AB1591P Sigma-AldrichAnti-Dopamine Transporter Antibody

L-DOPA is therapeutically efficacious in patients with Parkinson's disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.

DYT11 myoclonus-dystonia (M-D) syndrome is a neurological movement disorder characterized by myoclonic jerks and dystonic postures or movement that can be alleviated by alcohol. It is caused by mutations in SGCE encoding ε-sarcoglycan (ε-SG); the mouse homolog of this gene is Sgce. Paternally-inherited Sgce heterozygous knockout (Sgce KO) mice exhibit myoclonus, motor impairment and anxiety- and depression-like behaviors, modeling several clinical symptoms observed in DYT11 M-D patients. The behavioral deficits are accompanied by abnormally high levels of dopamine and its metabolites in the striatum of Sgce KO mice. Neuroimaging studies of DYT11 M-D patients show reduced dopamine D2 receptor (D2R) availability, although the possibility of increased endogenous dopamine, and consequently, competitive D2R occupancy cannot be ruled out.The protein levels of striatal D2R, dopamine transporter (DAT), and dopamine D1 receptor (D1R) in Sgce KO mice were analyzed by Western blot. The striatal dopamine release after amphetamine injection in Sgce KO mice were analyzed by microdialysis in vivo. The striatal D2R was significantly decreased in Sgce KO mice without altering DAT and D1R. Sgce KO mice also exhibited a significant increase of dopamine release after amphetamine injection in comparison to wild-type (WT) littermates.The results suggest ε-SG may have a role in the regulation of D2R expression. The loss of ε-SG results in decreased striatal D2R, and subsequently leads to increased discharge of dopamine which could contribute to the behavioral impairment observed in DYT11 dystonia patients and in Sgce KO mice. The results suggest that reduction of striatal D2R and enhanced striatal dopamine release may contribute to the pathophysiology of DYT11 M-D patients.

Prenatal systemic inflammation has been implicated in neurological diseases, but optimal animal models have not been developed. We investigated whether a partial genetic deletion of glial cell line-derived neurotrophic factor (Gdnf(+/-)) increased vulnerability of dopamine (DA) neurons to prenatal lipopolysaccharide (LPS). LPS [0.01 mg/kg intraperitoneal (i.p.)] or saline was administered to wild-type (WT) or Gdnf(+/-) pregnant mice on gestational day 9.5. Male offspring were examined at 3 weeks, 3 and 12 months of age. There was a progressive degeneration of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) with age in Gdnf(+/-) but not in WT mice, with no observed effects on locus coeruleus (LC) noradrenergic neurons or DA neurons of the ventral tegmental area. Inflammatory markers were elevated in SN of LPS treated offspring, with exacerbation in Gdnf(+/-) mice. Intracellular accumulation of α-synuclein (α-syn) immunoreactivity in DA neurons of SN was observed in all groups of Gdnf(+/-) and in WT mice with prenatal LPS, with altered distribution between pars reticulata (pr) and pars compacta (pc). The findings suggest that prenatal LPS leads to accelerated neuropathology in the SN with age, and that a partial loss of GDNF exacerbates these effects, providing a novel model for age-related neuropathology of the nigrostriatal DA system.

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.

The aim of this study was to investigate the role of gamma-synuclein in the rewarding effects of chronic cocaine administration and its putative interaction with the dopamine transporter (DAT). For this purpose, regulatable lentiviruses driving overexpression of the rat gamma-synuclein or DAT have been prepared, as well as lentiviruses expressing siRNAs, aimed at silencing either DAT or gamma-synuclein mRNA expression. Overexpression of DAT in the nucleus accumbens (NAc) induced a 35% decrease in locomotor activity, which could be abolished when the same animal was fed doxycycline. Furthermore, local inhibition of DAT in the NAc, using lentiviruses expressing siRNAs targeted against DAT, resulted in significant hyperlocomotion activity (72% increase over controls). By contrast, overexpression of gamma-synuclein in the NAc alone had no effect, while local silencing lead to a significant decrease in cocaine-induced locomotor activity (47% decrease compared with controls). Surprisingly, coinjection lentiviruses expressing DAT and gamma-synuclein - leading to overexpression of both proteins in the NAc - resulted in a strong increase in cocaine-induced rat locomotor activity (52% increase compared with controls), which was abolished upon locally silencing these genes, suggesting a synergetic role of both proteins, possibly mediated through a direct interaction.

