How Serotonin Receptors Can Shape Drug Effects from LSD to Migraine Medication

A team including scientists from The Scripps Research Institute (TSRI), the University of North Carolina at Chapel Hill and the Chinese Academy of Sciences has determined and analyzed the high-resolution atomic structures of two kinds of human serotonin receptor. The new findings help explain why some drugs that interact with these receptors have had unexpectedly complex and sometimes harmful effects.

“Understanding the structure-function of these receptors allows us to discover new biology of serotonin signaling and also gives us better ideas about what biological questions to probe in a more intelligent manner,” said TSRI Professor Raymond Stevens, who was a senior investigator for the new research. The studies were published in two papers on March 21, 2013 in Science Express [1 , 2], the advance online version of the journal Science.

Pioneering Important Molecular Structures

Stevens’s laboratory at TSRI has pioneered the development of techniques for determining the 3D atomic structures of cellular receptors—particularly the large receptor class known as G protein-coupled receptors (GPCRs). GPCRs sit in the cell membrane and sense various molecules outside cells. When certain molecules bind to them, the receptor’s respond in a way to transmit a signal inside the cell.

“Because G protein-coupled receptors are the targets of nearly 50 percent of medicines, they are the focus of several major National Institutes of Health (NIH) initiatives,” said Jean Chin of the NIH’s National Institute of General Medical Sciences, which partly funded the work through the Protein Structure Initiative. “These detailed molecular structures of two serotonin receptor subfamilies bound to antimigraines, antipsychotics, antidepressants or appetite suppressants will help us understand how normal cellular signaling is affected by these drugs and will offer a valuable framework for designing safer and more effective medicines.”

In the past several years, using X-ray crystallography, the Stevens laboratory has determined the high-resolution structures of 10 of the most important GPCRs for human health—including the β2 adrenergic receptor, the A2a adenosine receptor (the target of caffeine), HIV related CXCR4 receptor, the pain-mediating nociceptin receptor, S1P1 receptor important for inflammatory diseases, H1 histamine receptor (antihistamine medications) and the D3 dopamine receptor which is involved in mood, motivation and addiction.

Serotonin receptors are no less important. “Nearly all psychiatric drugs affect serotonin receptors to some extent, and these receptors also mediate a host of effects outside the brain, for example on blood coagulation, smooth muscle contraction and heart valve growth,” said Bryan Roth, a collaborator on both studies who is professor of pharmacology at the University of North Carolina (UNC).

Untangling Two Serotonin Receptors

Roth’s laboratory teamed up with Stevens’s as part of the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative. For this project the two labs also worked with the laboratories of Professors Eric Xu and Hualiang Jiang at the Shanghai Institute of Materia Medica, part of the Chinese Academy of Sciences. “By collaborating with the Chinese teams we were able to complete a much more thorough study and get the most out of our fundamental structural results,” said Stevens.

In the first of the new studies, co-lead author Chong Wang, a graduate student in the Stevens laboratory, and his colleagues determined the structure of the serotonin receptor subtype 5-HT_1B, the principal target of several drug classes. (5-HT, or 5-hydroxytryptamine, is a technical term for serotonin.) The team produced the 5-HT_1B receptor while it was bound by either ergotamine or dihydroergotamine—two old-line anti-migraine drugs that work in part by activating 5-HT_1B receptors.

With the help of the special fusion protein, nicknamed BRIL (apocytochrome b562RIL), Wang and colleagues were able to stabilize these structures and coax them to line up in a regular ordering known as a crystal. X-ray crystallography revealed, at high resolution, an atomic structure of 5-HT_1B with a main binding pocket and a separate, extended binding pocket.

Harmful Off-Target Effects

In the second study, TSRI graduate student and lead author Daniel Wacker and colleagues used similar techniques to determine the structure of the 5-HT_2B receptor bound to ergotamine. The 5-HT_2B receptor was chiefly of interest because drug developers want to avoid activating it.

“Drugs that are meant to target other serotonin receptors in the brain can have harmful off-target effects on 5-HT_2B receptors, which are found abundantly on heart valves, for example,” said Roth. The weight-loss drug fenfluramine and closely related dexfenfluramine were withdrawn from the US market in 1997 after being linked to heart valve disease. Roth’s laboratory later showed that this side effect was mediated by heart valve 5-HT_2B receptors.

