Dopamine and vesicular monoamine transport loss supports incidental Lewy body disease as preclinical idiopathic Parkinson.

Incidental Lewy body disease (ILBD) is a neuropathological diagnosis of brains with Lewy bodies without clinical neuropsychiatric symptoms. Dopaminergic deficits suggest a relationship to preclinical Parkinson's disease (PD). We now report a subregional pattern of striatal dopamine loss in ILBD cases, with dopamine found significantly decreased in the putamen (-52%) and only to a lower extent in the caudate (-38%, not statistically significant); this is similar to the pattern in idiopathic PD in various neurochemical and in vivo imaging studies. We aimed to find out if our recently reported impaired storage of dopamine in striatal synaptic vesicles prepared from striatal tissue of cases with idiopathic PD might be an early or even causative event. We undertook parallel measurements of [3H]dopamine uptake and vesicular monoamine transporter (VMAT)2 binding sites by the specific label [3H]dihydrotetrabenazine on vesicular preparation from caudate and putamen in ILBD. Neither specific uptake of dopamine and binding of [3H]dihydrotetrabenazine, nor mean values of the calculated ratios of dopamine uptake and VMAT2 binding, a measure of uptake rate per transport site, were significantly different between ILBD and controls. ATP-dependence of [3H]dopamine uptake revealed significantly higher rates in putamen than in caudate at saturating concentrations of ATP in controls, a subregional difference lost in ILBD. Our findings support a loss of the normally higher VMAT2 activity in putamen as a contributing factor to the higher susceptibility of the putamen to dopamine depletion in idiopathic PD. Moreover, we suggest ILBD postmortem tissue as a valuable source for testing hypotheses on processes in idiopathic PD.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

The psychostimulant (±)-cis-4,4'-dimethylaminorex (4,4'-DMAR) interacts with human plasmalemmal and vesicular monoamine transporters.

(±)-cis-4,4'-Dimethylaminorex (4,4'-DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4'-DMAR on human high-affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4'-DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4'-DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC50 values < 2 µM). Release assays identified 4,4'-DMAR as a substrate type releaser, capable of inducing transporter-mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4'-DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4-methylenedioxymethylamphetamine (MDMA). This study identified 4,4'-DMAR as a potent non-selective monoamine releasing agent. In contrast to the known effects of aminorex and 4-methylaminorex, 4,4'-DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4'-DMAR may result in long-term neurotoxicity.

Early Paradoxical Increase of Dopamine: A Neurochemical Study of Olfactory Bulb in Asymptomatic and Symptomatic MPTP Treated Monkeys.

Parkinson's disease (PD) is a neurodegenerative disease with both motor and non-motor manifestations. Hyposmia is one of the early non-motor symptoms, which can precede motor symptoms by several years. The relationship between hyposmia and PD remains elusive. Olfactory bulb (OB) pathology shows an increased number of olfactory dopaminergic cells, protein aggregates and dysfunction of neurotransmitter systems. In this study we examined tissue levels of dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and their metabolites, of noradrenaline (NA) and of the amino acid neurotransmitters aspartate, glutamate, taurine and γ-aminobutyric acid in OBs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated Macaca fascicularis in different stages, including monkeys who were always asymptomatic, monkeys who recovered from mild parkinsonian signs, and monkeys with stable moderate or severe parkinsonism. DA was increased compared to controls, while neither NA and 5-HT nor the amino acid neurotransmitters were significantly changed. Furthermore, DA increased before stable motor deficits appear with +51% in asymptomatic and +96% in recovered monkeys. Unchanged DA metabolites suggest a special metabolic profile of the newly formed DA neurons. Significant correlation of homovanillic acid (HVA) with taurine single values within the four MPTP groups and of aspartate with taurine within the asymptomatic and recovered MPTP groups, but not within the controls suggest interactions in the OB between taurine and the DA system and taurine and the excitatory neurotransmitter triggered by MPTP. This first investigation of OB in various stages after MPTP administration suggests that the DA increase seems to be an early phenomenon, not requiring profound nigrostriatal neurodegeneration or PD symptoms.

