SHORT COMMUNICATIONRegulation of striatal tyrosine hydroxylase phosphorylationby acute and chronic haloperidol

Kerstin Hakansson,1 Laura Pozzi,1,* Alessandro Usiello,1 John Haycock,2 Emiliana Borrelli3 and Gilberto Fisone11Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden2Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA, USA3Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP 163, 67404 Illkirch, Cedex, Strasbourg, France

Keywords: basal ganglia, clozapine, dopamine D2 receptors, extracellular signal-regulated protein kinases, striatum

Abstract

The typical neuroleptic haloperidol increases the state of phosphorylation and activity of tyrosine hydroxylase (TH), the rate-limitingenzyme in the synthesis of catecholamines. Here we show that the increases in TH phosphorylation produced by haloperidol at Ser31and Ser40, two sites critically involved in the regulation of enzymatic activity, are abolished in dopamine D2 receptor-null mice andmimicked by the selective dopamine D2 receptor antagonist, eticlopride. Moreover, the ability of haloperidol and eticlopride tostimulate phosphorylation at both seryl residues is prevented by treatment with SL327, a compound that blocks activation ofextracellular signal-regulated protein kinases 1 and 2 (ERK1 ⁄ 2). We also show that chronic administration of haloperidol reduces thebasal levels of phosphoSer31-TH and decreases the ability of the drug to stimulate Ser40 phosphorylation. These results provide amodel accounting for the stimulation exerted by haloperidol on dopamine synthesis. According to this model, haloperidol increasesTH activity via blockade of dopamine D2 receptors, disinhibition of dopaminergic projection neurons and ERK1 ⁄ 2-dependentphosphorylation of TH at Ser31 and Ser40. These studies also show that lower levels of phosphorylated TH are associated withchronic neuroleptic treatment and may be related to depressed dopaminergic transmission in nigrostriatal neurons.

Introduction

Neuroleptic drugs used in the treatment of schizophrenia and otherpsychotic disorders are known to act as antagonists at dopamine D2-like receptors (Creese et al., 1976; Seeman et al., 1976), a group ofheptahelical proteins that comprise the D2, D3 and D4 receptors. In thestriatum, the dopamine D2 receptor subtype is expressed in a largesubpopulation of projection neurons, where it mediates a variety ofpostsynaptic effects produced by dopamine, including changes inmotor activity, regulation of protein phosphorylation and expression ofimmediate-early genes. Dopamine D2 receptors are also present on theterminals and cell bodies of midbrain dopaminergic neurons, wherethey reduce firing rate and decrease neurotransmitter release (Picettiet al., 1997).The inhibitory feedback exerted by D2 autoreceptors on dopamin-

ergic transmission is particularly evident in the striatum. Administra-tion of haloperidol, a conventional neuroleptic with high affinity fordopamine D2 receptors, stimulates dopamine release (Imperato & DiChiara, 1985; Zetterstrom et al., 1985; Moghaddam & Bunney, 1990;Rayevsky et al., 1995). This effect is most likely mediated viadisinhibition of nigrostriatal and mesolimbic dopaminergic neurons(Bunney & Grace, 1978; Chiodo & Bunney, 1983). In addition,haloperidol promotes dopamine turnover, as indicated by its ability to

increase the levels of homovanillic acid (HVA), dihydroxyphenylace-tic acid (DOPAC) and 3-methoxytyramine (Carlsson & Lindqvist,1963; Asper et al., 1973; Zivkovic et al., 1975; Zetterstrom et al.,1985; Nissbrandt et al., 1989).Changes in the state of phosphorylation of tyrosine hydroxylase

(TH), the rate-limiting enzyme in the synthesis of catecholamines, arecritically involved in the regulation of dopamine synthesis. Inparticular, increases in the phosphorylation of Ser31 and Ser40accelerate TH activity, thereby stimulating production of neurotrans-mitter (Harada et al., 1996; Lindgren et al., 2000; Jedynak et al.,2002). Previous work showed that administration of haloperidolincreases the state of phosphorylation of TH in the rat striatum(Salvatore et al., 2000). In this study, we have identified a mechanismresponsible for the ability of haloperidol to increase TH phosphory-lation at Ser31 and Ser40 in the mouse striatum. Furthermore, we haveexamined changes in the regulation of TH phosphorylation producedby chronic treatment with haloperidol.

