Girault Et Al., (2004)

7/21/2019 Girault Et Al., (2004)

http://slidepdf.com/reader/full/girault-et-al-2004 1/5

SECTION EDITOR: HASSAN M. FATHALLAH-SHAYKH, MD

The Neurobiology of Dopamine Signaling

Jean-Antoine Girault, MD, PhD; Paul Greengard, PhD

The brain contains 2 major groups of dopamine neurons. One is located in the arcuate

nucleus of the hypothalamic median eminence and is involved in neuroendocrine regu-

lation. The other, which is the subject of this article, is located in the ventral mesen-

cephalon and projects to the forebrain. Although dopamine neurons are few (1/

100000 brain neurons), they play an important role in regulating several aspects of basic brain

function. They are necessary for the normal tasks of the regions they innervate, including motorbehavior, motivation, and working memory. Dopamine neurons are also a central element in the

brain reward system that controls the learning of many behaviors. Disappearance of nigrostriatal

neurons results in Parkinson disease, whereas blockade of dopamine receptors has therapeutic ef-

fects in psychosis. Finally, artificial increase in dopamine transmission is the common mechanism

of action of drugs of abuse that leads to addiction. Understanding how dopamine works is a major

goal of neurobiology. Much progress has been accomplished in identifying the intracellular sig-

naling pathways that underlie the immediate actions of dopamine and account for its long-term

effects on brain properties. Recent findings allow us to identify molecules that may represent fu-

ture therapeutic targets in neurology and psychiatry. Arch Neurol. 2004;61:641-644

Dopamine wasidentified as a potentialneu-rotransmitter in the brain in the late 1950sby Carlsson.1 Soon thereafter, it was foundthatParkinsondisease wasaccompanied bya dramatic decrease in the dopamine con-tent of the putamen and caudate nucleus.This led to the use of levodopa, the meta-bolic precursor of dopamine, as an effi-cientreplacement therapy for alleviating thesymptoms of Parkinson disease. The firstdopamine receptorswere identified by theirability to stimulate cyclic adenosine mono-phosphate (cAMP) production and wereshown to be the targets of neuroleptic

agents.2

This discovery defined a class of slow-actingneurotransmittersthat exerttheireffects through cascadesof biochemicalre-actions, as opposed to fast synaptic neuro-transmitters like glutamate or -aminobu-tyric acid that open ligand-gated ion

channels.3

Although fast neurotransmis-sion accounts for most of the quasi-instantaneousfunctioning of the brain, slowneurotransmission is essential for neuro-modulation and long-term regulation. Neu-roleptic agents were found to block a sec-ond type of dopamine receptor.4 Thisdefined 2 classes of dopamine receptors, D1and D2, which respectively stimulate andinhibit adenylyl cyclase. Later, cloning of thesereceptors demonstrated 2 D1-like re-ceptors (D1 and D5) and 3 D2-like recep-tors (D2, D3, and D4). D1 and D2 recep-tors areabundant in theneostriatum,which

comprises the caudate nucleus, putamen,and nucleus accumbens. These receptorsareenriched in distinct populations of stria-tal neurons, although there is a degree of coexpression. While D1 and D2 receptorshave opposite effects at the molecular level,they often have a synergistic action whenmore complex outputs are considered. Al-though D3 receptors seem to play a minorrole in normal circumstances, they appearto be up-regulated in pathologic condi-

From the Institut National de la Sante et de la Recherc he Medicale and Universite Pierre et Marie Curie Unit 536, Institut du Fer à Moulin, Paris, France (Dr Girault);and the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY (Dr Greengard).

