The serotonin transporter gene and Parkinson's disease.

Dysfunction in the serotonin (5-hydroxytryptamine) system and reduced serotonin concentrations have been reported in patients with Parkinson's disease (PD). Serotonin concentrations in neural tissue are controlled by a presynaptic serotonin transporter protein that is encoded by a single gene. Therefore, we investigated whether a polymorphic region in the serotonin transporter gene is associated with PD. Three variable-number tandem repeat (VNTR) elements of the serotonin transporter gene were detected by polymerase chain reaction, those with 9, 10, 11 and 12 copies of the repeat element. The 10-copy VNTR element was significantly less common in patients with PD than controls in the univariate analysis (p < 0.05). Logistic regression analysis revealed no significant differences between patients (n = 198) and controls (n = 200) in the distribution frequencies of 9- and 12-copy alleles and combined genotypes (odds ratio = 1.20; p = 1.71). A positive family history of PD was a strong predictor of disease risk (odds ratio = 2.98; 95% confidence interval 1.51-5.87; p = 0.001). Although slight differences were observed between patient and control groups, these data suggest that defects in serotonin concentrations in patients with PD are unlikely to be due to polymorphisms in the serotonin transporter gene in this large Australian cohort; however, the inverse association observed with the 10-copy allele warrants further investigation.

Further characterisation of the interaction of haloperidol metabolites with neurotransmitter transporters in rat neuronal cultures and in transfected COS-7 cells.

It has been proposed that the extrapyramidal symptoms such as tardive dyskinesia developed by patients on long-term haloperidol treatment may be the result of uptake of haloperidol metabolites into neurons via the monoamine neurotransmitter transporters followed by neurotoxic events, as occurs for MPP+, the pyridinium metabolite of MPTP. We recently showed that haloperidol and its metabolites are inhibitors of the human noradrenaline transporter (NAT), dopamine transporter (DAT) and serotonin transporter (SERT), and determined their Ki values for inhibition of the three transporters expressed in transfected COS-7 cells. In this study, we extended the investigation of these compounds to their inhibitory effects on DAT, SERT and the high affinity choline uptake (HACU) in neuronal cultures from embryonic rat brain, and investigated whether the compounds are substrates or non-transported inhibitors of the NAT, DAT and SERT in transfected COS-7 cells and DAT and SERT in the neuronal cultures. Haloperidol and its metabolites inhibited DAT, SERT and HACU in the neuronal cultures, indicating that they are not specific inhibitors of the monoamine neurotransmitter transporters. The ratio of the Ki values of the least and most potent inhibitors were found to be 2.8 for DAT, 24 for SERT and 7.6 for HACU. The compounds were more potent inhibitors of DAT and SERT in neuronal cultures than we found previously in transfected COS-7 cells. The question of whether the compounds are substrates or non-transported inhibitors of the monoamine transporters was investigated by determining whether they caused an increase in efflux of [3H]amine in transfected COS-7 cells or neuronal cultures preloaded with [3H]amine. Haloperidol metabolites were weak substrates for SERT, but not for NAT or DAT, in transporter-transfected COS-7 cells. In neuronal cultures, the metabolites appeared to be non-transported inhibitors or very weak substrates of DAT and SERT. Despite inhibition of the monoamine transporters by haloperidol and its metabolites, there is little evidence to support the proposal that these compounds are likely to cause neurotoxic effects via neuronal uptake using the monoamine transporters. The mechanisms of the side effects of haloperidol therapy, such as tardive dyskinesia, are still unclear, but are unlikely to depend on interactions of the drug or its metabolites with NAT, DAT or SERT.

Mitochondrial ultrastructure and density in a primate model of persistent tardive dyskinesia.