Materials and methods

Drugs

Haloperidol and clozapine were purchased from Sigma (St. Louis,MO, USA) and dissolved in saline containing 1% acetic acid; pH wasadjusted to 6.0 with 1 m NaOH. Eticlopride was purchased fromSigma (St. Louis, MO, USA) and dissolved in saline. SL 327, a giftfrom Dr James Trzaskos (DuPont Pharmaceuticals, Wilmington, DE,USA), was dissolved in 40% dimethylsulphoxide (DMSO).

Correspondence: Dr G. Fisone, as above.E-mail: gilberto.fisone@neuro.ki.se

*Present address: Department of Neuroscience, Mario Negri Institute for Pharma-

cological Research, Milan, Italy.

Received 28 February 2004, revised 25 May 2004, accepted 7 June 2004

European Journal of Neuroscience, Vol. 20, pp. 1108–1112, 2004 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2004.03547.x

Treatment and tissue extraction

Male C57BL ⁄ 6 mice (25–35 g) were purchased from B & K(Stockholm, Sweden). Dopamine D2 receptor knockout mice weregenerated as previously described (Baik et al., 1995). All studies wereapproved by the Swedish Animal Welfare Agency. In the acutestudies, the animals were injected i.p. with drugs or vehicle and killedby decapitation at various times as described. In the chronic studies,animals were divided into four groups and treated daily for 14 dayswith vehicle (groups 1 and 2) or haloperidol (0.5 mg ⁄ kg, i.p.; groups3 and 4). Twenty-four hours after the last chronic injection, the mice ingroups 1 and 3 were injected with vehicle and those in groups 2 and 4were injected with haloperidol (0.5 mg ⁄ kg, i.p.). The animals werethen killed after 15 min. Immediately after decapitation, the heads ofthe mice were immersed in liquid nitrogen for 6 s. The striata wererapidly dissected out on an ice-cold surface, sonicated in 1% sodiumdodecylsulphate and heated for 10 min. Previous studies show thatthis extraction procedure prevents protein phosphorylation anddephosphorylation, which occur very rapidly post-mortem (Sven-ningsson et al., 2000).

Determination of phosphoproteins

Aliquots (5 lL) of the homogenate were used for protein determin-ation using the BCA (bichinchoninic acid) assay kit (Pierce, OudBeijerland, the Netherlands). Equal amounts of protein (30 lg;corresponding to equal amounts of TH, cf. Results) from each samplewere loaded onto polyacrylamide gels (7.5 or 10%). The proteins wereseparated by sodium dodecyl sulphate–polyacrylamide gel electro-phoresis and transferred to polyvinylidine difluoride membranes(Amersham Pharmacia Biotech, Uppsala, Sweden), as described(Towbin et al., 1979). The membranes were immunoblotted usingaffinity-purified polyclonal antibodies that selectively detect phospho-Ser31-TH and phosphoSer40-TH (Salvatore et al., 2000). Antibodyagainst TH (a gift from Prof. Tomas Hokfelt, Karolinska Institutet,Stockholm, Sweden) that is not phosphorylation state-specific wasused to measure the total amount of TH protein. Antibody binding wasrevealed by incubation with goat anti-rabbit horseradish peroxidase-linked IgG (Pierce Europe, Oud Beijerland, the Netherlands) and theEnhanced Chemiluminescence Plus immunoblotting detection kit(Amersham Pharmacia Biotech). Chemiluminescence was detected byautoradiography and immunoreactivities were quantified by densi-tometry, using the NIH Image software (version 1.61). To determinethe relative increases in protein phosphorylation, immunoreactivitiesof the samples were interpolated to standard curves generated by arange of aliquots of pooled tissue standard (striatal tissue fromhaloperidol-treated mice) run on each gel.