Analyses of the 5-HT_1B and 5-HT_2B receptor structures revealed a subtle difference between them. “Although their main binding pockets look very similar, their extended binding pockets are not as similar—the one for 5-HT_2B is narrower and in a slightly different position,” said Wang.

With the two receptor structures in hand, the Xu and Jiang team simulated the bindings of various drugs. They showed, for example, that anti-migraine drugs called triptans should bind well to 5-HT_1B receptors but poorly to 5-HT_2B receptor structures, in which the extended binding pocket is less accessible. Similarly, the team’s calculations confirmed that fenfluramine’s active metabolite should bind very tightly to the 5-HT_2B receptor.

Delving Deeper

In the second study, the researchers used the 5-HT_2B and 5-HT_1B structural data to better understand a recently discovered GPCR signaling pathway.

When a neurotransmitter such as serotonin binds to its GPCR receptor and triggers the primary, G protein-mediated activation signal, it also usually triggers another signal, often mediated by a protein called β-arrestin. This second signaling cascade may simply have the effect of “arresting” or inhibiting the primary, G protein-mediated signaling. But it can also have other effects on the cell, and although most molecules bind to their target GPCRs in a way that activates these primary and secondary signals equally, others preferentially activate one or the other. “Such functional selectivity, as we call it, adds another layer of complexity to drug effects on GPCRs,” said Roth, a co-senior author of the study.

Roth’s laboratory produced several 5-HT receptor subtypes in test cells, and compared the strength of G-protein and β-arrestin signaling when these receptors were bound by ergotamine or various other drugs, including the ergotamine-derived hallucinogen LSD (lysergic acid diethylamide). Most of the tested drugs showed no bias. However, ergotamine, LSD and some of their relatives turned out to be clearly biased in favor of β-arrestin signaling at the 5-HT_2B receptor. Comparison of the ergotamine-bound 5-HT_2B structure with the ergotamine-bound 5-HT_1B structure revealed the likely reason. “We could see that when ergotamine is bound to the 5-HT_2B receptor it stabilizes the receptor structure in a conformation that interferes with G protein signaling,” said Wacker.

The findings allow scientists to start probing this arrestin-mediated signaling pathway and its downstream effects in a more targeted manner. “These structural data are teaching us to ask better questions about receptor biology,” said Stevens.

Breaking down the Parkinson’s pathway

The key hallmark of Parkinson’s disease is a slowdown of movement caused by a cutoff in the supply of dopamine to the brain region responsible for coordinating movement. While scientists have understood this general process for many years, the exact details of how this happens are still murky.

“We know the neurotransmitter, we know roughly the pathways in the brain that are being affected, but when you come right down to it and ask what exactly is the sequence of events that occurs in the brain, that gets a little tougher,” says Ann Graybiel, an MIT Institute Professor and member of MIT’s McGovern Institute for Brain Research.

A new study from Graybiel’s lab offers insight into some of the precise impairments caused by the loss of dopamine in brain cells affected by Parkinson’s disease. The findings, which appear in the March 12 online edition of the Journal of Neuroscience, could help researchers not only better understand the disease, but also develop more targeted treatments.

The neurons responsible for coordinating movement are located in a part of the brain called the striatum, which receives information from two major sources — the neocortex and a tiny region known as the substantia nigra. The cortex relays sensory information as well as plans for future action, while the substantia nigra sends dopamine that helps to coordinate all of the cortical input.

“This dopamine somehow modulates the circuit interactions in such a way that we don’t move too much, we don’t move too little, we don’t move too fast or too slow, and we don’t get overly repetitive in the movements that we make. We’re just right,” Graybiel says.

Parkinson’s disease develops when the neurons connecting the substantia nigra to the striatum die, cutting off a critical dopamine source; in a process that is not entirely understood, too little dopamine translates to difficulty initiating movement. Most Parkinson’s patients receive L-dopa, which can substitute for the lost dopamine. However, the effects usually wear off after five to 10 years, and complications appear.

The zebrafish revealed a central regulator for the development of the brain histamine system

Research has shown that mutations in the psen1 gene are common in the familial forms of Alzheimer’s disease, and the Presenilin-1 protein that the gene encodes is known to be involved in the cleavage of the amyloid precursor protein. In Alzheimer’s disease the amyloid precursor protein is not cleaved the normal way, and the protein accumulates in the brain damaging neuronal tracts and neurons. It is still unknown if the psen1 gene is involved in the etiology of Alzheimer’s disease via another mechanism.