The use of neuroleptic drugs to treat schizophrenia is almost invariably associated with extrapyramidal movement disorders. One of these disorders, tardive dyskinesia (TD), can persist long after neuroleptic withdrawal suggesting that permanent neurological damage is produced. However, there appears to be no convincing pathology of TD and its pathogenesis remains unknown. Findings that neuroleptics interfere with normal mitochondrial function and produce mitochondrial ultrastructural changes in the basal ganglia of patients and animals suggest that mitochondrial dysfunction plays a role in TD. We have established a model for persistent TD in baboons that appears to involve compromised mitochondrial function. In this study, we evaluated two animals treated for 41 weeks with a derivative of haloperidol and two treated with vehicle only. Treatment was then withdrawn and the animals observed for a further 17-18 weeks. Treated animals developed abnormal orofacial signs that were consistent with TD. These symptoms persisted during the drug-free period. The animals were euthanased, the brains perfused-fixed then post-fixed in 4% paraformaldehyde and the caudate and putamen prepared for electron microscopy. Regardless of whether mitochondria were located in neural soma, excitatory terminals, glia or in non-somal neuropil there was no consistent difference either in size or number between treated and control animals. Thus, even if mitochondria in striatal neurons undergo ultrastructural alterations during neuroleptic therapy, these changes do not persist after drug withdrawal.

Potencies of haloperidol metabolites as inhibitors of the human noradrenaline, dopamine and serotonin transporters in transfected COS-7 cells.

Extrapyramidal symptoms, such as tardive dyskinesia, often develop in patients on long-term treatment with haloperidol. It has been proposed that these symptoms could be caused by neurotoxic effects of haloperidol metabolites following uptake by monoamine transporters, in an analogous mechanism to the neurotoxic effect of MPP+ (1-methyl-4-phenylpyridinium) metabolised from MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In this study, the hypothesis was partially investigated by determining the potencies of haloperidol and reduced haloperidol and the corresponding pyridinium and tetrahydropyridine metabolites, compared with MPP+ and MPTP, as inhibitors of the noradrenaline transporter (NAT), dopamine transporter (DAT) and 5-HT transporter (SERT). Two days after COS-7 cells were transiently transfected with the cDNA for the human NAT, DAT or SERT (Lipofectamine method), the cells were incubated with 10 nM [3H]noradrenaline, dopamine or 5-HT, respectively, for 2 min at 37 C, in the absence or presence of various concentrations of the eight compounds or a specific uptake inhibitor (NAT: nisoxetine 1 microM; DAT: GBR 12909 1 microM; SERT: citalopram 10 microM). Specific amine uptake (fmol/ mg protein) was calculated as the difference in uptake in the absence and presence of the specific uptake inhibitor. Ki values were calculated for the eight compounds for inhibition of NAT, DAT and SERT. Haloperidol, its five metabolites and MPP+ and MPTP all inhibited NAT, DAT and SERT. For the pyridinium and tetrahydropyridine metabolites of haloperidol, there were not marked differences between their potencies as inhibitors between each other for NAT or DAT or between NAT and DAT, with all of the Ki values in the range of 5.8-16 microM. However, there were more marked differences for SERT, with all but one of the metabolites showing selectivity for inhibition of SERT relative to NAT and DAT. Haloperidol and reduced haloperidol had similar inhibitory potencies for all three transporters, and were clearly less potent than the other haloperidol metabolites only for inhibition of SERT. The lack of correlation between the inhibitory potencies of the haloperidol metabolites and their structural analogues, MPTP and MPP+, suggests that they are not likely to cause neurotoxicity by a mechanism analogous to that of the latter neurotoxin.

Clinical and neuropathological abnormalities in baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

Tardive dyskinesia (TD) is relatively common among psychiatric patients on maintenance therapy with typical neuroleptics and persists in more than 20% even after withdrawal of the medication. Such persistence suggests an underlying pathology due to neurotoxicity. We present evidence for such a neurotoxic mechanism in a baboon model of TD. Four baboons were treated chronically with the dehydration product of haloperidol, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HPTP), which is metabolized, similarly to haloperidol, to two neurotoxic pyridinium species. The animals developed orofacial dyskinesia which persisted after HPTP was ceased. Serial sections of the entire brain from the four treated animals and four vehicle-treated controls revealed volume loss in the basal forebrain and hypothalamus. Histological evaluation demonstrated a reduction in the density of magnocellular neurons in the anterior region of the nucleus basalis of Meynert (NbM). We speculate that the loss of these NbM neurons may be associated with the persistent orofacial dyskinesia observed in the HPTP-treated animals. These findings may contribute to a better understanding of neuroleptic-induced TD.