The profile of mephedrone on human monoamine transporters differs from 3,4-methylenedioxymethamphetamine primarily by lower potency at the vesicular monoamine transporter.

Mephedrone (4-methylmethcathinone, MMC) and 3,4-methylenedioxymethamphetamine (MDMA) are constituents of popular party drugs with psychoactive effects. Structurally they are amphetamine-like substances with monoamine neurotransmitter enhancing actions. We therefore compared their effects on the human monoamine transporters using human cell lines stably expressing the human noradrenaline, dopamine and serotonin transporter (NET, DAT and SERT); preparations of synaptic vesicles from human striatum in uptake experiments; and a superfusion system where releasing effects can be reliably measured. MMC and MDMA were equally potent in inhibiting noradrenaline uptake at NET, with IC50 values of 1.9 and 2.1 µM, respectively. Compared to their NET inhibition potency, both drugs were weaker uptake inhibitors at DAT and SERT, with MMC being more potent than MDMA at DAT (IC50: 5.9 vs 12.6 µM) and less potent than MDMA at SERT (IC50: 19.3 vs 7.6 µM). MMC and MDMA both induced concentration-dependently [(3)H]1-methyl-4-phenylpyridinium-release from NET-, DAT or SERT-expressing cells which was clearly transporter-mediated release as demonstrated by the selective inhibitory effects of nmolar to low µmolar concentrations of desipramine, GBR 12909 and fluoxetine, respectively. MMC and MDMA differed most in their inhibition of [(3)H]dopamine uptake by synaptic vesicles from human striatum with MDMA being 10-fold more potent than MMC (IC50: 20 vs 223 µM) and their ability to release [(3)H]dopamine from human vesicular monoamine transporter expressing SH-SY5Y neuroblastoma cells in which MDMA seems to have a stronger effect. Our findings give a molecular explanation to the lower long-term neurotoxicity of MMC compared to MDMA.

Is Parkinson's disease a vesicular dopamine storage disorder? Evidence from a study in isolated synaptic vesicles of human and nonhuman primate striatum.

The cause of degeneration of nigrostriatal dopamine (DA) neurons in idiopathic Parkinson's disease (PD) is still unknown. Intraneuronally, DA is largely confined to synaptic vesicles where it is protected from metabolic breakdown. In the cytoplasm, however, free DA can give rise to formation of cytotoxic free radicals. Normally, the concentration of cytoplasmic DA is kept at a minimum by continuous pumping activity of the vesicular monoamine transporter (VMAT)2. Defects in handling of cytosolic DA by VMAT2 increase levels of DA-generated oxy radicals ultimately resulting in degeneration of DAergic neurons. Here, we isolated for the first time, DA storage vesicles from the striatum of six autopsied brains of PD patients and four controls and measured several indices of vesicular DA storage mechanisms. We found that (1) vesicular uptake of DA and binding of the VMAT2-selective label [(3)H]dihydrotetrabenazine were profoundly reduced in PD by 87-90% and 71-80%, respectively; (2) after correcting for DA nerve terminal loss, DA uptake per VMAT2 transport site was significantly reduced in PD caudate and putamen by 53 and 55%, respectively; (3) the VMAT2 transport defect appeared specific for PD as it was not present in Macaca fascicularis (7 MPTP and 8 controls) with similar degree of MPTP-induced nigrostriatal neurodegeneration; and (4) DA efflux studies and measurements of acidification in the vesicular preparations suggest that the DA storage impairment was localized at the VMAT2 protein itself. We propose that this VMAT2 defect may be an early abnormality promoting mechanisms leading to nigrostriatal DA neuron death in PD.

The noradrenaline transporter as site of action for the anti-Parkinson drug amantadine.