Professor Pertti Panula’s research team at the University of Helsinki has elucidated the role of psen1 gene in the development of the neuronal histamine system and its modulation. Histamine is one of the neurotransmitters, which all are essential for cognitive functions, which in turn are impaired in Alzheimer’s disease. The histamine system is altered during the progression of Alzheimer’s disease.

In the study the zebrafish was used as a model organism. The rapidly developing zebrafish is suitable as a model organism, as its transparency allows researchers to study the development and function of vital organs. To study the function of psen1 gene, zebrafish that did not produce functional Presenilin-1 protein were generated. Despite the fact that the fish lacked functional Presenilin-1 they were viable and developed until adulthood.

The lack of Presenilin-1 protein induced a change in the behavior of the larval zebrafish, they did not as normal fish react to fast changes in the light intensity. “Based on previous research we know that this change in behavior is associated with lack of histamine in the brain”, Panula explains.

In adulthood the motor behavior of the mutant zebrafish differed from the normal fish: the fish swam by the edges of the arena that was available and avoided the inner part. Previous studies from the group have shown that this behavioral alteration also is due to changes in the histamine system.

The researchers found that larval fish lacking Presenilin-1 protein had significantly fewer histamine neurons; in adulthood the histamine neuron number was significantly increased in these fish when compared with normal fish.

"These results reveal that the psen1 gene is a central regulator of the development of the histamine neurons and that the mutation can cause a persistent lifelong change in the neuronal histamine system. This is a very interesting finding", Panula states.

One interesting remaining question is from where the new histamine neurons arise in the brains of adult zebrafish. Are they newly differentiated stem cells or do other cells become histamine neurons? The answer is not known, but based on these results it is advisable to elucidate the role of Presenilin-1 protein in differentiation of stem cells also in the brains of mammals. “Mammals have stem cells in the hypothalamus, in the same area where the histamine neurons are located in all studied vertebrates”, Panula comments.

Panula empathizes that the published study does not tell about an Alzheimer’s disease mechanism in humans. The new knowledge on the function of psen1 gene and the development of the brain histamine system provided by the study is one step forward to understanding the etiology of the disease.

"We perform basic research on molecular level, from where it is a long way to treatment of human diseases. This type of research provides the findings on which the treatments are finally based", Panula says.

Journal of Neuroscience published the study that was conducted at University of Helsinki Neuroscience center, and Institute of Biomedicine.

(Image: Charles Badland, Florida State University)

Dopamine regulates the motivation to act

The widespread belief that dopamine regulates pleasure could go down in history with the latest research results on the role of this neurotransmitter. Researchers have proved that it regulates motivation, causing individuals to initiate and persevere to obtain something either positive or negative.

The neuroscience journal Neuron publishes an article by researchers at the Universitat Jaume I of Castellón that reviews the prevailing theory on dopamine and poses a major paradigm shift with applications in diseases related to lack of motivation and mental fatigue and depression, Parkinson’s, multiple sclerosis, fibromyalgia, etc. and diseases where there is excessive motivation and persistence as in the case of addictions.

"It was believed that dopamine regulated pleasure and reward and that we release it when we obtain something that satisfies us, but in fact the latest scientific evidence shows that this neurotransmitter acts before that, it actually encourages us to act. In other words, dopamine is released in order to achieve something good or to avoid something evil", explains Mercè Correa.

Studies had shown that dopamine is released by pleasurable sensations but also by stress, pain or loss. These research results however had been skewed to only highlight the positive influence, according to Correa. The new article is a review of the paradigm based on the data from several investigations, including those conducted over the past two decades by the Castellón group in collaboration with the John Salamone of the University of Connecticut (USA), on the role of dopamine in the motivated behaviour in animals.

The level of dopamine depends on individuals, so some people are more persistent than others to achieve a goal. “Dopamine leads to maintain the level of activity to achieve what is intended. This in principle is positive, however, it will always depend on the stimuli that are sought: whether the goal is to be a good student or to abuse of drugs” says Correa. High levels of dopamine could also explain the behaviour of the so-called sensation seekers as they are more motivated to act.