The epidemiology of Parkinson's disease in an Australian population.

A prevalence study of Parkinson's disease (PD) was conducted in the rural town of Nambour, Australia. There were 5 cases of PD in a study population of 1207, yielding a crude prevalence ratio of 414 per 100,000 (95% confidence interval; 53-775). We performed a separate case-control study involving 224 patients with PD and 310 controls from South East Queensland and Central West New South Wales, to determine which factors increase the risk for PD in Australia. A positive family history of PD was the strongest risk factor for the development of the disease (odds ratio = 3.4; p < 0.001). In addition, rural residency was a significant risk factor for PD (odds ratio = 1.8, p < 0.001). Hypertension, stroke and well water ingestion were inversely correlated with the development of PD. There was no significant difference between patients and controls for exposure to herbicides and pesticides, head injury, smoking or depression. The high prevalence of PD in Nambour may be explained by rural residency. However, the most significant risk factor for PD was a positive family hisotry. This demonstrates the need for improved understanding of the genetic nature of the disease.

Chirality of reduced haloperidol in humans.

In vitro, cytosolic human ketone reductases catalyse the stereospecific (i.e. >99%) formation of S(-) reduced haloperidol (RHP) from haloperidol (HP). Whether this situation is reflected in patients taking the drug is unknown. In this study in nine patients taking HP, only 73.2+/-18.2% of the RHP excreted in urine was the S(-) enantiomer. Thus, enzymes other than cytosolic ketone reductases must be responsible for the formation of the minor enantiomer.

Microscale high-performance liquid chromatography-electrospray tandem mass spectrometry assay for cyclosporin A in blood.

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC-electrospray tandem mass spectrometry assay. Blood samples (100 microl) were prepared by acetonitrile precipitation and C18 solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5-1000 microg/l (r> or =0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 microg/l and 2.8% at 800 microg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x + 10.0, r=0.996). This HPLC-ESI-MS-MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.

The midbrain dopaminergic cell groups in the baboon Papio ursinus.

The present study evaluates the cytoarchitecture of midbrain dopaminergic regions in baboons using similar methodology to that recently applied to compare humans and rats. This information is relevant for the interpretation of nonhuman primate models of Parkinson's disease (PD). The midbrains of four alpha male baboons were serially sectioned into 10 evenly spaced series of 50 microm sections. Series were stained with either cresyl violet or immunohistochemically reacted for tyrosine hydroxylase, substance P, calbindin-D28k, or parvalbumin. The organization of dopaminergic cell groups and the distribution of proteins within these groups were found to be very similar to that previously described in humans [McRitchie et al., J. Comp. Neurol. 364:121-150; 1996]. Dorsal and ventral tiers of the A9 substantia nigra (SN) pars compacta and all divisions of the A8 and A10 cell groups were identified revealing a high degree of homology in the arrangement of chemically distinct midbrain neurons between primates. The major difference between the organization of human and baboon midbrain dopaminergic neurons is the anteroposterior extent of the dense cell clusters within the SN pars compacta. In baboons the dorsomedial cell cluster is absent at posterior levels. The ventral tier cell clusters, which are targeted by PD in humans, are restricted to the posterior and ventral regions of the SN pars compacta of the baboon. In humans these cell clusters are found throughout the rostrocaudal extent of the SN. These ventral cell clusters have been previously shown to have reciprocal connections with sensorimotor regions of the putamen.

The dopamine transporter gene and Parkinson's disease in a Chinese population.

We studied a variable number tandem repeat polymorphism within the dopamine transporter gene (DAT) for an association with Parkinson's disease in a Chinese population. Five alleles were detected, consisting of 6, 8, 9, 10, and 11 copies of the 40 base pair repeat sequence. The 10-copy allele was most common, accounting for 90% of alleles. There were no significant differences between the patients and the control subjects in the distribution frequencies of the alleles or genotypes. Therefore, this polymorphism is not associated with Parkinson's disease in Chinese populations.

Association of a polymorphism in the dopamine-transporter gene with Parkinson's disease.

