Posts tagged glutamate
Articles and news from the latest research reports.
Identification of a protein that may increase the currently short therapeutic window in stroke
A new study published in the prestigious publication The EMBO Journal shows that the mitochondrial protein Mfn2 may be a future therapeutic target for neuronal death reduction in the late phases of an ischemic stroke. The study has been coordinated by Dr Francesc Soriano, Ramón y Cajal researcher at the Department of Cell Biology of the University of Barcelona (UB) and member of the Research Group Celltec UB. The study, funded by the Fundació La Marató de TV3, is part of the PhD thesis developed by Àlex Martorell Riera (UB), first author of the article. Experts Antonio Zorzano and Manuel Palacín, from the Department of Biochemistry and Molecular Biology of UB and the Institute for Research in Biomedicine (IRB Barcelona), and Jesús Pérez Clausell and Manuel Reina, from the Department of Cell Biology of UB, also collaborated in the study.
When blood flow is blocked in the brain
According to the World Health Organization (WHO), strokes are the second leading cause of death in the world. A stroke occurs when a blood vessel is blocked interrupting blood flow in the brain. Ictus damage is progressive: it begins some minutes after the attack. Recommended treatment consists in restoring blood flow to the brain, but it must be done during the first four hours after the stroke.
According to researcher Francesc Soriano, “one of the main causes of brain death in ictus events is glutamate increase; glutamate is the main excitatory neurotransmitter in the central nervous system. Glutamate extracellular concentrations remain low due to the activity of membrane transporters, which require energy to work”.
When blood flow is blocked, energy levels are reduced in the affected area. This phenomenon leads glutamate transporters to work inversely, so glutamate is expelled to the extracellular space. Glutamate activates its receptors —particularly, the N-methyl-D-aspartate receptor (NMDA)— on neurons’ surface, a process that triggers an excessive flux of calcium, the activation of a series of reactions and neuronal death, in a process known as excitotoxicity. “Many of these excitotoxic cascades —points out Soriano— converge on the mitochondrion, an organelle which plays a major role not only in energy production, but also in apoptosis”.
New therapeutic strategies against ischemic ictus
Specifically, Mfn2 is a mitochondrial protein involved in the regulation of organelles’ morphology and function. The team led by Dr Francesc Soriano has just discovered that the reduction in Mfn2 protein levels occurs four hours after the initiation of the excitotoxic process in in vitro and in vivo animal models.
In vivo experiments proved that if Mfn2 reduction is stopped, delayed excitotoxic cell death is blocked. The research team from the Department of Cell Biology of UB found that the Mfn2 reduction is triggered by a genetic transcription mechanism (DNA is transcribed into RNA molecules). UB experts also discovered that MEF2 is the transcription factor involved in this process. Authors affirm that these findings are essential to find a strategy to reverse Mfn2 reduction.
Currently, the team led by Dr Francesc Soriano are researching on brain damage in excitotoxic conditions in animal models where the gene Mfn2 has been removed. The main objective is to design therapeutic strategic in order to reduce damage.
Researchers discover that Klotho is neuroprotective against Alzheimer’s disease
Boston University School of Medicine researchers may have found a way to delay or even prevent Alzheimer’s disease (AD). They discovered that pre-treatment of neurons with the anti-aging protein Klotho can prevent neuron death in the presence of the toxic amyloid protein and glutamate. These findings currently appear in the Journal of Biological Chemistry.
Alzheimer’s disease is the most frequent age-related dementia affecting 5.4 million Americans including 13 percent of people age 65 and older and more than 40 percent of people over the age of 85. In AD the cognitive decline and dementia result from the death of nerve cells that are involved in learning and memory. The amyloid protein and the excess of the neurotransmitter, glutamate are partially responsible for the neuronal demise.
Nerve cells were grown in petri dishes and treated with or without Klotho for four hours. Amyloid or glutamate then were added to the dish for 24 hours. In the dishes where Klotho was added, a much higher percentage of neurons survived than in the dishes without Klotho.
