg i.p.) and KW6002 (1.5 and 3 mg/kg i.p.) given once daily for 6 days and on the 7th day 2 h before and 4 h after LPS injection reversed the LPS-induced changes in extracellular levels of DA, adenosine, glutamate, and hydroxyl radical production. Moreover, LPS-induced decrease in the striatal A2A receptor density was increased by caffeine and KW6002. In order to show the late LPS effect on oxidative damage of DA neurons, the contents of DA, DOPAC, HVA, and hydroxyl radical were determined 72 h after LPS (10 μg) administration into both striata. LPS decreased striatal and substantia nigra content of DA, DOPAC, and HVA while increased striatal but not nigral content of hydroxyl radical. Caffeine (20 mg/kg) and KW60002 (3 mg/kg) given once daily for 6 days and on the 7th day 2 h before and 4 h after intrastriatal injection of LPS normalized the content of DA and its metabolites in both brain regions as well as decreased LPS-induced increase in the striatal level of hydroxyl radical. In conclusion, our data demonstrated antioxidant effects of caffeine and KW6002 in the inflammatory model of PD.

The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson’s disease (PD). In our previous study, we demonstrated that adenosine A2A receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A2A receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. l-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A2A receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage mechanism in early PD.

Investigating Bacterial Sources of Toxicity as an Environmental Contributor to Dopaminergic Neurodegeneration

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as α-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD.

Increased Vesicular Monoamine Transporter Binding during Early Abstinence In Human Methamphetamine Users: Is VMAT2 a Stable Dopamine Neuron Biomarker?

Animal data indicate that methamphetamine can damage striatal dopamine terminals. Efforts to document dopamine neuron damage in living brain of methamphetamine users have focused on the binding of [(11)C]dihydrotetrabenazine (DTBZ), a vesicular monoamine transporter (VMAT2) positron emission tomography (PET) radioligand, as a stable dopamine neuron biomarker. Previous PET data report a slight decrease in striatal [(11)C]DTBZ binding in human methamphetamine users after prolonged (mean, 3 years) abstinence, suggesting that the reduction would likely be substantial in early abstinence. We measured striatal VMAT2 binding in 16 recently withdrawn (mean, 19 d; range, 1-90 d) methamphetamine users and in 14 healthy matched-control subjects during a PET scan with (+)[(11)C]DTBZ. Unexpectedly, striatal (+)[(11)C]DTBZ binding was increased in methamphetamine users relative to controls (+22%, caudate; +12%, putamen; +11%, ventral striatum). Increased (+)[(11)C]DTBZ binding in caudate was most marked in methamphetamine users abstinent for 1-3 d (+41%), relative to the 7-21 d (+15%) and >21 d (+9%) groups. Above-normal VMAT2 binding in some drug users suggests that any toxic effect of methamphetamine on dopamine neurons might be masked by an increased (+)[(11)C]DTBZ binding and that VMAT2 radioligand binding might not be, as is generally assumed, a "stable" index of dopamine neuron integrity in vivo. One potential explanation for increased (+)[(11)C]DTBZ binding is that VMAT2 binding is sensitive to changes in vesicular dopamine storage levels, presumably low in drug users. If correct, (+)[(11)C]DTBZ might be a useful imaging probe to correlate changes in brain dopamine stores and behavior in users of methamphetamine.

Microglial activation is not prevented by tacrolimus but dopamine neuron damage is reduced in a rat model of Parkinson's disease progression

A progressive model of Parkinson's disease has been recently developed in the rat where a unilateral excitotoxic injection into the globus pallidus leads to a gradual loss of dopaminergic neurons in the ipsilateral substantia nigra over a period of at least 6 weeks. In this model microglial activation is observed in the ipsilateral substantia nigra 3 weeks after the lesion and could contribute to neuronal death at this time. The immunosuppressant drug tacrolimus (FK506) reduces dopamine cell death at 3 weeks following a globus pallidus lesion, but not thereafter. Tacrolimus-mediated neuroprotection could result from suppression of microglial activation but the microglial activation at three weeks post-lesion was not much reduced. Microglial activation was observed even in the apparent absence of neuronal death, prompting the suggestion that tacrolimus may prevent, or at least delay, the release of toxic cytokines from activated microglia. By 6 weeks after the GP lesion, even this mechanism fails to protect the dopamine cells from damage.