Results and discussion

As shown in Fig. 1, the state of phosphorylation of TH at Ser31 andSer40 is significantly increased 15 min after injection of 0.5 mg ⁄ kg ofhaloperidol. The lower dose of 0.1 mg ⁄ kg produced a similar increasein Ser31 phosphorylation, but did not affect phosphorylation at Ser40(data not shown). These results provide a possible mechanismexplaining the ability of haloperidol to promote TH activity anddopamine turnover in the striatum (Carlsson & Lindqvist, 1963; Asperet al., 1973; Zivkovic et al., 1975; Scatton, 1977; Tissari et al., 1979;Zetterstrom et al., 1985; Nissbrandt et al., 1989; Ichikawa & Meltzer,1991; Cho et al., 1999).

It has been shown that a high dose (30 mg ⁄ kg) of the atypicalneuroleptic clozapine stimulates the phosphorylation of striatal TH,

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Fig. 1. Effects of haloperidol and clozapine on tyrosine hydroxylase (TH)phosphorylation at Ser31 and Ser40. Mice were treated i.p. with vehicle,haloperidol (0.5 mg ⁄ kg) or clozapine (5.0 mg ⁄ kg) and killed 15 min later.Upper panels show representative autoradiograms of Western blots obtainedusing polyclonal antibodies against phosphoSer31-TH (PSer31-TH; left) andphosphoSer40-TH (PSer40-TH; right). Lower panels show summaries of dataexpressed as means ± SEM (n ¼ 5–10). The amount of phosphorylated TH isexpressed as a percentage of that determined after vehicle administration.*P < 0.001 vs. respective vehicle-treated group (Student’s t-test).

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Fig. 2. Haloperidol fails to stimulate tyrosine hydroxylase (TH) phosphory-lation in dopamine D2 receptor knockout (D2R-KO) mice. Wild-type and D2R-KO mice were treated i.p. with vehicle or haloperidol (0.5 mg ⁄ kg) and killed15 min later. TH phosphorylation was measured using polyclonal antibodiesagainst phosphoSer31-TH (A) or phosphoSer40-TH (B). Upper panels showrepresentative autoradiograms; lower panels show summaries of data expressedas means ± SEM (n ¼ 6–9). The amount of phosphorylated TH is expressed asa percentage of that determined after vehicle administration to wild-type mice.*P < 0.001 vs. vehicle (one-way anova followed by Newman–Keuls test).�The interaction between treatment (haloperidol) and genotype (D2R-KO) issignificant (P < 0.001; two-way anova). (C) Representative autoradiogram ofa Western blot obtained using a polyclonal antibody against total TH.

Regulation of striatal tyrosine hydroxylase phosphorylation 1109

ª 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 20, 1108–1112

particularly at Ser19 and Ser40 (Salvatore et al., 2000). In this study,we have tested clozapine at 5 mg ⁄ kg, a more clinically relevant dose,able to induce high dopamine D2 receptor occupancy in the striatum ofprimates (Suhara et al., 2002). In the mouse striatum, this dose ofclozapine affects postsynaptic transmission, as demonstrated by itsability to stimulate the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), which is selec-tively localized in striatal medium spiny neurons (Pozzi et al., 2003).Nevertheless, we did not observe any changes in TH phosphorylationduring a period of time ranging from 15 min (Fig. 1) to 120 min (datanot shown) after injection of 5 mg ⁄ kg of clozapine. These results arein line with previous studies showing that, in the striatum, clozapine isless efficient than haloperidol in stimulating dopamine turnover(Anden et al., 1970; Zivkovic et al., 1975; Rayevsky et al., 1995).The increases in Ser31 and Ser40 phosphorylation produced by

