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

owadays, whether people suffer the atomic bombs that he changed his ca- from depression, mental health reer path. “I didn’t want to go into theo- disorders, or addictions, medi- retical physics and risk making more N ” cation can help. Researchers potent bombs, Greengard said. Instead, have learned how to fine tune the brain’s he entered the nascent field of biophysics functions, often by targeting chemicals and focused on trying to understand called neurotransmitters and the many the brain. signaling pathways through which they act. During his graduate research at Johns Although certain neurotransmitters had Hopkins University, Greengard used been identified more than 40 years ago, electrophysiology, or the study of electric scientists knew relatively little about how currents in nerve cells, to try to understand these chemicals actually functioned. The how the brain functioned. He started basis for much of what neuroscientists know with this approach, because it was the today about brain signaling was shaped by major tool of neuroscientists at the time. National Academy of Sciences member As he progressed through his postdoctoral Paul Greengard’s 1972 PNAS Classic pa- work, however, Greengard increasingly per, “Dopamine-sensitive Adenylate cy- turned to biochemistry, believing that it clase in caudate nucleus of rat brain, and its could provide unique insights. His research similarity to the ‘dopamine receptor’” (1). led him to pioneer many biochemical In the PNAS Classic paper, Kebabian approaches prevalent today. et al. (1) identified enzyme activity in the These approaches soon bore fruit. In brain that was triggered by the neuro- 1968, Greengard became a professor in the transmitter dopamine. This understanding Department of Pharmacology at Yale and of how dopamine works has formed our “within a year to a year and a half, gen- basic understanding of brain signaling and erated papers that still have continuing helped discover therapies for brain dis- Fig. 1. Dopamine release and binding to re- impact today,” said Angus Nairn, a neuro- orders. Dopamine continues to be the ceptor. Dopamine is released by a neuron into the scientist at Yale who did his postdoctoral junction between two nerve cells called the syn- research in Greengard’s laboratory. “It’s subject of much research and has been apse. There, it can bind to dopamine receptors on implicated in addictions, attention deficit really quite remarkable how much they neighboring neurons, and the receptors activate ” hyperactivity disorder (ADHD), mood the second messenger and downstream signaling generated in a short time. disorders, schizophrenia, and movement pathways. Huge Conceptual Barrier disorders such as Parkinson’s disease. By the 1970s, many scientists had studied Greengard realized that the enzyme ’ activity that he discovered probably in- Greengard s discovery of the dopamine neurotransmitters, but no one had a clear dicated the presence of a dopamine re- receptor opened up many additional ave- idea of how these chemicals exert their nues for clinical treatments. Many anti- fl ceptor that bound to the neurotransmitter. myriad in uences on the brain. The con- psychotic drugs, particularly those drugs Subsequent identification and purification cept of receptors that neuroscientists take used to treat schizophrenia, act by block- “ of the proteins that form this receptor for granted today was still mostly a black ing dopamine receptors. Greengard’s re- ” revealed that Greengard had figured out box, said Snyder, a neuroscientist at sults helped explain how these drugs acted the critical first steps of neurotransmitter Johns Hopkins University and author of and how they could be improved. Dopa- signaling. He went on to show that the the PNAS Classic Perspective on Green- mine signaling pathways have also been gard’s paper, “What dopamine does in receptor-mediated enzyme activates a sec- implicated in the reward mechanisms of the brain” (5). ond messenger, which then triggers a cas- the brain, and drugs that modulate these “Receptors were still a conceptual cade of other signaling events that regu- pathways can help with mood disorders thing,” said Richard Huganir, a neurosci- late complex neurological traits and affect and addictions. entist at Johns Hopkins University and how nerve cells grow and function. However, the implications of Green- ’ former postdoctoral researcher with Dopamine s role as a neurotransmitter gard’s work extended beyond clinical “ ’ fi Greengard. It wasn t even clear if re- was rst uncovered by Arvid Carlsson, who treatments. They also helped lay the ceptors were proteins, believe it or not.” shared the 2000 Nobel Prize in Physiology foundations of modern neuroscience. His – Researchers studying other signaling and Medicine for this discovery (2 4). use of classical biochemistry to study the molecules such as hormones had revealed Carlsson et al. (2, 3) noticed that patients brain influenced many other researchers ’ that they bound to receptors on cell sur- with Parkinson s disease have particularly and helped discover new signaling path- faces, which triggered other signaling low dopamine concentrations in certain ways and regulatory proteins. In recogni- events inside the cell. One of Greengard’s parts of the brain. Dopamine depletion tion of his work’s wide-ranging influence, insights was that neurotransmitters might in the brains of animals also caused Greengard was awarded the Nobel Prize work in the same way in the brain. Parkinson’s-like movement symptoms, and in Physiology and Medicine in 2000, the Carlsson was able to treat these symptoms same year that Carlsson was honored. using L-DOPA, which gets converted to Despite his contributions to neurosci- ’ dopamine in the brain. His experiments ence, Greengard s initial interest was in See Classic Article “Dopamine-sensitive adenylate cyclase helped develop L-DOPA as a drug for mathematics and theoretical physics. It in caudate nucleus of rat brain, and its similarity to the Parkinson’s disease, and it remains one of was only after he served in the US Navy in ‘dopamine receptor’” on page 2145 in issue 8 of volume 69. the disease’s most important treatments. World War II and after the dropping of See Classic Perspective on page 18869.