"Finding a neuroprotective agent that will protect nerve cells from amyloid that accumulates as a function of age in the brain is novel and of major importance," explained corresponding author Carmela R. Abraham, PhD, professor of biochemistry and pharmacology at BUSM. "We now have evidence that if more Klotho is present in the brain, it will protect the neurons from the oxidative stress induced by amyloid and glutamate.
According to the researchers, Klotho is a large protein that cannot penetrate the blood brain barrier so it can’t be administered by mouth or injection. However in a separate study the researchers have identified small molecules that can enter the brain and increase the levels of Klotho. “We believe that increasing Klotho levels with such compounds would improve the outcome for Alzheimer’s patients, and if started early enough would prevent further deterioration. This potential treatment has implications for other neurodegenerative diseases such as Parkinson’s, Huntington’s, ALS and brain trauma, as well,” added Abraham.
(Source: eurekalert.org)
(Image caption: Granule cells connect with other cells via long projections (dendrites). The actual junctions (synapses) are located on thorn-like protuberances called “spines”. Spines are shown in green in the computer reconstruction. Credit: DZNE/Michaela Müller)
A protein couple controls flow of information into the brain’s memory center
Neuroscientists in Bonn and Heidelberg have succeeded in providing new insights into how the brain works. Researchers at the DZNE and the German Cancer Research Center (DKFZ) analyzed tissue samples from mice to identify how two specific proteins, ‘CKAMP44’ and ‘TARP Gamma-8’, act upon the brain’s memory center. These molecules, which have similar counterparts in humans, affect the connections between nerve cells and influence the transmission of nerve signals into the hippocampus, an area of the brain that plays a significant role in learning processes and the creation of memories. The results of the study have been published in the journal Neuron.
Brain function depends on the active communication between nerve cells, known as neurons. For this purpose, neurons are woven together into a dense network where they constantly relay signals to one another. However, neurons do not form direct contacts with each other. Instead they are separated by an extremely narrow gap, known as the synapse. This gap is bridged by ‘neurotransmitters’, which carry nerve signals from one cell to the next.
Docking stations
Specific molecular complexes in the cell’s outer shell, so-called ‘receptors’, receive the signal by binding the neurotransmitters. This triggers an electrical impulse in the receptor-bearing cell and thus the nerve signal has moved on one neuron further.
In the current study, a team led by Dr Jakob von Engelhardt focused on the AMPA receptors. These bind the neurotransmitter glutamate and are particularly common in the brain. “We looked at AMPA receptors in an area of the brain, which constitutes the main entrance to the hippocampus,” explains von Engelhardt, who works for the DZNE and DKFZ. “The hippocampus is responsible for learning and memory formation. Among other things it processes and combines sensory perception. We therefore asked ourselves how the flow of information into the hippocampus is controlled.”
A pair of helpers
Dr von Engelhardt’s research team specifically focused on two protein molecules: ‘CKAMP44’ and ‘TARP Gamma-8’. These proteins are present, along with AMPA receptors, in the ‘granule’ cells, which are neurons that receive signals from areas outside of the hippocampus. It was already known that these proteins form protein complexes with AMPA receptors. “We have now found out that they exert a significant influence on the functioning of glutamate receptors. Each in its own way, as chemically they are completely different,” says the neuroscientist. “We identified that the ability of a nerve cell to receive signals doesn’t depend solely on the actual receptors; CKAMP44 and TARP Gamma-8 are just as important. Their function cannot be separated from that of the receptors.”
This was the result of an analysis in which the researchers compared brain tissue from mice with a natural genotype with brain tissue from genetically modified mice. Neurons in the genetically modified animals were not able to produce either CKAMP44 or TARP Gamma-8 or both.
Long-term effect
The researchers discovered, among other things, that both proteins promote the transportation of glutamate receptors to the cell surface. “This means they influence how receptive the nerve cell is to incoming signals,” says von Engelhardt.