Application for depression and addiction

To know the neurobiological parameters that make people be motivated by something is important to many areas such as work, education or health. Dopamine is now seen as a core neurotransmitter to address symptoms such as the lack of energy that occurs in diseases such as depression. “Depressed people do not feel like doing anything and that’s because of low dopamine levels,” explains Correa. Lack of energy and motivation is also related to other syndromes with mental fatigue such as Parkinson’s, multiple sclerosis or fibromyalgia, among others.

In the opposite case, dopamine may be involved in addictive behaviour problems, leading to an attitude of compulsive perseverance. In this sense, Correa indicates that dopamine antagonists which have been applied so far in addiction problems probably have not worked because of inadequate treatments based on a misunderstanding of the function of dopamine.

Why good resolutions about taking up a physical activity can be hard to keep

The collective appraisal conducted by Inserm in 2008 highlighted the many preventive health benefits of regular physical activity. Such activity is limited, however, by our lifestyle in today’s industrial society. While varying degrees of physical inactivity may be partly explained by social causes, they are also rooted in biology.

“The inability to experience pleasure during physical activity, which is often quoted as one explanation why people partially or completely drop out of physical exercise programmes, is a clear sign that the biology of the nervous system is involved”, explains Francis Chaouloff.

But how exactly? The neurobiological mechanisms underlying physical inactivity had yet to be identified.

Francis Chaouloff (Giovanni Marsicano’s team at the NeuroCentre Magendie; Inserm joint research unit, Université Bordeaux Ségalen) and his team have now begun to decipher these mechanisms. Their work clearly identifies the endogenous cannabinoid (or endocannabinoid) system as playing a decisive role, in particular one of its brain receptors. This is by no means the first time that data has pointed to interactions between the endocannabinoid system, which is the target of delta9-tetrahydrocannabinol (the active ingredient of cannabis), and physical exercise. It was discovered ten years ago that physical exercise activated the endocannabinoid system in trained sportsmen, but its exact role remained a mystery for many years. Three years ago, the same research team in Bordeaux observed that when given the opportunity to use a running wheel, mutant mice lacking the CB1 cannabinoid receptor, which is the principal receptor of the endocannabinoid system in the brain, ran for a shorter time and over shorter distances than healthy mice. The research published in Biological Psychiatry this month seeks to understand how, where and why the lack of CB1 receptor reduces voluntary exercise performance (by 20 to 30%) in mice allowed access to a running wheel three hours per day.

The researchers used various lines of mutant mice for the CB1 receptor, together with pharmacological tools. They began by demonstrating that the CB1 receptor controlling running performance is located at the GABAergic nerve endings. They went on to show that the receptor is located in the ventral tegmental area of the brain, which is an area involved in motivational processes relating to reward, whether the reward is natural (food, sex) or associated with the consumption of psychoactive substances.

The Nerve-Growth Factor: A New Tool for Manipulating Neurons

The human nervous system is a vast network of several billion neurons, or nerve cells, endowed with the remarkable ability to receive, store and transmit information. In order to communicate with one another and with non-neuronal cells the neurons rely on the long extensions called axons, which are somewhat analogous to electrically conducting wires. Unlike wires, however, the axons are fluid-filled cylindrical structures that not only transmit electrical signals but also ferry nutrients and other essential substances to and from the cell body. Many basic questions remain to be answered about the mechanisms governing the formation of this intricate cellular network. How do the nerve cells differentiate into thousands of different types? How do their axons establish specific connections (synapses) with other neurons and non-neuronal cells? And what is the nature of the chemical messages neurons send and receive once the synaptic connections are made?

This article will describe some major characteristics and effects of a protein called the nerve-growth factor (NGF), which has made it possible to induce and analyze under highly favorable conditions some crucial steps in the differentiation of neurons, such as the growth and maturation of axons and the synthesis and release of neurotransmitters: the bearers of the chemical messages. The discovery of NGF has also promoted an intensive search for other specific growth factors, leading to the isolation and characterization of a number of proteins with the ability to enhance the growth of different cell lines.

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Definitive proof for receptor’s role in synapse development

Jackson Laboratory researchers led by Associate Professor Zhong-wei Zhang, Ph.D., have provided direct evidence that a specific neurotransmitter receptor is vital to the process of pruning synapses in the brains of newborn mammals.

Faulty pruning at this early developmental stage is implicated in autism-spectrum disorders and schizophrenia. The definitive evidence for N-methyl-D-aspartate receptor (NMDAR) in pruning has eluded researchers until now, but in research published in the Proceedings of the National Academy of Sciences, Zhang’s lab had serendipitous help in the form of a mouse model containing brain cells lacking NMDAR side-by-side with cells containing the receptor.