18872–18874 | PNAS | November 22, 2011 | vol. 108 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1116714108 Downloaded by guest on September 24, 2021 NSCASCPROFILE CLASSIC PNAS

Gathering the biochemical evidence to was cemented by what was to come. “The support this theory brought its own chal- paper sets the stage pretty well for what lenges. The only neurotransmitter receptor followed,” said Nairn. “The details got discovered until then had been in the more complex, but the direction was re- electric eel, where it constituted 20% of the ally set up by this paper.” weight of the eel’s electric organ (7). This Greengard, Kebabian, and others went breakthrough result only made scientists on to characterize dopamine receptor more convinced “that it would be impos- activity in more detail, finding that do- Fig. 2. Dopamine receptor availability in the sible to isolate receptors in the human pamine actually had multiple different brain. Brain images show a decrease in the avail- brain, where they would constitute a mil- receptors to which it could bind. They able dopamine receptors in the brain of a person lionth of its weight at most,” said Snyder. and other researchers used similar addicted to cocaine compared with a nondrug However, Greengard was careful in his approaches to study the receptors and user. A similar decrease is seen with many other selection of the neurotransmitter and signaling of other neurotransmitters, such addictive substances, including alcohol and heroin. the target location to study. He picked as serotonin or noradrenaline. Their work The lower level of receptors is thought to occur dopamine and looked for its effects in a led to more studies of the slow, bio- because of their repeated overstimulation, and it brain area where Carlsson’s previous chemical transmission of signals by these can lead to a reduced activation of natural reward pathways that contributes to the tendency to studies had shown that it was not only neurotransmitters, which has prolonged abuse these addictive substances. Adapted from abundant but where its loss elicited a effects on nerve cells and helps regulate Volkow et al. (9). syndrome similar to Parkinson’s disease basal nervous system functions, including (2–4). “If he had looked at the whole moods. This slow transmission can also brain, he would probably have missed this open ion channels to mediate the fast As Greengard started putting various phenomenon, because everything would transmission of nerve signals. As pieces of evidence together, he was in- be diluted out,” said Nairn. Greengard and others were to find out, spired by the discovery that cAMP medi- The researchers had to carefully extract slow, chemical nerve signaling is an ex- ated the action of certain hormones in the specific brain regions from multiple rats tremely important and complicated liver (6). cAMP acted as a second mes- and pool them together, while ensuring process, involving a whole cascade of senger that transmitted a signal from a that they did not destroy the enzyme ac- downstream agents. hormone’s receptor to other proteins tivity in the process. Isolating the appro- within the cell, triggering downstream priate brain area using the techniques of Setting the Stage for Modern signaling pathways. Greengard started the time required “heavy slogging,” said Neuroscience looking for a similar interaction in the Snyder. “They used very classical bio- As a follow-up to the Classic paper, brain mediated by adenylate cyclase, the chemical techniques that required big Greengard’s laboratory systematically enzyme that converts ATP into cAMP. time careful work to perform success- tracked down the events that occur after However, this search was far from an fully,” he said. dopamine binds to its receptor and acti- obvious train of thought. Most neuro- However, the hard work paid off. When vates an adenylate cyclase enzyme. In scientists still believed that any signaling Greengard and his graduate student John other organs, cAMP had been shown to in the brain was electrical and not bio- Kebabian added dopamine to specific regulate enzymes called kinases, which chemical. “There was a huge conceptual brain fractions, they saw a production of add phosphate groups to molecules in barrier,” said Nairn. adenylate cyclase cAMP as a second messenger, indicating a process called phosphorylation (8). took many seconds to minutes to generate that the neurotransmitter was signaling Phosphorylation can change the structure a signal, a seemingly lethargic process through an adenylate cyclase. “To actually and function of proteins and cause them compared with the rapid electrical sig- find an adenylate cyclase that was coupled to trigger signaling events. “[Greengard] naling that neuroscientists were familiar to neurotransmitter—nobody had done figured out that neurotransmitters could with in the brain. That Greengard pushed that,” said Snyder. work in the same way,” said Huganir. past this paradigm and explored such a Kebabian said that Greengard and he Greengard showed that, after dopa- counterintuitive theory was a major factor had anticipated that this research would mine acted through its receptor to in- in his receipt of the Nobel Prize, Nairn be an important piece of work, and they crease the amount of cAMP in a nerve said. “He kept pushing at it, gathering were pleased when they succeeded. cell, the cAMP activated a kinase, which biochemical evidence for it.” However, the importance of their results phosphorylated other proteins in the cell. Some of these proteins could form ion channels in the cell membrane, thus controlling the nerve cell’s ability to send electrical impulses. Greengard also found a phosphorylated protein, Dopamine- and cAMP-regulated phosphoprotein (DARPP)-32, which is a central regulator of neuronal function and affects a large number of other proteins. DARPP-32 has been implicated in several neurological disorders and the action of multiple addictive drugs. Greengard’s work showed that phos- phorylation was an important signaling Fig. 3. Dopamine signals through a cAMP second messenger. After dopamine binds to its receptor, the receptor then triggers the activation of an adenylate cyclase enzyme, which produces cAMP from ATP. mechanism in the brain. The discovery cAMP acts as a second messenger that transmits a signal from the receptor to other proteins within the cell that neurotransmitter signals are medi- by binding to them and altering their activity. After dopamine increases the level of cAMP in nerve cells, the ated by the addition and removal of cAMP activates a kinase that adds phosphate groups to other proteins in the cell in a process known as phosphate groups from proteins in nerve phosphorylation. Phosphorylation of these other proteins triggers downstream signaling pathways. cells continues to have an impact today.