However, the number of receptors and thus the signal reception can be altered by neuronal activity. The von Engelhardt group found that in this regard the auxiliary molecules have different effects: TARP Gamma-8 is essential to ensure that more AMPA receptors are integrated into the synapse following a plasticity induction protocol, whereas CKAMP44 plays no role in this context. “Synapses alter their communication depending on their activity. This ability is called plasticity. Some of the changes involved are only temporary, others may last longer,” explains von Engelhardt. “TARP Gamma-8 influences long-term plasticity. It makes the cell able to strengthen synaptic communication for a prolonged time-period. The larger the number of receptors on the receiving side of the synapse, the better the neuronal connection.”
The number of receptors doesn’t change suddenly, but remains largely stable for a certain amount of time. “This condition may last for hours, days or even longer. This long-term effect is essential for the creation of memories. We can only remember things if the connections between neurons undergo a long-lasting change,” says the scientist.
Fast sequence of signals
However, CKAMP44 and TARP Gamma-8 also act over shorter periods of time. The research team discovered that the molecules affect how quickly the AMPA receptors return to a receptive state. “If glutamate has docked on to a receptor, it takes a while until the receptor can react to the next neurotransmitter. CKAMP44 lengthens this period. In contrast, TARP Gamma-8 helps the receptor to recover more quickly,” says von Engelhardt.
Hence, CKAMP44 temporarily weakens the synaptic connection, while TARP Gamma-8 strengthens it. Through the interplay of these proteins the synapse is able to tune its sensitivity to a specific level. This condition can last from milliseconds to a few seconds before the strength of the connection is again adapted. Specialists refer to this as “short-term plasticity”.
“These molecules ultimately influence how well the nerve cell is able to react to a rapid succession of signals,” the scientist summarises the findings. “Such a rapid firing enables neuronal networks to synchronize their activity, which is a common process in the brain.”
Sensitive balance
Much to the researchers’ surprise, it turned out that the two proteins influence not only the synapse but also the shape of the nerve cells. In the absence of these auxiliary molecules, the neurons have fewer dendrites to establish contact with other nerve cells. “The organism can use CKAMP44 and TARP Gamma-8 molecules to regulate neuronal connections in a number of ways,” von Engelhardt says. “This ability depends on the balance between the partners, as to some extent they have a contrary effect. The way in which the neurons of the hippocampus react to signals from other regions of the brain is therefore highly dependent on the presence and the expression ratio of these molecules.”
Since the two molecules act directly on the structure and function of synapses of granule cells, Jakob von Engelhardt considers it probable that they also have an influence on learning and memory.
A tiny molecule may help battle depression
Levels of a small molecule found only in humans and in other primates are lower in the brains of depressed individuals, according to researchers at McGill University and the Douglas Institute. This discovery may hold a key to improving treatment options for those who suffer from depression.
Depression is a common cause of disability, and while viable medications exist to treat it, finding the right medication for individual patients often amounts to trial and error for the physician. In a new study to be published in the journal Nature Medicine, Dr. Gustavo Turecki, a psychiatrist at the Douglas and professor in the Faculty of Medicine, Department of Psychiatry at McGill, together with his team, discovered that the levels of a tiny molecule, miR-1202, may provide a marker for depression and help detect individuals who are likely to respond to antidepressant treatment.
“Using samples from the Douglas Bell-Canada Brain Bank, we examined brain tissues from individuals who were depressed and compared them with brain tissues from psychiatrically healthy individuals, says Turecki, who is also Director of the McGill Group for Suicide Studies, “We identified this molecule, a microRNA known as miR-1202, only found in humans and primates and discovered that it regulates an important receptor of the neurotransmitter glutamate”.
The team conducted a number of experiments that showed that antidepressants change the levels of this microRNA. “In our clinical trials with living depressed individuals treated with citalopram, a commonly prescribed antidepressant, we found lower levels in depressed individuals compared to the non-depressed individuals before treatment,” says Turecki. “Clearly, microRNA miR-1202 increased as the treatment worked and individuals no longer felt depressed.”
Antidepressant drugs are the most common treatment for depressive episodes, and are among the most prescribed medications in North America. “Although antidepressants are clearly effective, there is variability in how individuals respond to antidepressant treatment,” says Turecki, “We found that miR-1202 is different in individuals with depression and particularly, among those patients who eventually will respond to antidepressant treatment”.