Treatment of Parkinson’s Disease with Trophic Factors

Trophic factors are proteins that support and protect subpopulations of cells. A number have been reported to act on dopaminergic neurons in vitro and in vivo, making them potential therapeutic candidates for Parkinson's disease. All of these candidate factors protect dopaminergic neurons if given prior to, or with, selective neurotoxins. Fewer trophic factors, primarily glial-derived neurotrophic factor (GDNF) and its relative, neurturin (NRTN; also known as NTN), have been shown to restore function in damaged dopamine neurons after the acute effects of neurotoxins have subsided. A major barrier to clinical translation has been delivery. GDNF delivered by intracerebroventricular injection in patients was ineffective, probably because GDNF did not reach the target, the putamen, and intraputaminal infusion was ineffective, probably because of limited distribution within the putamen. A randomized clinical trial with gene therapy for NRTN is underway, in an attempt to overcome these problems with targeting and distribution. Other strategies are available to induce trophic effects in the CNS, but have not yet been the focus of human research. To date, clinical trials have focused on restoration of function (i.e., improvement of parkinsonism). Protection (i.e., slowing or halting disease progression and functional decline) might be a more robust effect of trophic agents. Laboratory research points to their effectiveness in protecting neurons and even restoring dopaminergic function after a monophasic neurotoxic insult. Utility for such compounds in patients with Parkinson's disease and ongoing loss of dopaminergic neurons remains to be proven.

Paraquat and Parkinson's Disease: Response by Dr. Miller

Paraquat and Parkinson's Disease: Response by Dr. Miller

Paraquat: The Red Herring of Parkinson's Disease Research

After the discovery that 1-methyl 4-phenyl 1,2,3,6tetrahydropyridine (MPTP) induced a parkinsonian syndrome in individuals who inadvertently injected the by-product of illicit drug manufacturing (Langston et al., 1983; Ramachandiran et al., 2007; Richardson et al., 2005), numerous investigators made the observation that the herbicide paraquat (N,N#dimethyl-4,4#-bipyridinium dichloride, CAS# 1910-42-5) bore a degree of structural similarity to the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPPþ; CAS# 3979499-5; see Fig. 1). Since that time, well over 100 papers have been published using paraquat as a cellular or animal model of Parkinson’s disease under the premise that the two compounds are structurally similar. Direct injections of paraquat into the brain demonstrated its ability to damage the dopamine system and helped to identify some potential toxicological manifestations of the compound, but at the same time such studies largely ignored the physical obstacle presented by the blood-brain barrier and the complicated architecture of the brain (Barbeau et al., 1985; Corasaniti et al., 1992; Iannone et al., 1989; Liou et al., 1996). Indeed, it is intuitively obvious that an injection of a strong oxidant directly into nigral neurons or direct application to isolated mitochondria will cause damage, but is this toxicologically relevant? When paraquat is administered systemically, the results have been equivocal (Brooks et al., 1999; Corasaniti et al., 1992; McCormack et al., 2002; Widdowson et al., 1996). Some laboratories observe a consistent loss in nigral neurons (of no more than 25%), but not loss of striatal dopamine, which is considered a hallmark of the disease. Other laboratories have failed to demonstrate toxicity after systemic administration. In contrast, systemically administered MPTP kills a much higher percentage of the neurons (> 90%) and leads to a consistent loss of dopamine, both of which exhibit a clear dose response. Thus, while it does appear that after i.p. injection paraquat enters the brain of certain rodent species and strains, it is not clear if this is at all relevant to the human condition. Many of these widely cited studies, on which the ability of paraquat to damage dopamine neurons is based, are quite artificial from an exposure standpoint and only served to perpetuate the fishy and tenuous connection between the compounds. In retrospect, evidence suggests that the aforementioned research efforts have been somewhat misguided. It has become a veritable dogma that since paraquat looks like MPPþ that it must act like MPPþ (coincidentally, the structure of the related cyperquat is truly identical to that of MPPþ, see Fig. 1). Recent studies cast serious doubt upon this assertion. It has been assumed that paraquat is transported into the dopamine neuron by the dopamine transporter, like MPPþ. Further, it has been assumed (and stated in numerous reviews) that paraquat is a complex I inhibitor. Recent data demonstrate neither to be physiologically plausible. Studies using cell lines expressing the dopamine transporter reveal that paraquat is not transported via the dopamine transporter, nor does it impair dopamine uptake (Ramachandiran et al., 2007; Richardson et al., 2005). Paraquat can inhibit complex I, but only at an IC50 of 7mM, which would be biologically impossible, especially given the fact that in actively respiring mitochondria paraquat does not display further accumulation. Meanwhile, MPPþ inhibits complex I in the 30lM range, which considering the ability of MPPþ to be specifically accumulated in the dopamine neuron by the dopamine transporter and that actively respiring mitochondria do accumulate the toxic compound, is indeed physiologically relevant (Richardson et al., 2005, 2007). For comparison’s sake, the lipophilic piscicide rotenone inhibits complex I in the low nanomolar range. While paraquat can induce redox cycling in the cytosol, which could alter mitochondrial function indirectly, the data suggest that when compared to MPPþ or rotenone, paraquat 1 For correspondence via. E-mail: Gray.miller@emory.edu.

Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage

Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

Nitrotyrosine as a marker for peroxynitrite‐induced neurotoxicity: the beginning or the end of the end of dopamine neurons?

This review examines the involvement of nitrotyrosine as a marker for peroxynitrite-mediated damage in the dopamine neuronal system. We propose that the dopamine neuronal phenotype can influence the cytotoxic signature of peroxynitrite. Dopamine and tetrahydrobiopterin are concentrated in dopamine neurons, and both are essential for their proper neurochemical function. It is not well appreciated that dopamine and tetrahydrobiopterin are also powerful blockers of peroxynitrite-induced tyrosine nitration. What is more, the reaction of peroxynitrite with either dopamine or tetrahydrobiopterin forms chemical species (i.e. o-quinones and pterin radicals, respectively) whose cytotoxic effects may be manifested far earlier than nitrotyrosine formation in the course of dopamine neuronal damage. A better understanding of how the dopamine neuronal phenotype modulates the effects of reactive nitrogen species could reveal early steps in drug- and disease-induced damage to the dopamine neuron and form the basis for rational, protective therapies.

Tetrahydrobiopterin prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine. TH is inhibited and nitrated at tyrosine residues in vitro by the reactive nitrogen species peroxynitrite and nitrogen dioxide (NO2) and in vivo by drugs that damage dopamine neurons. Tetrahydrobiopterin, which is the essential cofactor for TH and is concentrated in dopamine neurons, completely blocks nitration of tyrosine residues in TH caused by peroxynitrite or NO2. Various tetrahydro- and dihydro-analogs of tetrahydrobiopterin, including 6,7-dimethyl-tetrahydropterin, 6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, tetrahydropterin, 7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin, also prevent nitration of tyrosines caused by the reactive nitrogen species. Biopterin and pterin, the fully oxidized forms of the pterin molecule, fail to block peroxynitrite- or NO2-induced nitration of TH. Reduced pterins prevent neither the inhibition of TH activity nor cysteine modification caused by peroxynitrite or NO2, despite blocking tyrosine nitration. However, dithiothreitol prevents and reverses these effects on TH of tetrahydrobiopterin and reactive nitrogen species. Using an enhanced green fluorescent protein-TH fusion construct as a real-time reporter of intracellular tyrosine nitration, tetrahydrobiopterin was found to prevent NO2-induced tyrosine nitration in intact cells but to leave TH activity inhibited. These results indicate that tetrahydrobiopterin prevents the tyrosine-nitrating properties of peroxynitrite and NO2. Tetrahydrobiopterin-derived radical species formed by reaction with reactive nitrogen species may account for inhibition of TH via mechanisms that do not involve tyrosine nitration.