Soon after birth, mammals’ brains undergo significant development and change. Initially, large numbers of synapses form between neurons. Then, in response to stimuli, the synaptic connections are refined—some synapses are strengthened and others eliminated, or pruned.

In most synapses, glutamate serves as the neurotransmitter, and NMDAR, a major type of post-synaptic glutamate receptor, was previously known to play an important role in neural circuit development. Previous research has implicated the importance of NMDARs in pruning, but it remained unclear whether they played a direct or indirect role.

Zhang and colleagues focused on the thalamus, a brain region where synapse pruning and strengthening can be monitored and quantified with relative ease. They got unexpected help when they realized the mouse model they were using had thalamus cells lacking NMDARs right next to cells with normal NMDAR levels.

The researchers showed that the refinement process was disrupted in the absence of NMDARs. At the same time, neighboring neurons with the receptors proceeded through normal synaptic strengthening and pruning, clearly establishing the necessity of NMDARs in postsynaptic neurons for synaptic refinement.

"Whenever I give a talk or meet colleagues," Zhang says, "the first question that comes up is whether the NMDA receptor is important. It’s good that this is now settled definitively."

There has been extensive research into synaptic strengthening, and most of these studies indicate that the presence of NMDARs may support the recruitment of larger numbers of another kind of glutamate receptor to strengthen the synaptic connections. How NMDARs regulate the pruning process remains largely unknown, however.

The ethical minefield of using neuroscience to prevent crime

On the evening of March 10, 2007, Abdelmalek Bayout, an Algerian citizen living in Italy, brutally stabbed to death Walter Perez, a fellow immigrant from Colombia. Bayout admitted to the crime, saying he was provoked by Perez, who ridiculed him for wearing eye makeup.

According to Nature magazine, Bayout’s defence argued that he was mentally ill at the time of the offence. The court accepted that argument and, although it found Bayout guilty of the crime, imposed on him a reduced prison sentence of nine years and two months.

Bayout nevertheless appealed the judgment, and the Court of Appeal ordered a new psychiatric report. That report showed, among other things, that Bayout had low levels of the neurotransmitter monoamine oxidase A (MAO-A) — an important development given that previous research discovered that men who had low MAO-A levels and who had been abused as children were more likely to be convicted of violent crimes as adults.

Ultimately, the Court of Appeal further reduced Bayout’s sentence by a year, with Judge Pier Valerio Reinotti describing the MAO-A evidence as “particularly compelling.”

Upon a brief review of the scientific evidence, certain glaring problems with the court’s judgment quickly become apparent. Most obviously, the research showing an association between low MAO-A levels and violence tells us nothing about Bayout’s — or any specific individual’s — propensity for violence. Indeed, while a significant percentage of men with low MAO-A levels commit violent offences, the majority do not.

Yet the fact that the court allowed such evidence to influence its verdict suggests that neuroscience, while not eliminating criminal responsibility, might lead courts to conclude that defendants with certain neurological deficits are less responsible than those with “normal” brains.

There is, in fact, a precedent for this, and it’s one that few people question. Adolescents in virtually every country are subject to differential sentencing, and in many cases to an entirely separate system of justice, because their neurobiology renders them less blameworthy, less responsible than adults.

Indeed, while the limbic system, or emotional centre of the brain, is typically mature by the age of 16, the prefrontal cortex, which is associated with one’s capacity to control emotions, is not fully developed, in most people, until the early 20s. Hence according to what’s sometimes called the “two systems” theory, the imbalance in development of the limbic system and the PFC explains the risk taking and emotional behaviour that is characteristic of adolescence. And it justifies our treating adolescents as less responsible than adults.

There are, of course, substantial differences between adolescents and adults with neurological deficits, the most obvious being that most adolescents will outgrow the developmental imbalance. But the basic principle — that people who suffer from neurological aberrations that render them less capable of controlling their behaviour should be held less blameworthy — seems to have swayed the Italian Court of Appeal.

But not just the Italian Court of Appeal. While the “MAO-A defence” has been tried and failed in many courts around the world, recent research led by University of Utah psychologist Lisa Aspinwall suggests that many judges, when presented with neurobiological evidence, are inclined to reduce defendants’ sentences.

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