BASIC SCIENCE SEMINARS IN NEUROLOGY

(REPRINTED) ARCH NEUROL / VOL 61, MAY 2004 WWW.ARCHNEUROL.COM641

©2004 American Medical Association. All rights reserved.

wnloaded From: http://archneur.jamanetwork.com/ by a Centro de Investigaciones User on 02/09/2015

7/21/2019 Girault Et Al., (2004)


tions, making them prime targets fordrug development.5

The cell bodies of dopamineneurons innervating the caudatenucleus and the putamen are lo-cated in the substantia nigra, whilethose of neurons innervating theven-tral striatum and the prefrontal cor-texhavea more medial location in the

ventral tegmental area. Dopamine isnot a simple excitatory or inhibitoryneurotransmitter. It is a neuromodu-lator that alters the responses of tar-get neurons to other neurotransmit-ters in a manner that depends on thefunctional state of these neurons. Be-cause of this complex modulatoryrole, trying to elucidate the physi-ological role of dopamine has beenfrustrating for many years. Recentprogress in many areas of neurosci-ence, ranging from cellular electro-physiology to in vivo recordings and

behavioral studies, allows us to bet-ter understand the function of dopa-mine neurons. One important find-ingwasthe identification of their rolein reward systems.6 In monkeys, un-predicted rewards increase the fir-ing of dopamine neurons, whereastheabsenceof anexpectedrewardhasan inhibitory effect. This led to theproposal that dopamine neuronsfunction as detectors of reward pre-diction errors. Although complex,this hasprofound implications fortherole of dopamine in learning.6

Receptors mediate the actionof neurotransmitters on target neu-rons by altering the permeability of ion channels and thereby changingthe membrane potential, or by acti-vating signaling pathways that in-clude different biochemical reac-tions. These should notbe consideredindependent mechanisms becausecalcium influx through ion chan-nels activates signaling pathways,while signaling pathways modify theproperties of several ion channels.Dopamine receptors comprise

7-transmembrane domain recep-tors and are associated with guano-sine triphosphate–binding proteins(or G proteins)that mediate their ef-fects. One major effect of D1 recep-tors is to raise cAMP levels andthereby activate a cAMP-dependentprotein kinase (PKA). This enzymetransfers a phosphate group fromadenosine triphosphate to severalspecific protein substrates, modify-

ingtheir properties in many ways. D2receptors are coupled to differenttypes of G proteins that decreasecAMP levels andalter the permeabil-ity of potassium channels. Becauseregulation of cAMP levels is a majoreffect of dopamine receptors and be-cause PKA is the major target of cAMP, understanding the action of

dopamine at the molecular level re-quired the identification and charac-terization of PKA substrates in neu-rons innervated by dopamine.

EXPERIMENTAL METHODS

Most of the initial experiments doneto understand the action of dopa-mine at a molecular level have in-volved the striatum, which containshigh levels of dopamine receptors.Several approaches have been usedto identify the relevant PKA sub-

strates in striatal neurons, includingthe search for proteins that arephos-phorylated by this enzyme in vitro.7

These proteins were then purified,their genes cloned, and their prop-erties studied. Another differentstrat-egy started from well-characterizedproteins such as ion channels or neu-rotransmitter receptors. Neurobiolo-gists examined whether these pro-teins were phosphorylated and howthis phosphorylation altered theirproperties. In all cases, it was cru-cial to determinehow thephosphory-

lation of relevant proteins is regu-lated. To do so, a practical methodis to use antibodies that recognize agiven protein only when it is phos-phorylated at a precise location on aspecific amino acidresidue.8 Such an-tibodies are usually obtained by im-munizing rabbits or mice against ashort phosphorylated peptide thatcorresponds to the site of interest.The specificity of the antibodies canbe improved by selecting those thatreact with the phosphorylated anti-gen but not with its unphosphory-

lated form, using a method calledaf- finity purification. A similar strategycanbe used to generate antibodies re-acting only with the unphosphory-lated form of a protein. Phosphory-lation state–specific antibodies can beused in 2 types of experiments(Figure1). First, the antibodies al-low the identification of phosphory-lated proteins by immunoblot (alsotermed Western blot). Within limits,

this allows a quantitative measure-mentof specific proteins, phosphory-lated or not, depending on the selec-tivity of the antibodiesused. Anotheruseful application of such antibod-ies is to reveal the presence of phos-phorylated proteins in fixed cells ortissues using immunofluorescence.Onl y pho spho r y l ati o n state–

specificantibodies that have no cross-reactivity with any other antigen canbe used for immunofluorescencestudies because there isno prior sepa-ration of proteins.