Peroxynitrite inactivates the human dopamine transporter by modification of cysteine 342: potential mechanism of neurotoxicity in dopamine neurons.

Peroxynitrite (ONOO(-)) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease. ONOO(-) causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO(-) is manifested as a reduction in V(max). Cysteine, dithiothreitol, glutathione, and N-acetyl-cysteine, reagents that interact directly with ONOO(-), prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO(-), whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO(-) the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO(-) treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO(-) reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO(-). Sensitivity to ONOO(-) was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO(-) through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function in vivo (e.g., methamphetamine and MPTP) may exert their effects through ONOO(-)-mediated oxidative stress.

Peroxynitrite Inactivates the Human Dopamine Transporter by Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in Dopamine Neurons

Peroxynitrite (ONOO⁻) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson9s Disease. ONOO⁻ causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO⁻ is manifested as a reduction in<i>V</i>_max. Cysteine, dithiothreitol, glutathione, and <i>N</i>-acetyl-cysteine, reagents that interact directly with ONOO⁻, prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO⁻, whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO⁻ the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO⁻ treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO⁻ reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO⁻. Sensitivity to ONOO⁻ was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO⁻ through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function <i>in vivo</i> (e.g., methamphetamine and MPTP) may exert their effects through ONOO⁻-mediated oxidative stress.

Glial cell line derived neurotrophic factor promotes the recovery of dopamine neurons damaged by 6-hydroxydopamine in vitro

Glial cell line derived neurotrophic factor (GDNF) has been shown to be a potent neurotrophic factor for dopamine neurons in culture and to prevent the loss of substantia nigra dopamine neurons following in vivo lesions with 6-hydroxydopamine (6-OHDA). In this study we used mesencephalic cultures containing both neurons and glia to examine whether GDNF protects dopamine neurons from 6-OHDA toxicity in vitro. Our data show that GDNF does not prevent the loss of dopamine neurons caused by treatment with 6-OHDA in vitro. However, continuous exposure to GDNF increases the high affinity dopamine uptake in cultures treated with 6-OHDA, suggesting that it enhances the growth of damaged dopamine neurons. We also show that in vitro treatment with 6-OHDA causes widespread cell death in mesencephalic cultures, which is not restricted to dopamine neurons. The lack of selectivity of 6-OHDA toxicity when applied in vitro may explain the inability of GDNF to prevent the loss of dopamine neurons in mesencephalic cultures. The stimulation of the growth of 6-OHDA damaged dopamine neurons by GDNF, observed in our study, suggests that it may prove beneficial in the treatment of injured dopamine neurons.

Acidic fibroblast growth factor and catecholamines synergistically up-regulate tyrosine hydroxylase activity in developing and damaged dopamine neurons in culture.

Our previous studies indicate that, in certain non-catecholamine (CA) neurons, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the obligatory interaction of acidic fibroblast growth factor (aFGF) and a CA activator. In this study, we sought to determine whether these same differentiation factors also play a role in regulating existing TH expression in CA neurons. Thus, the effects of exogenous aFGF and CAs on TH were studied in developing or toxin-damaged dopamine (DA) neurons from the embryonic day 15 rat ventral midbrain, where it was likely to be at physiologically low levels. Cultures were incubated with various concentrations of aFGF, DA, or aFGF and DA. Some cultures were first damaged with 2.5 microM 1-methyl-4-phenylpyridinium. In developing DA neurons, an 80% increase in TH activity was found only after co-treatment with aFGF (100 ng/ml) and DA (1 microM) or other monoamines. Likewise, in damaged DA neurons, aFGF and DA reversed the 50% loss in TH activity caused by toxin. This was observed within 4 h of treatment and was not associated with changes in the number or appearance of DA neurons, suggesting a biochemical rather than a trophic effect. Pretreatment with protein or RNA synthesis inhibitors eliminated the increase. In PC12 cells, where TH is highly expressed, activity was unaltered by treatment. We conclude that the aFGF and CAs may be involved in not only the initiation but also the regulation of TH.