0.5 mg ⁄ kg of haloperidol were abolished in dopamine D2 receptorknockout mice (Fig. 2), further supporting the notion that the D2

receptor subtype is specifically involved in presynaptic dopaminergiccontrol. In addition, these results suggest that haloperidol increases THphosphorylation in the striatum by promoting firing rate in midbraindopaminergic neurons and depolarizing nigrostriatal nerve terminals(Bunney & Grace, 1978).It has recently been reported that, in rat striatal slices, high

potassium-induced depolarization stimulates TH activity and promotesphosphorylation at Ser19, Ser31 and Ser40 (Lindgren et al., 2002). It

has also been shown that the depolarization-induced phosphorylationat Ser31 and Ser40, two sites critically involved in the control of THactivity (Harada et al., 1996; Lindgren et al., 2000; Jedynak et al.,2002), depends on stimulation of the two mitogen-activated proteinkinases (MAPKs), extracellular signal-regulated protein kinases 1 and2 (ERK1 ⁄ 2; Lindgren et al., 2002). We therefore utilized theMAPK ⁄ ERK kinase (MEK) inhibitor, SL327 to check the involve-ment of ERK1 ⁄ 2 in the increase in TH phosphorylation produced bysystemic administration of haloperidol.

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Fig. 3. SL327 inhibits haloperidol- and eticlopride-induced phosphorylationof tyrosine hydroxylase (TH) at Ser31 and Ser40. Mice were treated i.p. withSL327 (100 mg ⁄ kg) 45 min before receiving haloperidol (0.5 mg ⁄ kg) (A andB) or eticlopride (0.5 mg ⁄ kg) (C and D) and killed 15 min later. THphosphorylation was measured using polyclonal antibodies against phospho-Ser31-TH (A and C) or phosphoSer40-TH (B and D). Upper panels showrepresentative autoradiograms; lower panels show summaries of data expressedas means ± SEM (n ¼ 7–8). The amount of phosphorylated TH is expressed asa percentage of that determined after vehicle administration. *P < 0.001 vs.respective vehicle (one-way anova followed by Newman–Keuls test). Theinteractions between SL327 and haloperidol or eticlopride are significant(�P < 0.05, ��P < 0.01 and ���P < 0.001; two-way anova).

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Fig. 4. Tyrosine hydroxylase (th) phoshorylation is reduced by chronicadministration of haloperidol. Chronic treatment consisted of 14 days oftreatment (one injection per day) with vehicle (Group 1 and 2) or haloperidol(0.5 mg ⁄ kg, i.p.; Group 3 and 4). On day 15, mice received one last injection(Challenge) of vehicle (Group 1 and 3), or haloperidol (0.5 mg ⁄ kg, i.p.)(Group 2 and 4), and were killed 15 min after. TH phosphorylation wasmeasured using polyclonal antibodies against phosphoSer31-TH (A) or phos-phoSer40-TH (B). Upper panels show representative autoradiograms; lowerpanels show summaries of data expressed as means ± SEM (n ¼ 9–20). Theamount of phosphorylated TH is expressed as a percentage of that determinedafter vehicle administration. *P < 0.05 and **P < 0.001 vs. Group 1 (one-wayanova followed by Newman–Keuls test). �P < 0.05 vs. Group 1 (one-wayanova followed by Newman–Keuls test). �The interaction betweeen chronicand acute haloperidol is significant (P < 0.01; two-way anova). (C) Repre-sentative autoradiogram of a Western blot obtained using a polyclonal antibodyagainst total TH.

1110 K. Hakansson et al.

ª 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 20, 1108–1112

We found that treatment with 100 mg ⁄ kg of SL327 (a dose thatselectively blocks MEK1 and 2; Pozzi et al., 2003) abolished theincrease in Ser31 produced by haloperidol (0.5 mg ⁄ kg) or eticlo-pride (0.5 mg ⁄ kg) (Fig. 3A and C). SL327 was also able to reduceby 50% the effects of the two drugs on Ser40 phosphorylation(Fig. 3B and D), indicating that ERK1 ⁄ 2 are, at least in part,responsible for phosphorylation at this site. In contrast, SL327 didnot affect the ability of haloperidol to stimulate Ser19 phosphoryla-tion (data not shown). These experiments suggest that haloperidolstimulates TH activity and accelerates dopamine metabolism via amechanism involving blockade of dopamine D2 receptors, increasedfiring rate of midbrain dopaminergic neurons, depolarization-dependent activation of ERK1 ⁄ 2 and phosphorylation of TH atSer31 and Ser40.