RELEVANCE TO THE STUDYOF NEUROSCIENCE

Using the methods herein and othermolecular and cellular techniques,many proteins and phosphorylationreactionsinvolved in theactionof do-pamine have been identified. Some

phosphorylated proteins are di-rectly responsible for dopamine’s ef-fects, eg, dopamine controls the ac-tivity of glutamate receptors thatmediate the corticostriatal neuro-transmission. It does this throughphosphorylation by PKA of 2 majorsubtypes of glutamate receptors, so-called AMPA (-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)and NMDA (N -methyl-D-aspartate)receptors, according to their selec-tive syntheticagonist. Dopamine alsoregulates the activity of voltage-

gated ion channels, including so-dium and calcium channels, bymodulating the phosphorylationstateof thesechannelsor of associated pro-teins. Another class of proteins iscritical for dopamine’s long-term ef-fects comprises transcription fac-tors that regulate the expression of specific genes. Through compli-cated phosphorylationcascades, do-pamine increases the phosphoryla-tion of specific transcription factors,leading to their increasedactivity andthe expression of immediate early

genes. In turn, immediateearly genesactivate the expression of late genesthat arethought to beessential for thelong-lasting modifications of synap-tic transmission controlled by dopa-mine.

In addition to these proteinsregulated by phosphorylation, whichmay be thought of as the effectorsof dopaminesignaling, another classof proteins plays a critical role in the




7/21/2019 Girault Et Al., (2004)


7/21/2019 Girault Et Al., (2004)


the traditional boundaries betweenneurology and psychiatry. Until re-cently, virtually all the drugs avail-able to neurologists or psychia-trists were acting at the level of theneuronal membranes, modifyingneurotransmitters’metabolism or in-teractions with receptors. Under-standing the intracellular signalingpathways underlying the action of neurotransmitters, especially dopa-

mine, suggests a newclass of poten-tial therapeutic agents that couldtar-get a specific protein kinase or aphosphatase. In principle, one ad-vantage of pharmacological manipu-lation of signaling pathways is thatit would not alter basic function butonly modify regulatory processes.Forexample, inhibitors of proteinki-nases modulating DARPP-32 couldhave applications in enhancing ordecreasing specific aspects of dopa-mine actions. Knowledge of the sig-naling pathways activated by dopa-

mine also allows us to look forchanges in these pathways in patho-logic conditions. At present, thisap-proach is limited in humans to thesearch for mutations in the genes of the relevant proteins and for varia-tion of their levels in postmortembrain samples. Therefore, it will bechallenging to design novel meth-

ods allowing the study of intracel-lular signaling pathways in vivo bynoninvasive approaches. The use of experimental animal models and thedesign of drugs capable of selec-tively altering these pathways shouldallow important progressto be made.

Accepted for publication January 8, 2004.

Author contributions: Study

concept and design (Drs Girault andGreengard); acquisition of data (DrsGirault and Greengard);analysis andinterpretation of data (DrsGirault andGreengard); drafting of the manu-script (Drs Girault and Greengard);critical revision of the manuscript for important intellectual content (DrsGirault and Greengard); obtained funding (Drs Girault and Green-gard); study supervision (Drs Giraultand Greengard).

We thank Denis Hervé PhD, andEmmanuel Valjent, PhD, for the illus-

tration in Figure 1.Corresponding author: Jean-

Antoine Girault, MD, PhD, InstitutNational de la Sante et de la Recher-che Medicale and Universite Pierre etMarie Curie Unit 536, Institut du Fer a Moulin, 17, rue du Fer a Moulin,7 5 0 0 5 P a r i s , F r a n c e ( e - m a i l :[email protected]).

