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Paul Greengard: Signals underlying moods, addictions,and brain disorders

Nowadays, whether people sufferfrom depression, mental healthdisorders, or addictions, medi-cation can help. Researchers

have learned how to ne tune the brain sfunctions, often by targeting chemicalscalled neurotransmitters and the many signaling pathways through which they act.

Although certain neurotransmitters hadbeen identi ed more than 40 years ago,scientists knew relatively little about howthese chemicals actually functioned. Thebasis for much of what neuroscientists knowtoday about brain signaling was shaped by National Academy of Sciences memberPaul Greengard s 1972 PNAS Classic pa-per, Dopamine-sensitive Adenylate cy-clase in caudate nucleus of rat brain, and itssimilarity to the dopamine receptor (1).

In the PNAS Classic paper, Kebabianet al. (1) identi ed enzyme activity in thebrain that was triggered by the neuro-transmitter dopamine. This understandingof how dopamine works has formed ourbasic understanding of brain signaling andhelped discover therapies for brain dis-orders. Dopamine continues to be thesubject of much research and has beenimplicated in addictions, attention de cithyperactivity disorder (ADHD), mooddisorders, schizophrenia, and movementdisorders such as Parkinson s disease.

Greengard realized that the enzymeactivity that he discovered probably in-dicated the presence of a dopamine re-ceptor that bound to the neurotransmitter.Subsequent identi cation and puri cationof the proteins that form this receptorrevealed that Greengard had gured outthe critical rst steps of neurotransmittersignaling. He went on to show that thereceptor-mediated enzyme activates a sec-ond messenger, which then triggers a cas-cade of other signaling events that regu-late complex neurological traits and affecthow nerve cells grow and function.

Dopamine s role as a neurotransmitter

was

rst uncovered by Arvid Carlsson, whoshared the 2000 Nobel Prize in Physiology and Medicine for this discovery (2 4).Carlsson et al. (2, 3) noticed that patients with Parkinson s disease have particularly low dopamine concentrations in certainparts of the brain. Dopamine depletionin the brains of animals also causedParkinson s-like movement symptoms, andCarlsson was able to treat these symptomsusing L -DOPA, which gets converted todopamine in the brain. His experimentshelped develop L -DOPA as a drug forParkinson s disease, and it remains one of the disease s most important treatments.

Greengard

s discovery of the dopaminereceptor opened up many additional ave-nues for clinical treatments. Many anti-psychotic drugs, particularly those drugsused to treat schizophrenia, act by block-ing dopamine receptors. Greengard s re-sults helped explain how these drugs actedand how they could be improved. Dopa-mine signaling pathways have also beenimplicated in the reward mechanisms of the brain, and drugs that modulate thesepathways can help with mood disordersand addictions.

However, the implications of Green-gard s work extended beyond clinicaltreatments. They also helped lay thefoundations of modern neuroscience. Hisuse of classical biochemistry to study thebrain in uenced many other researchersand helped discover new signaling path- ways and regulatory proteins. In recogni-tion of his work s wide-ranging in uence,Greengard was awarded the Nobel Prizein Physiology and Medicine in 2000, thesame year that Carlsson was honored.

Despite his contributions to neurosci-ence, Greengard s initial interest was inmathematics and theoretical physics. It was only after he served in the US Navy inWorld War II and after the dropping of

the atomic bombs that he changed his ca-reer path. I didn t want to go into theo-retical physics and risk making morepotent bombs, Greengard said. Instead,he entered the nascent eld of biophysicsand focused on trying to understandthe brain.

During his graduate research at JohnsHopkins University, Greengard usedelectrophysiology, or the study of electriccurrents in nerve cells, to try to understandhow the brain functioned. He started with this approach, because it was themajor tool of neuroscientists at the time. As he progressed through his postdoctoral work, however, Greengard increasingly turned to biochemistry, believing that itcould provide unique insights. His researchled him to pioneer many biochemicalapproaches prevalent today.

These approaches soon bore fruit. In1968, Greengard became a professor in theDepartment of Pharmacology at Yale and within a year to a year and a half, gen-erated papers that still have continuingimpact today, said Angus Nairn, a neuro-scientist at Yale who did his postdoctoralresearch in Greengard s laboratory. It sreally quite remarkable how much they generated in a short time.

Huge Conceptual BarrierBy the 1970s, many scientists had studied

neurotransmitters, but no one had a clearidea of how these chemicals exert theirmyriad in uences on the brain. The con-cept of receptors that neuroscientists takefor granted today was still mostly a black box, said Snyder, a neuroscientist atJohns Hopkins University and author of the PNAS Classic Perspective on Green-gard s paper, What dopamine does inthe brain (5).