Amantadine is an established antiparkinsonian drug with a still unclear molecular site of action. In vivo studies on rodents, in vitro studies on tissue of rodents as well as binding studies on post mortem human tissue implicate monoamine transporters and NMDA receptors. In order to re-examine its action at human variants of these proteins on intact cells we established cells stably expressing the human NR1/2A NMDA-receptor, noradrenaline transporter (NAT) or dopamine transporter (DAT) and tested the activity of amantadine in patch-clamp, uptake, release, and cytotoxicity experiments. Amantadine was less potent in blockade of NMDA-induced inward currents than in blockade of noradrenaline uptake and in induction of inward currents in NAT expressing cells. It was 30 times more potent in blocking uptake in NAT- than in DAT cells. Amantadine induced NAT-mediated release at concentrations of 10-100 µM in superfusion experiments and blocked NAT-mediated cytotoxicity of the parkinsonism inducing neurotoxin 1-methyl-4-phenyl-pyridinium (MPP(+)) at concentrations of 30-300 µM, whereas 300-1000 µM amantadine was necessary to block NMDA-receptor mediated cytotoxicity. Similar to amphetamine, amantadine was inactive at α(2A)-adrenergic receptors and induced reverse noradrenaline transport by NAT albeit with smaller effect size. Thus, amantadine acted as "amphetamine-like releaser" with selectivity for the noradrenergic system. These findings and differences with memantine, which had been reported as less efficient antiparkinsonian drug than amantadine but in our hands was significantly more potent at the NMDA-receptor, suggest contributions from a noradrenergic mechanism in the antiparkinsonian action of amantadine.

Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner.

The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.

Pharmacological characterization of ecstasy synthesis byproducts with recombinant human monoamine transporters.

Ecstasy samples often contain byproducts of the illegal, uncontrolled synthesis of N-methyl-3,4-methylenedioxy-amphetamine or 3,4-methylenedioxymethamphetamine (MDMA). MDMA and eight chemically defined byproducts of MDMA synthesis were investigated for their interaction with the primary sites of action of MDMA, namely the human plasmalemmal monamine transporters for norepinephrine, serotonin, and dopamine [(norepinephrine transporter (NET), serotonin transporter (SERT), and dopamine transporter (DAT)]. SK-N-MC neuroblastoma and human embryonic kidney cells stably transfected with the transporter cDNA were used for uptake and release experiments. Two of the eight compounds, 1,3-bis (3,4-methylenedioxyphenyl)-2-propanamine (12) and N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (13) had uptake inhibitory potencies with IC50 values in the low micromolar range similar to MDMA. Compounds with nitro instead of amino groups and a phenylethenyl instead of a phenylethyl structure or a formamide or acetamide modification had IC50 values beyond 100 microM. MDMA, 12, and 13 were examined for induction of carrier-mediated release by superfusion of transporter expressing cells preloaded with the metabolically inert transporter substrate [3H]1-methyl-4-phenylpyridinium. MDMA induced release mediated by NET, SERT, or DAT with EC50 values of 0.64, 1.12, and 3.24 microM, respectively. 12 weakly released from NET- and SERT-expressing cells with maximum effects less than one-tenth of that of MDMA and did not release from DAT cells. 13 had no releasing activity. 12 and 13 inhibited release induced by MDMA, and the concentration dependence of this effect correlated with their uptake inhibitory potency at the various transporters. These results do not support a neurotoxic potential of the examined ecstasy synthesis byproducts and provide interesting structure-activity relationships on the transporters.

Dopamine inhibits cell growth and cell cycle by blocking ribonucleotide reductase.

Dopamine (DA) is a classical neurotransmitter modulating various brain functions by acting on its specific receptors. In addition, DA is a reactive molecule that has been implicated in neurodegeneration, especially in Parkinson's disease. Here we show that DA inhibited cell growth of dopamine transporter transfected cells by intracellularly blocking cell cycle progression. To pinpoint the site of this effect, we measured DNA distribution and 5-bromo-2'-deoxyuridine (BrdU) incorporation, as well as the levels of the key cell cycle proteins. DA increased number of cells with a G1 DNA content, decreased BrdU incorporation and simultaneously increased cyclin A but had no effect on cyclin D2, D3, E, nor on cdk4 and p21. These results narrowed down the DA effect to the beginning of S phase, suggesting inhibition of the ribonucleotide reductase, an enzyme essential for DNA synthesis. Indeed, measurement of enzyme activity in situ revealed that DA, within 1h of addition to cells labelled with [3H]cytidine, strongly reduced the cell content of [3H]2'-deoxycytidine 5'-triphophate. The time course of this DA effect preceded the cell cycle progression. This novel molecular mechanism of intracellular DA action independent of plasmamembrane receptors may be involved in processes controlling the development and survival of brain dopaminergic neurons.