Ravindran PNAS | November 22, 2011 | vol. 108 | no. 47 | 18873 Downloaded by guest on September 24, 2021 Many current drug discovery efforts in- well as between different pathways. As it The principles and approaches in volve studying the phosphorylation of turns out, these interactions are not only Greengard’s Classic paper helped dis- different proteins in nerve cells. critical to understanding how the human cover many nervous system drugs used However, when these results were first body works but vital to our understanding today. The paper had major applications published, their novelty made them con- of many diseases. Disruptions in normal to drug discovery, said Huganir. As a re- troversial. “There was a point when I cell signaling are features of both cancer sult of it, “now we could do pharmacol- thought they would not be accepted in my and chronic diseases such as diabetes ogy,” he said. “It led to the ability to look lifetime,” said Greengard. “There was and heart disease, and many pathogens at antipsychotics and other drugs.” a lot of skepticism.” He remained un- hijack the body’s signaling mechanisms to According to Greengard, “the fact that deterred. “After we got the first data, I cause infections. it was possible to work out entire path- was positive I was correct,” he said. The ways used by nerve cells to communicate rapid rise of modern molecular biology Broader Impacts made it implicit that one could find in the 1980s helped validate his con- Apart from changing the way that neu- potential new targets for drug develop- victions. “Real acceptance came with the roscience research is done, Greengard has ment.” Researchers could identify chem- development of new techniques,” Green- also mentored and trained many now icals that modified brain signaling by gard said. “Gradually, the evidence prominent neuroscientists. For several of targeting the regulatory proteins involved. became so overwhelming that half the them, Greengard’s Classic dopamine re- Additionally, as more signaling proteins people in brain science today study ceptor paper inspired their choice of field and pathways were discovered, therapeu- signaling.” and approaches to research. “I read the tic agents could more finely modulate the With this Classic paper and his follow- paper when I was 19,” said Huganir. “It brain, Greengard said. up work came the realization that “sig- particularly struck a chord. It cemented Four decades later, Greengard remains naling pathways are cell function,” said my commitment to neuroscience.” fascinated by the intricacies of the brain’s Greengard. “It’s at the heart of how all Nairn said he first read the paper as signaling and continues to expand on the cells function, and researchers now use a postdoctoral researcher, and he appre- ideas introduced in his 1972 paper at his more and more sophisticated analyses of ciated its logical, straightforward ap- research laboratory at Rockefeller Uni- signaling to understand how nerve cells proach and clear data. “It was very typical versity. “That’s part of the thrill of it all,” function.” of Greengard,” he said. Having reread he noted. “Testing things and hoping you As researchers have developed a better the paper recently, Nairn said it was also uncover new worlds.” His Classic paper understanding of the various molecules, quite striking how it “really got into certainly opened up new worlds, with new receptors, and second messengers in- translational medicine. It has great rele- avenues of research and experimental volved in signaling pathways, their re- vance to Parkinson’s disease as well as in approaches for neuroscientists to follow. search has begun to focus on the in- other areas such as ADHD and the ad- teractions between these components as dictive actions of drugs of abuse.” Sandeep Ravindran, Science Writer

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