REFERENCES

1. CarlssonA. Theoccurrence, distributionandphysi-ologicalrole of catecholamines inthe nervous sys-tem. Pharmacol Rev. 1959;11:490-493.

2. Kebabian JW, Greengard P. Dopamine-sensitiveadenyl cyclase. Science . 1971;174:1346-1349.

3. Greengard P. The neurobiology of slow synaptictransmission. Science . 2001;294:1024-1030.

4. Creese I, Burt DR, Snyder SH. Dopamine recep-tor binding predicts clinical and pharmacologicalpotencies of antischizophrenic drugs. Science .1976;192:481-483.

5. LuedtkeRR, MachRH. Progressin developing D3

dopaminereceptorligandsas potentialtherapeu-tic agents for neurological and neuropsychiatricdisorders. Curr Pharm Des . 2003;9:643-671.

6. SchultzW. Dopamineneurons andtheir rolein re-ward mechanisms. Curr OpinNeurobiol. 1997;7:191-197.

7. Walaas SI, Nairn AC, Greengard P. Regional dis-tribution of calcium- and cyclic adenosine 3’:5’-monophosphate–regulatedprotein phosphoryla-tion systems in mammalian brain, II: solublesystems. J Neurosci. 1983;3:302-311.

8. Czernik AJ, Girault JA, Nairn AC, et al. Produc-tion of phosphorylation state–specific antibod-ies. Methods Enzymol. 1991;201:264-283.

9. SvenningsonP, NishiA, FisoneG, Girault JA,NairnAC,GreengardP. DARPP-32:an integratorof neu-rotransmission. Annu Rev Pharmacol Toxicol.2004;44:269-296.

10. Greengard P, Allen PB, Nairn AC. Beyond the do-

pamine receptor: the DARPP-32/protein phos-phatase-1 cascade. Neuron . 1999;23:435-447.

11. Fienberg AA, Hiroi N, Mermelstein PG, et al.DARPP-32: regulator of theefficacyof dopaminer-gic neurotransmission. Science . 1998;281:838-842.

12. Bibb JA, Snyder GL, Nishi A, et al. Phosphoryla-tion of DARPP-32 by Cdk5 modulates dopaminesignalling in neurons. Nature . 1999;402:669-671.

13. BibbJA,ChenJ, Taylor JR, etal.Effectsof chronicexposure to cocaine are regulated by the neuro-nal protein Cdk5. Nature . 2001;410:376-380.

DARPP-32DARPP-32

PhysiologicalEffects

P-Thr34-DARPP-32

P-Thr75-DARPP-32

Extracellular

Intracellular

Dopamine

Phospho-protein Protein

Inhibition

I n h i b i

t i o n

PP-1

V

VV

V

V

V

GTPATP cAM P

D1

G

AdenylylCyclase

Extracellular

Intracellular

Dopamine

GTPATP cAMP

D1

G

AdenylylCyclase

PKA PKA

CDK5

A B

Figure 2. The role of DARPP-32 in the action of dopamine. A, In basal conditions, dopamine stimulates the phosphorylation of DARPP-32 on threonine 34(P-Thr34–DARPP-32) through a signaling cascade that includes dopamine D1 receptors, a specific heterotrimeric G protein (G), adenylyl cyclase that raises cyclicadenosine monophosphate (cAMP) levels, and cAMP-dependent protein kinase (PKA). Because phosphorylated DARPP-32 is a potent inhibitor of proteinphosphatase 1 (PP-1), it increases the phosphorylation of numerous substrates and plays a major role in dopamine actions. B, When cyclin-dependent kinase 5

(CDK5) is activated (eg, following treatment for cocaine abuse), it phosphorylates DARPP-32 on threonine 75, turning it into an inhibitor of PKA and switching offthe regulations depicted in A. ATP indicates adenosine triphosphate; GTP, guanosine triphosphate.




7/21/2019 Girault Et Al., (2004)


REFERENCES

1. Scherzer CR, Offe K, Gearing M, et al. Loss of apolipoprotein E receptor LR11 in

Alzheimer disease. Arch Neurol . 2004;61:1200-1205.