The discovery may provide “a potential target for the development of new and more effective antidepressant treatments,” he adds.
(Source: mcgill.ca)
Promising approach to slow brain degeneration in a model of Huntington’s disease uncovered
Research presented by Dr. Lynn Raymond, from the University of British Columbia, shows that blocking a specific class of glutamate receptors, called extrasynaptic NMDA receptors, can improve motor learning and coordination, and prevent cell death in animal models of Huntington disease. As Huntington disease is an inherited condition that can be detected decades before any clinical symptoms are seen in humans, a better understanding of the earliest changes in brain cell (neuronal) function, and the molecular pathways underlying those changes, could lead to preventive treatments that delay the onset of symptoms and neurodegeneration. “After more than a decade of research on the pre-symptomatic phase of Huntington disease, markers are being developed to facilitate assessment of interventional therapy in individuals carrying the genetic mutation for Huntington disease, before they become ill. This will make it possible to delay onset of disease,” says Dr. Raymond. These results were presented at the 2014 Canadian Neuroscience Meeting, the 8th annual meeting of the Canadian Association for Neuroscience - Association Canadienne des Neurosciences (CAN-ACN), held in Montreal, May 25-28.
The neurotransmitter glutamate has long been known to promote cell death, and its toxic effects occur through the action of a family of receptors known as the NMDARs (N-methyl-D-Aspartate ionotropic glutamate receptors). Unfortunately, treating disorders of the nervous system by blocking NMDARs has not been successful because such treatments have numerous side effects. A recent hypothesis based on work from many scientists suggests that NMDARs located in different regions at the surface of neurons may have opposite effects, which would explain why blocking all NMDARs is not a good treatment option. A synapse is a structure that allows one neuron to connect to another neuron and pass an electrical or chemical signal between them. Many receptors for neurotransmitters are located in synapses, as these are the main area where these chemical signals are transmitted. However, receptors can also be found outside the synapse, and in this case are called extra-synaptic receptors. Many recent studies have revealed that NMDARs located at synapses act to increase survival signaling and promote learning and memory, whereas extra-synaptic NMDARs shut off survival signaling, interfere with learning mechanisms, and increase cell death pathways.
Dr. Raymond and her team were able, by using a drug that selectively blocks extra-synaptic NMDARs early, before the appearance of any symptoms, to delay the onset of Huntington-like symptoms in a mouse model of the disease. These promising results could lead to new treatment avenues for Huntington patients, and delay the appearance of symptoms. “The drug we used, memantine, is currently being used to treat moderate-stage Alzheimer disease patients. Our results suggest that clinical studies of memantine and similarly-acting drugs in Huntington disease, particularly in the pre-symptomatic stage, are warranted,”says Dr. Raymond.
Extra-synaptic NMDARs have also been shown to be involved in other neurodegenerative diseases, such as Alzheimer disease, and in damage caused by traumatic brain injury and some forms of stroke. These results therefore suggest novel treatment avenues for many conditions in which neurons degenerate and die, a new way to protect neurons before the appearance of symptoms of neurodegeneration.
(Source: eurekalert.org)
Eavesdropping on brain cell chatter
Everything we do — all of our movements, thoughts and feelings – are the result of neurons talking with one another, and recent studies have suggested that some of the conversations might not be all that private. Brain cells known as astrocytes may be listening in on, or even participating in, some of those discussions. But a new mouse study suggests that astrocytes might only be tuning in part of the time — specifically, when the neurons get really excited about something. This research, published in Neuron, was supported by the National Institute of Neurological Disorders and Stroke (NINDS), part of the National Institutes of Health.
For a long time, researchers thought that the star-shaped astrocytes (the name comes from the Greek word for star) were simply support cells for the neurons.
It turns out that these cells have a number of important jobs, including providing nutrients and signaling molecules to neurons, regulating blood flow, and removing brain chemicals called neurotransmitters from the synapse. The synapse is the point of information transfer between two neurons. At this connection point, neurotransmitters are released from one neuron to affect the electrical properties of the other. Long arms of astrocytes are located next to synapses, where they can keep tabs on the conversations going on between neurons.