Receptors were still a conceptualthing, said Richard Huganir, a neurosci-entist at Johns Hopkins University andformer postdoctoral researcher withGreengard. It wasn t even clear if re-ceptors were proteins, believe it or not. Researchers studying other signalingmolecules such as hormones had revealedthat they bound to receptors on cell sur-faces, which triggered other signalingevents inside the cell. One of Greengard sinsights was that neurotransmitters might work in the same way in the brain.

Fig. 1. Dopamine release and binding to re-ceptor. Dopamine is released by a neuron into the junction between two nerve cells called the syn-apse. There, it can bind to dopamine receptors onneighboring neurons, and the receptors activatethe second messenger and downstream signalingpathways.

See Classic Article Dopamine-sensitive adenylate cyclasein caudate nucleus of rat brain, and its similarity to the dopamine receptor on page 2145in issue 8 of volume69.

See Classic Perspective on page 18869.

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As Greengard started putting variouspieces of evidence together, he was in-spired by the discovery that cAMP medi-ated the action of certain hormones in theliver (6). cAMP acted as a second mes-senger that transmitted a signal from ahormone s receptor to other proteins within the cell, triggering downstreamsignaling pathways. Greengard startedlooking for a similar interaction in thebrain mediated by adenylate cyclase, theenzyme that converts ATP into cAMP.

However, this search was far from anobvious train of thought. Most neuro-scientists still believed that any signalingin the brain was electrical and not bio-chemical. There was a huge conceptual

barrier,

said Nairn. adenylate cyclasetook many seconds to minutes to generatea signal, a seemingly lethargic processcompared with the rapid electrical sig-naling that neuroscientists were familiar with in the brain. That Greengard pushedpast this paradigm and explored such acounterintuitive theory was a major factorin his receipt of the Nobel Prize, Nairnsaid. He kept pushing at it, gatheringbiochemical evidence for it.

Gathering the biochemical evidence tosupport this theory brought its own chal-lenges. The only neurotransmitter receptordiscovered until then had been in theelectriceel, where it constituted 20% of the weight of the eels electric organ (7). Thisbreakthrough result only made scientistsmore convinced that it would be impos-sible to isolate receptors in the humanbrain, where they would constitute a mil-lionth of its weight at most, said Snyder.

However, Greengard was careful in hisselection of the neurotransmitter andthe target location to study. He pickeddopamine and looked for its effects in abrain area where Carlsson s previousstudies had shown that it was not only abundant but where its loss elicited asyndrome similar to Parkinson s disease(2 4). If he had looked at the wholebrain, he would probably have missed thisphenomenon, because everything wouldbe diluted out, said Nairn.

The researchers had to carefully extractspeci c brain regions from multiple ratsand pool them together, while ensuringthat they did not destroy the enzyme ac-tivity in the process. Isolating the appro-priate brain area using the techniques of the time required heavy slogging, saidSnyder. They used very classical bio-chemical techniques that required bigtime careful work to perform success-fully, he said.

However, the hard work paid off. WhenGreengard and his graduate student JohnKebabian added dopamine to speci cbrain fractions, they saw a production of

cAMP as a second messenger, indicatingthat the neurotransmitter was signalingthrough an adenylate cyclase. To actually nd an adenylate cyclase that was coupledto neurotransmitter nobody had donethat, said Snyder.

Kebabian said that Greengard and hehad anticipated that this research wouldbe an important piece of work, and they were pleased when they succeeded.However, the importance of their results

was cemented by what was to come. Thepaper sets the stage pretty well for whatfollowed, said Nairn. The details gotmore complex, but the direction was re-ally set up by this paper.

Greengard, Kebabian, and others wenton to characterize dopamine receptoractivity in more detail, nding that do-pamine actually had multiple differentreceptors to which it could bind. They and other researchers used similarapproaches to study the receptors andsignaling of other neurotransmitters, suchas serotonin or noradrenaline. Their work led to more studies of the slow, bio-chemical transmission of signals by theseneurotransmitters, which has prolongedeffects on nerve cells and helps regulatebasal nervous system functions, includingmoods. This slow transmission can alsoopen ion channels to mediate the fasttransmission of nerve signals. AsGreengard and others were to nd out,slow, chemical nerve signaling is an ex-tremely important and complicatedprocess, involving a whole cascade of downstream agents.

Setting the Stage for ModernNeuroscience As a follow-up to the Classic paper,Greengard s laboratory systematically tracked down the events that occur afterdopamine binds to its receptor and acti- vates an adenylate cyclase enzyme. Inother organs, cAMP had been shown toregulate enzymes called kinases, whichadd phosphate groups to molecules in

a process called phosphorylation (8).Phosphorylation can change the structureand function of proteins and cause themto trigger signaling events. [Greengard] gured out that neurotransmitters could work in the same way, said Huganir.