Effect of dopaminergic drugs on the in vivo binding of [3H]WIN 35,428 to central dopamine transporters

[11C]WIN 35,428 (also designated [11C]CFT) is now being used in several positron emission tomography (PET) centers to image dopamine (DA) transporter sites in the mammalian brain. Whether and to what extent in vivo WIN 35,428 binding is influenced by intra- and extrasynaptic dopamine levels are largely unknown. The purpose of the present study was to evaluate the effects of various drugs, known to affect DA levels and release, on the binding of [3H]WIN 35,428 to central DA transporters in the mouse brain. D-Amphetamine, which releases DA from neurons and blocks the DA transporter directly, inhibited striatal [3H]WIN 35,428 binding in dose-dependent manner. Similarly, alpha-methyl-DL-p-tyrosine, an inhibitor of tyrosine hydroxylase, blocked [3H]WIN 35,428 binding, possibly via competitive inhibition by the metabolite p-hydroxyamphetamine. Specific binding of [3H]WIN 35,428 was insensitive to changes in synaptic DA levels caused by pretreatment of the animals with high doses of D2 receptor agonists (apomorphine, bromocriptine), antagonists (haloperidol) or the inhibitor of dopaminergic neuron firing gamma-butyrolactone (GBL). High doses (> 50 mg/kg) of L-DOPA (in combination with benserazide), however, reduced [3H]WIN 35,428 binding significantly, yet for a relatively short time (approximately 2.5 h). Chronic treatment with L-deprenyl elicited no changes in in vivo [3H]WIN 35,428 accumulation in the striatum. Neurotoxic damage of DA neurons caused by administration of high doses of amphetamine was detected in the striatum by a significant reduction in [3H]WIN 35,428 binding 7 days after cessation of amphetamine treatment. Thus, [3H]WIN 35,428 binding was only affected by neurotoxic loss of neurons, by administration of uptake inhibitors, or by some treatments which significantly elevate DA levels. Compounds which inhibit DA release or deplete DA acutely do not increase [3H]WIN 35,428 binding, suggesting that normal or "resting" levels of DA are not sufficient to alter [3H]WIN 35,428 binding in vivo. These findings are important for our understanding of the function and regulation of the DA transporter, as well as the in vivo binding of the radioligand [3H/11 C]WIN 35,428. Moreover, they will be important for the interpretation of PET studies in which [11C]WIN 35,428 is used to assess the integrity of dopaminergic neurons.

GM1 ganglioside rescues substantia nigra pars compacta neurons and increases dopamine synthesis in residual nigrostriatal dopaminergic neurons in MPTP-treated mice.

GM1 ganglioside has been shown to stimulate recovery of the damaged dopamine system under a number of different circumstances. In addition to rescue of damaged dopamine neurons, the present study assessed the ability of GM1 to enhance the synthesis of dopamine in remaining nigrostriatal neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure. There was a significantly greater accumulation of L-dopa 30 min after aromatic amino acid decarboxylase inhibition with NSD-1015 (100 mg/kg) and an increase in the ratio of L-dopa to dopamine in MPTP+GM1-treated mice than in mice that received only MPTP. This effect of GM1 on dopamine synthesis was dependent upon the degree of initial damage to the nigrostriatal dopamine system. That is, the GM1 effect on dopamine synthesis could not be demonstrated in mice with greater than 95% striatal dopamine loss and 75% substantia nigra dopamine neuron loss. These results suggest that in addition to previously reported effects of GM1 on rescue and repair of dopaminergic neurons, GM1 may also have the ability to enhance dopamine synthesis in residual dopaminergic neurons. Direct effects on dopamine neurochemistry may contribute to functional improvement seen after GM1 treatment in various models of parkinsonism.