In recent years, it has been shown that astrocytes may also play a role in neuronal communication. When neurons release neurotransmitters, levels of calcium change within astrocytes. Calcium is critical for many processes, including release of molecules from the cell, and activation of a host of proteins within the cell. The role of this astrocytic calcium signaling for brain function remains a mystery.
In this study, Baljit S. Khakh, Ph.D., of the University of California, Los Angeles and his colleagues wanted to know when astrocytes responded to neuron activity with changes in their internal calcium levels. Using calcium indicator dyes, the researchers were able to image, for the first time, changes in calcium levels in the entire astrocyte. Previously, it was only possible to look at certain areas of the cell at one time, which provided an incomplete picture of what was happening.
Dr. Khakh said one of the most important outcomes of this work was in the methods that were used. “What our use of these calcium indicators shows is that we can image calcium throughout the entire astrocyte. This provides a new set of tools for the research community to use and to extend these findings,” he said.
“There has been intense interest in understanding how astrocytes facilitate communication between neurons, but it is only recently that studies with this level of precision have been possible,” said Edmund Talley, Ph.D., program director at NINDS. “Dr. Khakh’s study is an example of an exciting basic, or fundamental, research project that could have an important contribution to the shifting field of astrocyte biology,” he added.
For these experiments, researchers focused on the mossy fiber pathway, which connects two areas of the hippocampus, the structure involved in learning and memory. “This pathway has a unique architecture and although it has been very well studied, the role of astrocytes in this circuit has not been previously explored. This study provides one of the first really detailed understandings of astrocytes within this particular circuit,” said Dr. Khakh.
Dr. Khakh’s team activated neurons (getting them to release neurotransmitter by a variety of techniques) and then looked for a response in the neighboring astrocyte. As calcium levels rose, the astrocyte would light up quickly. They discovered that two neurotransmitters, glutamate and GABA, triggered the astrocytes to release calcium from their internal stores. Importantly, the researchers discovered that calcium levels increased through the entire astrocyte only if there was a large burst of neurotransmitter being released.
“We found that astrocytes in the mossy fiber pathway do not listen to the constant, millisecond by millisecond synaptic chatter that neurons engage in. Instead, they listen when neurons get excessively excited during bursts of activation,” said Dr. Khakh.
These findings suggest that astrocytes in the mossy fiber system may act as a switch that reacts to large amounts of neuronal activity by raising their levels of calcium. These calcium increases occur over multiple seconds, a relatively long time period compared to that seen in neurons. The spatial extent of the astrocyte calcium increases was also relatively large in comparison to the size of the synapse.
“Astrocytes may be sitting there quietly and when there is excessive activation in the neuronal circuit, they immediately respond with an increase in calcium which we could detect. And the next big question becomes, what they do with that calcium?” said Dr. Khakh.
Dr. Khakh’s results in the mossy fiber system differ from those others have described in other brain regions. This raises the intriguing possibility that astrocytes are not all the same and may serve various roles throughout the brain.
“It would be really interesting and important to find that astrocytes function differently in different areas of the brain, in a circuit-specific manner. This study gives a hint that this might be true,” said Dr. Talley.
(Image caption: During the learning processes, extensions grow on neurons. Synapses are located at the end of these extensions (left: as seen in nature; right: reconstruction). When the synapse growth is based on the correlated development of all synaptic components, it can remain stable for long periods of time. Credit: © MPI of Neurobiology/ Meyer)
Synapses – stability in transformation
Nothing lasts forever. This principle also applies to the proteins that make up the points of contact between our neurons. It is due to these proteins that the information arriving at a synapse can be transmitted and then received by the next neuron. When we learn something, new synapses are created or existing ones are strengthened. To enable us to retain long-term memories, synapses must remain stable for long periods of time, up to an entire lifetime. Researchers at the Max Planck Institute of Neurobiology in Martinsried near Munich have found an explanation as to how a synapse achieves remaining stable for a long time despite the fact that its proteins must be renewed regularly.