We examined the expression of the dopamine transporter in rat and bullfrog retinas by immunohistochemistry. In both species, the dopamine transporter was strongly expressed in somata and processes of all dopaminergic amacrine cells. In contrast, no immunoreactivity for dopamine transporter was observed in cholinergic amacrine cells. In rat dopaminergic interplexiform cells, dopamine transporter immunoreactivity was also observed on the ascending processes terminating in the outer plexiform layer. Furthermore, the labeling for dopamine transporter diffusely appeared in both outer and inner plexiform layers. This expression profile of the dopamine transporter suggests that dopamine may be taken up not only in the synapses but also extrasynaptically by dopamine transporter, diffusely distributed in both plexiform layers.

RATIONALE: Repeated maternal separations profoundly alter the adult stress response, the development of the hypothalamic-pituitary-adrenal axis, and prominently, the GABAergic and monoaminergic systems. These neural changes are postulated to influence the vulnerability to drugs of abuse implicating glucocortocoids in the behavioral responses to psychomotor stimulants.OBJECTIVE: To investigate whether repeated brief maternal separation stress increases behavioral sensitization to cocaine in adult male and female mice, and to assess any concurrent changes in hippocampal glucocorticoid receptors and accumbal dopamine transporters.METHODS: Half of the litters were separated from the nest for 1 h/day from post-natal days 1 to 13. Starting on post-natal day 50, all mice were injected with either cocaine (10.0 mg/kg) or saline for 10 consecutive days. Locomotor activity was assessed in an open field on days 50, 54 and 59 via a tracking system. Approximately 10 and 40 days later, all mice were challenged with 7.5 mg/kg cocaine.RESULTS: Repeated maternal separation increased the hyperlocomotor response to 10.0 mg/kg cocaine regardless of gender. During expression tests (days 69/71, 99), male, but not female, mice with a history of maternal separation exhibited significant sensitized hyperactivity in response to cocaine. Male mice that were maternally separated and had no history of cocaine sensitization, demonstrated cross-sensitization to 7.5 mg/kg cocaine. Immunohistochemical analysis revealed that the hippocampal CA1 glucocorticoid receptor and nucleus accumbens dopamine transporter proteins were expressed more in females than in males, regardless of maternal separation experience.CONCLUSIONS: Repeated maternal separation is a stressor that can induce heightened sensitivity to low doses of cocaine, as expressed by hyperactivity. Furthermore, sex differences in glucocorticoid receptor and dopamine transporter expression may be responsible for the sexual dimorphic expression of behavioral sensitization to cocaine.

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.

Alpha-synuclein accumulates in Lewy bodies in idiopathic Parkinson's disease. Neither the normal function nor contribution of alpha-synuclein to the pathophysiology of neurodegeneration is known. Here we show that a normal function of alpha-synuclein is the negative modulation of human dopamine transporter (hDAT) activity. In cotransfected Ltk(-) cells, alpha-synuclein attenuated the reuptake of dopamine by hDAT, in a manner dependent on expression levels of alpha-synuclein. Alpha-synuclein-mediated inhibition of hDAT activity was independent of expression vectors, cell types and methods of transfection. The alpha-synuclein-mediated decrease in DAT activity occurred through diminished uptake velocity of dopamine, without changes in the affinity of hDAT for dopamine. Co-immunoprecipitation studies confirmed the formation of a stable complex between alpha-synuclein and DAT, through direct protein:protein interactions. Thus, under normal (non-toxic) expression conditions, alpha-synuclein negatively modulates dopamine uptake by DAT.

Oxidative stress has been recently considered as a mediator of nerve cell death in several neurodegenerative diseases. We studied the effect of the parkinsonism-inducing toxine 1-methyl-4-phenyl-pyridine (MPP+) on several parameters of cell distress using native and neuronal PC12 cells. Then, since estrogens have been reported to prevent neuronal degeneration caused by oxidative damage, we investigated the ability of 17beta- estradiol (E2); two Ginkgo biloba extracts, EGb 761 and Cp 202; as well as two flavonoids, quercetin and kaempferol, to rescue PC12 cells submitted to MPP+- induced oxidative stress. Our results consistently show that both Ginkgo biloba extracts could prevent cell death in native and neuronal PC12 cells, while in neuronal PC12 cells also quercetin and E2 could reverse MPP+ neurotoxic effet. Western blot analysis demonstrated that MPP+ injuries might modulate dopamine transporter (DAT) protein expression but not estrogen receptor beta (ERbeta) protein expression. EGb 761 and Cp 202 also modulate DAT and ERbeta protein expression in neuronal cells. From these studies, we outline the importance of testing estrogen-like plant-derived molecules as potent antioxidants and examine their effect on protein expression.