Greengard showed that, after dopa-mine acted through its receptor to in-crease the amount of cAMP in a nervecell, the cAMP activated a kinase, whichphosphorylated other proteins in the cell.Some of these proteins could form ionchannels in the cell membrane, thuscontrolling the nerve cell s ability to sendelectrical impulses. Greengard also founda phosphorylated protein, Dopamine-and cAMP-regulated phosphoprotein(DARPP)-32, which is a central regulatorof neuronal function and affects a largenumber of other proteins. DARPP-32 hasbeen implicated in several neurologicaldisorders and the action of multipleaddictive drugs.

Greengard s work showed that phos-phorylation was an important signalingmechanism in the brain. The discovery that neurotransmitter signals are medi-ated by the addition and removal of phosphate groups from proteins in nervecells continues to have an impact today.

Fig. 2. Dopamine receptor availability in thebrain. Brain images show a decrease in the avail-able dopamine receptors in the brain of a personaddicted to cocaine compared with a nondruguser. A similar decrease is seen with many otheraddictive substances, including alcohol and heroin.The lower level of receptors is thought to occurbecause of their repeated overstimulation, and itcan lead to a reduced activation of natural rewardpathways that contributes to the tendency toabuse these addictive substances. Adapted fromVolkow et al. (9).

Fig. 3. Dopamine signals through a cAMP second messenger. After dopamine binds to its receptor, thereceptor then triggers the activation of an adenylate cyclase enzyme, which produces cAMP from ATP.cAMP acts as a second messenger that transmits a signal from the receptor to other proteins within the cellby binding to them and altering their activity. After dopamine increases the level of cAMP in nerve cells, thecAMP activates a kinase that adds phosphate groups to other proteins in the cell in a process known asphosphorylation. Phosphorylation of these other proteins triggers downstream signaling pathways.

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Many current drug discovery efforts in- volve studying the phosphorylation of different proteins in nerve cells.

However, when these results were rstpublished, their novelty made them con-troversial. There was a point when Ithought they would not be accepted in my lifetime, said Greengard. There wasa lot of skepticism. He remained un-deterred. After we got the rst data, I was positive I was correct, he said. Therapid rise of modern molecular biology in the 1980s helped validate his con- victions. Real acceptance came with thedevelopment of new techniques, Green-gard said. Gradually, the evidencebecame so overwhelming that half thepeople in brain science today study signaling.

With this Classic paper and his follow-up work came the realization that sig-naling pathways are cell function, saidGreengard. It s at the heart of how allcells function, and researchers now usemore and more sophisticated analyses of signaling to understand how nerve cellsfunction.

As researchers have developed a betterunderstanding of the various molecules,receptors, and second messengers in- volved in signaling pathways, their re-search has begun to focus on the in-teractions between these components as

well as between different pathways. As itturns out, these interactions are not only critical to understanding how the humanbody works but vital to our understandingof many diseases. Disruptions in normalcell signaling are features of both cancerand chronic diseases such as diabetesand heart disease, and many pathogenshijack the body s signaling mechanisms tocause infections.Broader Impacts Apart from changing the way that neu-roscience research is done, Greengard hasalso mentored and trained many nowprominent neuroscientists. For several of them, Greengard s Classic dopamine re-ceptor paper inspired their choice of eldand approaches to research. I read thepaper when I was 19, said Huganir. Itparticularly struck a chord. It cementedmy commitment to neuroscience.

Nairn said he rst read the paper asa postdoctoral researcher, and he appre-ciated its logical, straightforward ap-proach and clear data. It was very typicalof Greengard, he said. Having rereadthe paper recently, Nairn said it was alsoquite striking how it really got intotranslational medicine. It has great rele- vance to Parkinson s disease as well as inother areas such as ADHD and the ad-dictive actions of drugs of abuse.

The principles and approaches inGreengard s Classic paper helped dis-cover many nervous system drugs usedtoday. The paper had major applicationsto drug discovery, said Huganir. As a re-sult of it, now we could do pharmacol-ogy, he said. It led to the ability to look at antipsychotics and other drugs.

According to Greengard, the fact thatit was possible to work out entire path- ways used by nerve cells to communicatemade it implicit that one could ndpotential new targets for drug develop-ment. Researchers could identify chem-icals that modi ed brain signaling by targeting the regulatory proteins involved. Additionally, as more signaling proteinsand pathways were discovered, therapeu-tic agents could more nely modulate thebrain, Greengard said.

Four decades later, Greengard remainsfascinated by the intricacies of the brain ssignaling and continues to expand on theideas introduced in his 1972 paper at hisresearch laboratory at Rockefeller Uni- versity. That s part of the thrill of it all, he noted. Testing things and hoping youuncover new worlds. His Classic papercertainly opened up new worlds, with newavenues of research and experimentalapproaches for neuroscientists to follow.

Sandeep Ravindran, Science Writer

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6. Sutherland EW, Rall TW (1958) Fractionation and char-acterization of a cyclic adenine ribonucleotide formedby tissue particles. J Biol Chem 232:1077 1091.

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18874 | www.pnas.org/cgi/doi/10.1073/pnas.1116714108 Ravindran