Christian Pifl

Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d‐ and l‐amphetamine as well as substrate‐induced release of preloaded N‐methyl‐4‐[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium‐dependent and was inhibited by ouabain and cocaine, which also prevented substrate‐induced release of MPP+. Patch‐clamp recordings revealed that all four substrates elicited voltage‐dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch‐clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d‐amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate‐induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.

Central control of fever and female body temperature by RANKL/RANK

Receptor-activator of NF-κB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rankfloxed deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rankfloxed deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1β and TNFα. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE2/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rankfloxed mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation.

Globus pallidus dopamine and Parkinson motor subtypes Clinical and brain biochemical correlation

Background: Patients with Parkinson disease (PD) may be akinetic/rigid, be tremor dominant, or have comparable severity of these motor symptoms (classic). The pathophysiologic basis of different PD phenotypes is unknown. This study assessed pallidal and striatal dopamine level patterns in different motor subgroups of PD and normal control brains. Methods: Globus pallidus and striatum dopamine (DA) levels were measured with high performance liquid chromatography in eight autopsy confirmed PD and five control frozen brains. Results: DA levels in the external globus pallidus (GPe) of normal brains were nearly six times greater than in the internal pallidum (GPi). In PD, the mean loss of DA was marked (−82%) in GPe and moderate (−51%) in GPi. DA loss of variable degree was seen in different subdivisions of GPe and GPi in PD; however, DA levels were near normal in the ventral (rostral and caudal) GPi of PD cases with prominent tremor. There was marked loss of DA (−89%) in the caudate and severe loss (−98.4%) in the putamen in PD. The pattern of pallidal DA loss did not match the putaminal DA loss. Conclusion: There is sufficient loss of dopamine (DA) in external globus pallidus and the internal globus pallidum (GPi) as may contribute to the motor manifestations of Parkinson disease (PD). The possible functional disequilibrium between GABAergic and DAergic influences in favor of DA in the caudoventral parts of the GPi may contribute to resting tremor in tremor dominant and classic PD cases. GLOSSARY: CN = caudate nucleus; DA = dopamine; GPe = external globus pallidus; GPi = internal globus pallidum; LB = Lewy body; MDCS = Movement Disorder Clinic in Saskatoon; PD = Parkinson disease; PUT = putamen; VTA = ventral tegmental area.

Striatal biopterin and tyrosine hydroxylase protein reduction in dopa-responsive dystonia

Objective: To determine the mechanism leading to striatal dopamine (DA) loss in dopa-responsive dystonia (DRD). Background: Although mutations in the gene GCH1, coding for the tetrahydrobiopterin (BH4) biosynthetic enzyme guanosine triphosphate–cyclohydrolase I, have been identified in some patients with DRD, the actual status of brain BH4 (the cofactor for tyrosine hydroxylase [TH]) is unknown. Methods: The authors sequenced GCH1 and measured levels of total biopterin (BP) and total neopterin (NP), TH, and dopa decarboxylase (DDC) proteins, and the DA and vesicular monoamine transporters (DAT, VMAT2) in autopsied brain of two patients with typical DRD. Results: Patient 1 had two GCH1 mutations but Patient 2 had no mutation in the coding region of this gene. Striatal BP levels were markedly reduced (<20% of control subjects) in both patients and were also low in two conditions characterized by degeneration of nigrostriatal DA neurons (PD and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated primate), whereas brain NP concentrations were selectively decreased (<45%) in the DRD patients. In the putamen, both DRD patients had severely reduced (<3%) TH protein levels but had normal concentrations of DDC protein, DAT, and VMAT2. Conclusions: The data suggest that 1) brain BH4 is decreased substantially in dopa-responsive dystonia, 2) dopa-responsive dystonia can be distinguished from degenerative nigrostriatal dopamine deficiency disorders by the presence of reduced brain neopterin, and 3) the striatal dopamine reduction in dopa-responsive dystonia is caused by decreased TH activity due to low cofactor concentration and to actual loss of TH protein. This reduction of TH protein, which might be explained by reduced enzyme stability/expression consequent to congenital BH4 deficiency, can be expected to limit the efficacy of acute BH4 administration on dopamine biosynthesis in dopa-responsive dystonia.

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 Parkinsons 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 [3H]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.

Extrastriatal dopamine in symptomatic and asymptomatic rhesus monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

We analyzed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys, with and without parkinsonian symptoms, the regional changes in dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and tyrosine hydroxylase activity (TH). Symptomatic monkeys had wide-spread DA loss in subcortical and cortical regions. However, the magnitude of this MPTP-induced DA reduction was markedly smaller than the DA loss in the caudate nucleus and putamen, where only less than 1% DA remained. No comparable loss of DA was found in the subcortical extrastriatal regions of asymptomatic MPTP monkeys, despite more than 90% DA loss in the striatal nuclei. The most pronounced difference in DA levels between the symptomatic and the asymptomatic group was observed in nucleus accumbens, nucleus of the stria terminalis, ventral tegmental area, globus pallidus and the cingulate gyrus. Levels of DOPAC and TH activity paralleled the behavior of DA. In contrast, the concentration of HVA was reduced in many brain regions of both symptomatic and asymptomatic monkeys. These effects of MPTP on extrastriatal DA levels in the rhesus monkey are compared with DA and HVA changes in the brain of patients with Parkinsons disease, and their possible contributory role for the production, by MPTP, of a permanent parkinsonian condition is discussed.

