Plasticity of extrapyramidal dopamine system in Parkinson\'s disease - A postmortem study

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PLASTICITY

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VOL. 25, NO. 2

DOPAMINE SYSTEM IN PARKINSON’S

DISEASE - A

POSTMORTEM STUDY

Lucyna Antkiewicz-Michaluk’,

Anna Krygowska-Wajs2, Jerzy Michaluk’, Irena Romanska’,

Andrzej Szczudlik2, Jerzy Vet&&*

‘Department of Biochemistry, Institute of Pharmacology Polish Academy of Sciences, Smetna 12,3 l-343 Krakow, Poland *Neurology Clinic, Collegium Medicum, Jagiellonian University, Krakow, Poland

(accepted

July

20,

1999)

SUMMARY Investigating the compensatory changes developing in the course of Parkinson’s disease, we assayed the concentrations of amine neurotransmitters and their metabolites, and dopamine D, and D, and cq noradrenergic receptors in various areas of parkinsonian and control brains obtained by autopsy Those data were used to calculate the [metabolite]/[amine]

and [receptor density]/[amine]

ratios, regarded as

measures of pre- and postsynaptic adaptations, respectively. The metabolic rate of dopamine rose by 2000 400% in all basal ganglia; the metabolism of serotonin was elevated, by 150.200%, only in the caudate nucleus and locus coeruleus. The receptor/neurotransmitter

ratios for dopamine D, and D, receptor were

augmented several-fold in the basal ganglia, but not in the nucleus accumbens. . In the locus coeruleus the dramatic decline in the noradrenaline concentration was parallel to the decrease in g-adrenoceptor density. The results suggest that the dopaminergic system of the parkinsonian brain shows plasticity of both pre- and postsynaptic structures that permits adaptations to the progressive loss ofthe neurotransmitter.

0 1999 Wiley-Liss,

Inc.

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Key words:

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Parkinson’s disease, adaptive changes, postmortem study, biogenic amine metabolism,

receptor density

INTRODUCTION Plasticity of the central nervous system is its basic characteristics and is essential for adaptation of the organism to changes in the external and internal environment. Several subsystems may have various capacity to adapt, and that property is generally regarded as declining with age. Catecholaminergic

systems were demonstrated to have a considerable adaptive potential. The

postsynaptic dopamine receptor density adapts to the availability of the neurotransmitter, increasing after a prolonged blockade with neuroleptics (11, 14, 17) or dopamine terminals destruction by Ghydroxydopamine (24). Similarly, p-noradrenergic receptor responsiveness increases after chemosympathectomy or reserpinization and declines after prolonged augmentation of noradrenaline concentration in the synaptic cleft due to action of monoamine oxidase inhibitors (33) or electroconvulsive

shock and

tricyclics antidepressants (34) While adaptation of receptors of catecholaminergic system seems to follow a straight negative feedback paradigm, the serotonergic receptor may behave differently. Thus, as shown by Leysen et al (20), antagonists as well as agonists may produce down-regulation of SHT, receptors. Most of the studies on receptor adaptation were carried out under experimental conditions, in which procedures applied caused relatively rapid development of changes, investigated over a period of few days or weeks. Much less is known about the adaptive processes operating in the course of neurodegenerative diseases of slow onset and development. From this point of view an interesting model may be the Parkinson’s disease, which is a neurodegenerative illness characterized by an insidious onset and slow but progressive deterioration of motor function and characteristic personality changes, involving cognitive and emotional disturbances (10, 13, 19). The etiology of Parkinson’s disease is not known yet, but there are indications that endogenous tetrahydroisoquinolines may be involved (16,26,27). that the level of a tetrahydroquinoline

It has been shown

derivative, salsolinol, is elevated in the cerebrospinal fluid of

parkinsonian patients, proportionally to the degree of motor impairment and, particularly, dementia (2). The slow course of neurodegenerative process gives the organism the time for development of adaptive changes that may further slow down the appearance of the clinical symptoms of the disease. Therefore, in the present study we investigated some biochemical indices of adaptation of dopaminergic,