alpha-Synuclein selectively increases manganese-induced viability loss in SK-N-MC neuroblastoma cells expressing the human dopamine transporter.

The established or potentially toxic agents implicated in the nigral cell death in Parkinson's disease, dopamine, 1-methyl-4-phenylpyridinium (MPP(+)), iron, and manganese, were examined as to their effects on the viability of cells overexpressing alpha-synuclein. SK-N-MC neuroblastoma cells stably expressing the human dopamine transporter were transfected with human alpha-synuclein and cell clones with and without alpha-synuclein immunoreactivity were obtained. Cells were exposed for 24-72 h to 1-10 microM dopamine, 0.1-3 microM MPP(+), 0.1-1 mM FeCl(2) or 30-300 microM MnCl(2) added to the culture medium. There was no difference between cells expressing alpha-synuclein and control cells after exposure to dopamine, MPP(+) or FeCl(2). However, MnCl(2) resulted in a significantly stronger decreased viability of cells overexpressing alpha-synuclein after 72 h. These findings suggest that manganese may co-operate with alpha-synuclein in triggering neuronal cell death such as seen in manganese parkinsonism. The relevance of our observations for the pathoetiology of Parkinson's disease proper remains to be determined.

Zn2+ modulates currents generated by the dopamine transporter: parallel effects on amphetamine-induced charge transfer and release.

The psychostimulant drug amphetamine increases extracellular monamines in the brain acting on neurotransmitter transporters, especially the dopamine transporter. Mediated by this plasmalemmal pump, amphetamine does not only induce release but also charge transfer which might be involved in the release mechanism. To study a potential link between the two phenomena, we used Zn(2+) as an acute regulatory agent which modulates dopamine uptake by a direct interaction with the transporter protein. Charge transfer was investigated in patch-clamp experiments on HEK 293 cells stably expressing the human dopamine transporter, release was studied in superfusion experiments on cells preloaded with the metabolically inert transporter substrate [(3)H]1-methyl-4-phenylpyridinium. Ten micromoles of Zn(2+) had only minor effects in the absence of amphetamine but stimulated release and inward currents induced by amphetamine depending on the concentration of the psychostimulant: the effect of 0.2 microM was not significantly modulated, whereas the effect of 1 and 10 microM amphetamine was stimulated, and the stimulation by Zn(2+) was significantly stronger at 10 microM than at 1 microM amphetamine. The stimulatory action of Zn(2+) on release and inward current was in contrast to its inhibitory action on dopamine uptake. This supports a release mechanism of amphetamine different from facilitated exchange diffusion but involving ion fluxes through the dopamine transporter.

Cellular effects of dopamine--beyond oxidative mechanisms.

Cell cycle blockers inhibit growth in dividing cells, but promote survival of differentiated cells, including neurons. Low micromolar dopamine profoundly inhibited cell growth in dopamine transporter transfected SK-N-MC neuroblastoma cells by cell cycle arrest at G(1). This effect was independent of oxy radical formation, antagonized by transporter block, abolished by FeCl(3) and mimicked by the iron chelator deferoxamine. We propose that dopamine inhibits cell growth by its ability to chelate intracellular iron. This novel biological action unrelated to neurotransmitter receptors, second messengers or oxidative stress, observed in human neuroblastoma cells of striatal origin, may be important for cell differentiation during neurodevelopment and survival of differentiated dopamine (nigral) neurons.