The nigrostriatal system in the presymptomatic and symptomatic stages in the MPTP monkey model: a PET, histological and biochemical study.

Parkinsons disease (PD) is diagnosed when striatal dopamine (DA) loss exceeds a certain threshold and the cardinal motor features become apparent. The presymptomatic compensatory mechanisms underlying the lack of motor manifestations despite progressive striatal depletion are not well understood. Most animal models of PD involve the induction of a severe dopaminergic deficit in an acute manner, which departs from the typical, chronic evolution of PD in humans. We have used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered to monkeys via a slow intoxication protocol to produce a more gradual development of nigral lesion. Twelve control and 38 MPTP-intoxicated monkeys were divided into four groups. The latter included monkeys who were always asymptomatic, monkeys who recovered after showing mild parkinsonian signs, and monkeys with stable, moderate and severe parkinsonism. We found a close correlation between cell loss in the substantia nigra pars compacta (SNc) and striatal dopaminergic depletion and the four motor states. There was an overall negative correlation between the degree of parkinsonism (Kurlan scale) and in vivo PET ((18)F-DOPA K(i) and (11)C-DTBZ binding potential), as well as with TH-immunoreactive cell counts in SNc, striatal dopaminergic markers (TH, DAT and VMAT2) and striatal DA concentration. This intoxication protocol permits to establish a critical threshold of SNc cell loss and dopaminergic innervation distinguishing between the asymptomatic and symptomatic parkinsonian stages. Compensatory changes in nigrostriatal dopaminergic activity occurred in the recovered and parkinsonian monkeys when DA depletion was at least 88% of control, and accordingly may be considered too late to explain compensatory mechanisms in the early asymptomatic period. Our findings suggest the need for further exploration of the role of non-striatal mechanisms in PD prior to the development of motor features.

Lower efficacy of the dopamine D1 agonist, SKF 38393, to stimulate adenylyl cyclase activity in primate than in rodent striatum

The selective D1 agonist, SKF 38393, stimulated adenylyl cyclase by about 40% of basal activity in rat striatum but by only about 10% in the striatum of rhesus monkeys. In contrast, dopamine stimulated striatal adenylyl cyclase in both species with equal efficiency (70-80%). SKF 38393 30 microM inhibited the effect of 30 microM dopamine by about 45% in rat and by about 75% in primate tissue. This difference may be due to a lower D1 receptor reserve in primate than in rodent tissue and suggests that only selective D1 agonists with full efficacy at D1 receptors can be expected to have beneficial effects in patients with Parkinsons disease.

Sensitization of dopamine-stimulated adenylyl cyclase in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- treated rhesus monkeys and patients with idiopathic parkinson's disease

Abstract: Dopamine‐stimulated adenylyl cyclase activity was measured in striatal homogenates of l‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated rhesus monkeys and humans with idiopathic Parkinsons disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine‐stimulated adenylyl cyclase activity was observed in the caudate of MPTP‐parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinsons disease. No such sensitization was seen in the MPTP‐treated rhesus putamen or human Parkinsons disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l‐sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP‐treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I‐SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP‐treated monkeys. Our results point to a region‐specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinsons disease.

Dopamine turnover is upregulated in the caudate/putamen of asymptomatic MPTP-treated rhesus monkeys

In Parkinsons disease (PD) and experimental parkinsonism, losses of up to 60% and 80%, respectively, of dopaminergic neurons in substantia nigra, and dopamine (DA) in striatum remain asymptomatic. Several mechanisms have been suggested for this functional compensation, the DA-mediated being the most established one. Since this mechanism was recently challenged by striatal DA analysis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, we present data on several DAergic parameters in three groups of rhesus monkeys: MPTP-treated asymptomatic animals; symptomatic MPTP-treated animals with stable parkinsonism; and untreated sex and age matched controls. We determined ratios of striatal and nigral 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA levels and tyrosine hydroxylase (TH) enzyme activity to DA levels, in addition to the commonly used homovanillic acid (HVA)/DA ratios which, as such, might be less reliable under the conditions of partial denervation. We found that in the asymptomatic MPTP monkeys the DOPAC/DA ratios in putamen and caudate nucleus were shifted with high statistical significance 1.9-5.8-fold, as compared to controls, the shifting of the ratios being in the same range as the 2.6-5.4-fold shifts in the symptomatic animals. Also TH/DA ratios were significantly increased in both, the asymptomatic and the symptomatic MPTP-treated monkeys, with shifts in the putamen and caudate nucleus of 3- and 2.7-7.0-fold, respectively. In the substantia nigra, DOPAC levels and TH activity were strongly decreased after MPTP (-77 to -97%), but the ratios DOPAC/DA and TH/DA were not changed in this brain region. Collectively, our findings support the concept of DAergic compensation of the progressive striatal DA loss in the presymptomatic stages of the parkinsonian disease process.