The presynaptic dopamine transporter in nigral dopaminergic neurons confers susceptibility to the cytotoxic effects of the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Polymorphisms in the dopamine transporter might influence the susceptibility to such toxins. Therefore, we investigated whether a polymorphic region in the 3'-untranslated region of the dopamine-transporter gene is associated with idiopathic Parkinson's disease (PD). The frequency distribution of the alleles was significantly different between the patients (n = 100) and controls (n = 200, p < 0.05). The rare 11-copy allele was more common in the patients (odds ratio = 10.2, 95% confidence interval - 1.2-87.9, p < 0.025). The susceptibility of some people to PD may be conferred by polymorphisms in the dopamine-transporter gene that could lead to increased cellular accumulation of neurotoxic compounds in dopaminergic neurons.

Cortisol metabolism and its inhibition by glycyrrhetinic acid in the isolated perfused human placental lobule.

We have previously reported the placental metabolism of prednisolone to prednisone, 20alpha- and beta-dihydroprednisone and 20beta-dihydroprednisolone. In this study, the disposition of cortisol was investigated in vitro in the dual perfused, isolated human placental lobule after the addition of cortisol (1.2 micromol, n = 3 and 12 micromol, n = 4) to the maternal compartment. Analysis of 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20alpha-dihydrocortisone, 20beta-dihydrocortisone, 20alpha-dihydrocortisol and 20beta-dihydrocortisol was also detected. Saturability of metabolism but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme inhibitor 18beta-glycyrrhetinic acid (GA). The disposition of GA was analysed using HPLC-atmospheric pressure chemical ionisation-MS/MS (HPLC-APCI-MS/MS). GA was found to transfer from the maternal to the fetal circulations without detectable metabolism during 6 h of perfusion.

Bound Tris confounds the identification of binding site residues in a paraquat single chain antibody.

We produced an anti-paraquat single chain antibody (scFv) to investigate its potential use in immunotherapy for paraquat poisoning. However, this scFv was expressed in an insoluble form and only displayed moderate binding affinity. An earlier examination of the pH dependence of antigen binding by the parent paraquat-specific mAb (7D7-3) suggested that the electrostatic effects of a tyrosine residue were important. The aims of the current study were to obtain expression of a soluble scFv (D10) and to increase its binding affinity. The former was achieved by expression in a phagemid vector. Site-directed mutagenesis of tyrosine residues in CDR H3 did not result in improved affinity for paraquat, suggesting that the original pH dependence required re-examination. Nuclear magnetic resonance studies of 7D7-3 Fab revealed that the original observation of the pH-dependent paraquat binding with a mid-point of approximately pH 8.9 was due to tightly bound Tris. It appears that as Tris is titrated to a neutral species the energetically unfavourable juxtaposition of its positive charge with that of paraquat is reduced. These findings have broad implications in the interpretation of the pH or salt dependence of any antibody-antigen interaction which should be made cautiously and with regard to the possible interference of buffer components introduced during the preparation of the antibody.

A high-performance liquid chromatography-electrospray-tandem mass spectrometry analysis of cortisol and metabolites in placental perfusate.

A reversed-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quantitate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha- and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6 alpha-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mM ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 microL/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100-2000 micrograms/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 micrograms/L was < or = 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20 beta-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 micrograms/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol.

Two pyridinium metabolites of haloperidol are present in the brain of patients at post-mortem.

We have shown in patients taking the antipsychotic drug haloperidol (HP) that two pyridinium metabolites (HPP+ and RHPP+) are present in blood and urine in nM concentrations. These metabolites are structurally analogous to MPP+, the neurotoxic metabolite of the well-known parkinsonian-producing protoxin, MPTP. In this study we measured the concentrations of HPP+ and RHPP+ in seven regions of the brain (putamen, substantia nigra, globus pallidus, caudate, hippocampus, cerebellum and occipital cortex) obtained at post-mortem from three patients who were taking HP before death. Blood, urine, and bile from one patient were analysed as well. HPP+ was present in all regions (except for substantia nigra in one patient and globus pallidus in another); the amount/g ranged from 1.6-8.3 pMol but there was no preferential sequestration of the metabolite in dopaminergic regions. Similarly, RHPP+ was present relatively uniformly in all regions; the amount/g ranged from 1.1-7.6 pMol. The concentrations of HPP+ and RHPP+ in one patient were 24 and 13 nM in blood, 660 and 230 nM in urine, and 13.0 and 1.4 microM in bile, respectively. The presence of these pyridinums in brain adds another important piece of information to the case that, at least for HP, metabolite-induced neurotoxicity could contribute to the extrapyramidal side-effects in patients receiving long-term therapy.