The residual phosphorylation on Ser40 observed in the presence ofSL327 is most likely due to activation of cAMP-dependent proteinkinase (PKA). Previous studies have indeed shown that Ser40 isphosphorylated by agents that increase cAMP levels (Lindgren et al.,2000). Thus, haloperidol and eticlopride may stimulate Ser40phosphorylation by removing the negative regulation exerted by D2

receptors on adenylyl cyclase (Stoof & Kebabian, 1981), increasingcAMP and activating PKA.

Prolonged administration of haloperidol reduces the ability of thisdrug to stimulate TH activity and dopamine turnover (Asper et al.,1973; Scatton, 1977; Tissari et al., 1979; Ichikawa & Meltzer, 1991;Cho et al., 1999). Moreover, chronic haloperidol is known to block thespontaneous firing of nigrostriatal dopaminergic neurons (Bunney &Grace, 1978; Chiodo & Bunney, 1983; Grace et al., 1997). This effectis regarded as a mechanism responsible for the generation ofextrapyramidal motor disturbances, which represent a severe limita-tion to the use of conventional antipsychotic drugs (Grace et al.,1997).

Chronic administration of haloperidol (0.5 mg ⁄ kg) for 14 dayslowered the basal levels of phosphoSer31-TH, measured 24 h after thelast injection of drug (Fig. 4A, cf. Group 3 and Group 1). Chronictreatment also reduced the ability of haloperidol to stimulatephosphorylation of TH at Ser40 (Fig. 4B, cf. Group 4 and Group2). The decrease in the basal levels of phosphoSer31-TH and theblunted stimulation of Ser40 phosphorylation may be a consequenceof the reduction in firing rate produced by chronic treatment withhaloperidol. They may also explain the tolerance to the stimulation ofdopamine synthesis produced by repeated administration of thisneuroleptic.

Our data show that chronic administration of haloperidol does notaffect the levels of striatal TH, measured 24 h after the last injection ofdrug (Fig. 4C, Group 3). In contrast, studies performed in the ratreported increased levels and activity of striatal TH, 18–24 h aftersuspension of chronic treatment with haloperidol (Lerner et al., 1977;Cho et al., 1999). This discrepancy may reflect a more rapidmetabolism of haloperidol in the mouse than in the rat. Indeed, THexpression and activity return to normal levels 48 h after terminatingchronic treatment in the rat (Guidotti et al., 1978; Tissari et al., 1979;Cho et al., 1999).

In summary, the present results demonstrate that, in the mousestriatum, the increases in TH phosphorylation at Ser31 and Ser40produced by haloperidol depend on concomitant activation of ERK1 ⁄ 2.This activation is most likely produced by increased firing rate ofmidbrain dopaminergic neurons, caused by blockade of D2 autorecep-tors and ⁄ or by regulation via striatonigral feedback loops, followingblockade of postsynaptic D2 receptors. Another finding of this study isthat chronic treatment with haloperidol blunts the stimulation exerted bythis drug on Ser40 phosphorylation. This effect may be responsible for

the parallel reduction in the ability of haloperidol to stimulate dopamineturnover (Asperet al., 1973; Scatton, 1977; Tissari et al., 1979; Ichikawa& Meltzer, 1991; Cho et al., 1999) and represents a specific molecularmarker indicating the occurrence of depressed dopaminergic transmis-sion following chronic haloperidol administration.

Acknowledgements

This work was supported by Swedish Research Council grants 13482 and14518 (to G.F.), the Foundation Blanceflor Boncompagni-Ludovisi, nee Bildt(to L.P.) and the Wenner-Gren Foundations (to A.U.).

Abbreviations

ERK 1 ⁄ 2, extracellular signal-regulated protein kinases 1 and 2; MAPK,mitogen-activated protein kinase; PKA, cAMP-dependent protein kinase; TH,tyrosine hydroxylase.

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