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noradrenergic and serotonergic system in the brain areas of individuals deceased in the course of Parkinson’s disease. Thanks to the classical studies of Homykiewicz

that demonstrated that the most prominent

biochemical change was a profound depletion of dopamine in the caudate, putamen, globus pallidus and substantia nigra (cf. 15), Parkinson’s disease became the first neurological disease whose neurochemical background had been described, allowing introduction of a rational .therapy with L-dopa. Apart of degeneration of nigro-striatal system, a prominent neuroanatomical and neurochemical feature of Parkinson’s disease is degeneration of the locus coeruleus. The degeneration of this, mainly noradrenergic, structure takes place in the cases of both Alzheimer’s and Parkinson’s disease (7, 8, 9). The degree of degeneration of the locus coeruleus in Parkinson’s disease seemed to be more prominent in the forms accompanied by dementia (5). Also, degeneration of serotonergic system was reported in the parkinsonian brains (23, 3 1). As Parkinson’s disease has a slow onset, a possibility of development of appropriate adaptive changes counteracting the effects of gradual neuronal loss is obvious. Such changes had already been described in animal experiments, in which a model parkinsonism is evoked in relatively short time (3, 18, 35). In the present study we investigated the changes in few parkinsonian brains, in which we measured the indices of adaptive processes in several brain structures. The ratio of the concentration of a metabolite of an amine to the concentration of the amine itself was taken as a measure of the rate of amine metabolism, while the ratio of the change of receptor density to the amine concentration (a number of receptors per unit of amine concentration) as the ratio of postsynaptic sensitivity. Our findings indicate that in the parkinsonian brain plastic adaptive changes occur in the dopaminergic system of the basal ganglia, both presynaptically (an excessive acceleration of dopamine turnover) and postsynaptically (an increased number of D, and D, receptors per a unitary concentration of dopamine), while no such adaptive changes were observed in the noradrenergic system of the locus coeruleus nor in the striatal serotonergic system.

SUBJECTS AND METHODS Subjects Brain tissue was obtained from four victims of Parkinson’s disease (mean age 78 years). All patients fulfilled the clinical diagnostic criteria for parkinsonism (at least two out of three symptoms of the parkinsonian triad: : resting tremor, rigidity, bradykinesia), and the neurological disorders other than Parkinson’s disease were excluded by a detailed history and examination including neuroimaging. One of them, assessedas an early stage (stage II of Hoehn-Yahr scale), had never been treated with any kind

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of antiparkinsonian drug, the others were in an advanced stage of the disease (stage III - IV) and were treated with levodopa in doses from 300 - 800 mg/day. The mean duration of illness was 5.5 years (range I - 8 years). No patient was demented according to DSM III criteria, nor showed depression nor anxiety according to the Hamilton’s Rating Scales for Depression. The clinical diagnosis was confirmed by postmortem histopathological study which disclosed typical changes including the presence of Lewy bodies in the substantia nigra. The control groups consisted of five subjects, mean age 52, who had died of non-neurological diseases. Tissues The autopsy was performed always 12 h post mortem. The brains were sectioned in the middle, one half was used for routine neuropathological examination, while from the second the tissue blocks (approx. 1 cm3; locus coeruleus approx. 0.5 cm3) were taken from the following structures: the substantia nigra (both pars compacta and reticulata), globus pallidus (both internal and external part), caudate, putamen, nucleus accumbens, and locus coeruleus. Care was taken to prepare samples without contamination from the neighbouring tissues. The tissue after dissection was immediately frozen at -70 “C for later biochemical analysis. Amines and their metabolites Dopamine, serotonin and noradrenaline together with their metabolites were assayed by means of high-performance liquid chromatography (HPLC). Dopamine, homovanillic acid (HVA) and 3 methoxytyramine (3-MT) were assayed in the substantia nigra, nucleus caudatus, putamen (PUT), globus pallidus, and nucleus accumbens. Serotonin and 5-hydroxy indoleacetic acid (5-HIAA) were assayed in the same structures and also in the locus coeruleus. Noradrenaline was assayed in the locus coeruleus only. The tissue samples were weighed and homogenized in ice-cold 0.1 M trichloroacetic acid containing 0.05 mM ascorbic acid. After centrifugation (I 0,000 x g, 5 min), the supematants were filtered through RC 58 0.2 pm cellulose membranes (Bioanalytical Systems, West Lafayette, IN), and injected into a Hewlett-Packard 1050 chromatograph with electrochemical detector 1049A and C-l 8 column (Hypersil, BDS, 100 x 4 mm). The mobile phase consisted of 0.05 M citrate-phosphate buffer, pH 3.5, 0.1 mM EDTA, 0.254 mM sodium octyl sulfonate, 2.5% methanol and 1% acetonitrile. The flow rate was maintained at 0.8 ml/min. The amines and their metabolites were quantified by peak height comparisons with standards run on the day of analysis with a sensitivity of 10 - 100 pg. Dopamine D, Receptors The tissue was homogenized in 40 vol of an ice-cold 50 mM Tris-HCl buffer, pH 7.4, using a Poly-tron disintegrator. The homogenate was centrifuged at 1000 x g for 15 min, the supernatant was decanted and recentrifuged at 25 000 x g for 30 min, and the resulting pellet was resuspended in the buffer and recentrifuged under the same conditions. The final pellet (fraction PJ was used for binding studies. For incubation it was reconstituted in the Tris-HCl buffer pH 7.4, to obtain a final protein concentration (measured according to Lowry et al (21)) of approximately 0.3 mg/ml. The radioligand, [3H]SCH-23390 ([3H](R+)7-chloro-8-hydroxy-3-methyl-l-phenyl-2,3,4,5tetrahydro- 1H-3-benzazepine hydrochloride, NEN, specific activity 85.5 Ci/mmol), was prepared in six concentrations from 0.06 - 2.0 r&I. The incubation mixture (final volume 550 ~1) consisted of 450 ~1 membrane suspension, 50 ~1 of [3H]SCH-23390 solution and 50 ~1 Tris-HCl buffer containing 1 PM serotonin, without (total binding) or with (unspecific binding) cold SCH-23390 (final concentration 5 PM). All assays were performed in duplicate and incubation proceeded in a shaking water bath, at 30°C for 60 min. The B,,, and K, values were calculated by Scatchard analysis. Dopamine D2 receptors. The procedure was very similar as above, but the initial homogenization took place in 20 vol of TrisHCl buffer, and the incubation medium was Tris-HCl buffer pH 7.1 supplemented with 120 mM NaCl, 5 mM KCl, 1 n&I CaCl,, 1 mM MgCl,, 10 PM pargyline and 0.1% ascorbic acid. The final concentration