Metabolic defects caused by treatment with the tetrahydropyridine analog of haloperidol (HPTP), in baboons.

Mounting evidence suggests that compromised cellular energy production is a major contributor to idiopathic and drug-induced degenerative processes. Our interest in neurotoxins have prompted us to examine in the baboon the effects of HPTP, the tetrahydropyridine dehydration product of haloperidol, on urinary chemical markers that reflect defects in mitochondrial respiration. Urinary dicarboxylic acid and conjugate profiles, similar to those seen in humans with inborn errors of mitochondrial metabolism and toxin-induced Jamaican vomiting sickness (JVS) were observed in the treated baboons. We interpret these results as evidence that HPTP and/or HPTP metabolites inhibit mitochondrial respiration in the baboon and speculate that analogous effects may occur in haloperidol-treated individuals.

Studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior.

A procedure for evaluating the thermodynamic equilibrium constant by kinetic analysis of sensorgrams which deviate from the pseudo-first-order kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis employs quantitative expressions that are used in conventional kinetic characterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitude. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomerization of the complex between ligate and affinity sites are used to explore the feasibility of the approach. Its application is then illustrated with BIAcore studies of the interaction between the Fab fragment of an anti-paraquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. Studies with an extremely high degree of antigen substitution on the sensor surface yielded sensorgrams that deviated markedly from pseudo-first-order kinetic behavior. However, they yielded the same binding constant (3 x 10(6) M-1) as the value deduced by conventional analysis of sensorgrams that conformed with pseudo-first-order kinetics because of a much lower concentration of immobilized antigen on the sensor surface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kinetic behavior.

Metabolism of haloperidol to pyridinium species in patients receiving high doses intravenously: is HPTP an intermediate?

The metabolism of haloperidol (HP) to the potentially neurotoxic pyridinium species, HPP+ and RHPP+, has been demonstrated in humans. In vitro studies in microsomes harvested from various animal species indicate that the tetrahydropyridines, HPTP and RHPTP, could be intermediates in this pathway. However, this has not yet been demonstrated in vivo in humans. In this study, plasma and urine collected from eight critically ill patients treated with high doses of intravenous HP were analyzed for HPTP and RHPTP using HPLC with electrochemical detection. However, neither HPTP nor RHPTP were detected despite plasma concentrations of HP and RHP higher than any previously reported. HPP+ and RHPP+ were both present in the urine in high concentrations and accounted for 1.1 +/- 0.5% and 5.3 +/- 3.6%, respectively, of the administered dose of HP. The apparent elimination half-lives of HPP+ and RHPP+ were 67.3 +/- 11.0 hr and 63.3 +/- 11.6 hr, respectively. The absence of HPTP and RHPTP in plasma and urine suggests that in humans these tetrahydropyridines either are insignificant intermediates in the metabolism of HP in vivo or are present only transiently at their site of formation and are not released into the circulation.

The association between polymorphisms in the cytochrome P-450 2D6 gene and Parkinson's disease: a case-control study and meta-analysis.

We performed a case-control study to investigate the association of the poor metaboliser genotype of the cytochrome P450 2D6 gene with Parkinson's disease (PD). Genotyping was determined by the polymerase chain reaction followed by digestion with restriction enzymes. The poor metaboliser genotype was more frequent in 112 patients with PD than in 206 matched controls (odds ratio 1.7, 95% CI: 0.94-2.45). A meta-analysis of these results together with ten other published studies gave a pooled odds ratio for the poor metaboliser genotype of 1.47 (95% CI: 1.18-1.96, P=0.01). Thus, the poor metaboliser genotype has a small but highly significant association with PD which would be easily missed in small studies. Research now should focus on the mechanism of this association.