2. Papassotiropoulos A, Streffer JR, Tsolaki M, et al. Increased brain beta-amyloid

load, phosphorylated tau, and risk of Alzheimer disease associated with an in-

tronic CYP46 polymorphism. Arch Neurol . 2003;60:29-35.

3. Kolsch H, Lutjohann D, Ludwig M, et al. Polymorphism in the cholesterol 24S-

hydroxylase gene is associated with Alzheimer’s disease. Mol Psychiatry . 2002;

7:899-902.

4. Wollmer MA, Streffer JR, Tsolaki M, et al. Genetic association of acyl-coenzyme

A: cholesterol acyltransferase with cerebrospinal fluid cholesterol levels, brainamyloid load, and risk for Alzheimer’s disease. Mol Psychiatry . 2003;8:635-

638.

5. Wollmer MA, Streffer JR, Lutjohann D, et al. ABCA1 modulates CSF cholesterol

levels and influences the age at onset of Alzheimer’s disease. Neurobiol Aging .

2003;24:421-426.

6. Kolsch H, Ptok U, Mohamed I, et al. Association of the C766T polymorphism of

the low-density lipoproteinreceptor-relatedprotein gene withAlzheimer’sdisease.

Am J Med Genet . 2003;121B:128-130.

7. Sanchez L, Alvarez V, Gonzalez P, et al. Variation in the LRP-associated protein

gene(LRPAP1) is associated withlate-onsetAlzheimer disease.AmJMedGenet .

2001;105:76-78.

8. Yau JL, Rasmuson S, Andrew R, et al. Dehydroepiandrosterone 7-hydroxylase

CYP7B: predominant expression in primate hippocampus and reduced expres-

sion in Alzheimer’s disease. Neuroscience . 2003;121:307-314.

9. BrownJ, Theisler C,Silberman S,et al.Differential expression of cholesterol hy-

droxylases in Alzheimer’s disease. J Biol Chem [serial online]. May 17, 2004.

Available at: http://www.jbc.org/cgi/reprint/M402324200v1. Accessed May 17,

2004.

10. Bogdanovic N, Bretillon L, Lund EG, et al. On the turnover of brain cholesterol in

patients with Alzheimer’s disease: abnormal induction of the cholesterol-

catabolic enzyme CYP46 in glial cells. Neurosci Lett . 2001;314:45-48.

11. Burns MP, Noble WJ, Olm V, et al. Co-localization of cholesterol, apolipoprotein

E and fibrillar Abeta in amyloid plaques. Brain Res Mol Brain Res . 2003;110:119-125.

12. Pietrzik CU,Busse T,MerriamDE, etal. Thecytoplasmic domainof theLDLreceptor-

related protein regulates multiple steps in APP processing. EMBO J . 2002;

21:5691-5700.

13. Sun Y, Yao J, Kim TW, Tall AR. Expression of liver X receptor target genes de-

creases cellular amyloid beta peptide secretion. J Biol Chem . 2003;278:27688-

27694.

14. KoldamovaRP, Lefterov IM, IkonomovicMD, et al. 22R-hydroxycholesterol and

9-cis-retinoic acid induce ATP-binding cassette transporter A1 expression and

cholesterol efflux in brain cells anddecreaseamyloid betasecretion. J Biol Chem .

2003;278:13244-13256.

Correction

Error in Figure Legend. In the article titled “The Neu-robiology of Dopamine Signaling,” published in the Mayissue of the ARCHIVES (2004;61:641-644), the legend toFigure 2 included an error. The description for part Bshould have read as follows: When cyclin-dependent ki-nase 5 (CDK5) is activated (eg, following long-term co-caine administration), it phosphorylates DARPP-32 onthreonine 75, turning it into an inhibitor of PKA andswitching off the regulations depicted in A.

(REPRINTED) ARCH NEUROL / VOL 61, AUG 2004 WWW.ARCHNEUROL.COM1180


Documents

Girault Et Al., (2004)