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of protein in the medium was adjusted to OS-O.6 mg/ml. The radioligand, [3H]spiperone (Amersham, specific activity 16.4 Cu/mmol) was used in six concentrations (0.06-4 nM), the incubation was carried out at 3’7°C for 10 min; the final volume of 1000 ~1 consisted of 500 ~1 of membrane suspension, 100 ~1of radioligand solution, 200 ~1of the buffer without (total binding) or with (unspecific binding) 10 PM of cold spiperone and 200 ~1 of the Tris buffer containing 1 PM serotonin. Adrenergic a2 receptors The procedure was similar as for dopamine D, receptor assay, but the initial homogenization took place in 20 vol of Tris-HCl buffer pH 7.6, the final incubation mixture (550 ~1) consisted of 450 ~1 membrane suspension, 50 ~1of [3H]clonidine (NEN, specific activity 26 Ci/mmol) solution ( 0.1 - 6 nM), and 50 ~1 Tris-HCl buffer, without (total binding) or with (unspecific binding) cold clonidine (final concentration 10 PM), and the incubation was carried out at 25°C for 30 min. Calculations and statistics No significant differences that could be related to the stage of the disease and drug history were observed among the samples taken from the parkinsonian patients and thus the group was treated as homogenous. The rate of dopamine metabolism was assessed as the ratio of concentration of its final metabolite, HVA, to dopamine [HVA]/[DA] from individual tissue samples (4). In the same manner the serotonin metabolism rate was assessedfrom [S-HIAA]/[S-HT] ratio. The relative receptor densities were assessed as the ratio of B,,, for the given receptor to the concentration of dopamine in the investigated structure (B ,,,/[amine]). To correlate the decrease in dopamine level in parkinsonian brains with the changes in relative dopamine receptor density the mean values for each brain structure from parkinsonian and control brains were compared. The correlation coefficients were calculated from the linear regression line. The statistical significance of differences was assessed with the Student’s T-test RESULTS. Dopamine The level of dopamine was significantly lower in parkinsonian brain structures than in the controls. The most dramatic changes the decrease more than 90%) were observed in the caudate nucleus and the globus pallidus, where the levels were only 6% of that of controls. A significant decrease in dopamine concentration was also observed in the putamen (16% of the control). In the substantia nigra the levels of dopamine were at 25% of the control but, owing to the great variability, the result did not reach the level of significance. The dopamine level in the nucleus accumbens was decreased by less than 60% and the result was not significant. The levels of dopamine metabolites, 3-MT and HVA , were also lower in the parkinsonian brains; the changes were, generally, parallel to those in dopamine levels, but were less accentuated (Fig. 1). As the decrease in dopamine concentration was more pronounced than that of its metabolites, the dopamine/metabolite

ratio was higher in the parkinsonian brains. The increase in the ratio of

HVA/dopamine concentration, an index of the rate of dopamine metabolism, was manifold increased in the substantia nigra (5=fold), caudate nucleus (-2.5fold)

and globus pallidus (-4-fold).

the metabolic index for dopamine were observed in the nucleus accumbens (Fig. 2).

No changes in

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DOPAMINE

Fig. 1. The postmortem levels of dopamine and its metabolites in the brain structures of parkinsonian patients. The bars represent the means h SEM from 5 (controls, open bars) or 4 (parkinsonians, crisscrossed bars) results. * P