Learning in the laboratory
“We were interested first of all in what happens to the different components of a synapse when it grows during a learning process,” explains study leader Volker Scheuss. An understanding of how the components grow could also provide information about the long-term stability of synapses. Hence, the researchers studied the growth of synapses in tissue culture dishes following exposure to a (learning) stimulus. To do this, they deliberately activated individual synapses using the neurotransmitter glutamate: scientists have long known that glutamate plays an important role in learning processes and stimulates the growth of synapses. Over the following hours, the researchers observed the stimulated synapses and control synapses under a 2-photon microscope. To confirm the observed effects, they then examined individual synapses with the help of an electron microscope. “When you consider that individual synapses are only around one thousandth of a millimetre in size, this was quite a Sisyphean task,” says Tobias Bonhoeffer, the Director of the department where the research was carried out.
Synaptic stability – a concerted effort
The scientists discovered that during synapse growth the different protein structures always grew coordinated with each other. If one structural component was enlarged alone, or in a way that was not correctly correlated with the other components, its structural change would collapse soon after. Synapses with such incomplete changes cannot store any long-term memories.
The study findings show that the order and interaction between synaptic components is finely tuned and correlated. “In a system of this kind, it should be entirely possible to replace individual proteins while the rest of the structure maintains its integrity,” says Scheuss. However, if an entire group of components breaks away, the synapse is destabilised. This is also an important process given that the brain could not function correctly without the capacity to forget things. Hence, the study’s results provide not only important insight into the functioning and structure of synapses, they also establish a basis for a better understanding of memory loss, for example in the case of degenerative brain diseases.
New clue to autism found inside brain cells
The problems people with autism have with memory formation, higher-level thinking and social interactions may be partially attributable to the activity of receptors inside brain cells, researchers at Washington University School of Medicine in St. Louis have learned.
(Image caption: Learning, social interactions and higher-level thinking in people with autism may be adversely affected by receptors inside brain cells, scientists at Washington University School of Medicine have learned. The type of receptor they studied glows green on the surface of this cell. Inside the cell, the receptor covers a membrane stained red. Credit: Yuh-Jiin I. Jong)
Scientists were already aware that the type of receptor in question was a potential contributor to these problems – when located on the surfaces of brain cells. Until now, though, the role of the same type of receptor located inside the cell had gone unrecognized. Such receptors inside cells significantly outnumber the same type of receptors on the surface of cells.
The receptor under study, known as the mGlu5 receptor, becomes activated when it binds to the neurotransmitter glutamate, which is associated with learning and memory. This leads to chain reactions that convert the glutamate’s signal into messages traveling inside the cell.
In the new study, scientists working with cells in a dish linked mGlu5 receptors inside cells to processes that turn down the volume at which brain cells talk to each other. These volume changes, essential for learning and memory, may become exaggerated in people with autism.
Pharmaceutical companies have developed therapeutic compounds to decrease signaling associated with the mGlu5 receptor, moderating its effects on brain cells’ volume knobs. But the compounds were designed to target mGlu5 surface receptors. In light of the new findings, the scientists question if those drugs will reach the receptors inside cells.
“Our results suggest that to have the greatest therapeutic benefit, we may need to make sure we’re blocking all of this type of receptor, both inside and on the surface of the cell,” said senior investigator Karen O’Malley, PhD, professor of neurobiology.
The findings, published March 25 in The Journal of Neuroscience, also add a significant new dimension to basic brain cell function. Scientists have long assumed that brain cell receptors are only active on the surface of cells. The new study shows that receptors can be active inside cells, and their effects can be considerably different from the same receptors located on the cell surface.
“The traditional wisdom was that receptors inside the cell were either waiting to go to work on the surface or had just finished working there,” said O’Malley. “But when we compared how much of the mGlu5 receptor was on the surface of cells to how much was inside it, we were seeing so much more receptor inside the cell – at least 50 percent, and in some cases as much as 90 percent – that we wondered if the interior receptors had separate functions.”
In earlier studies, O’Malley and her collaborators showed that mGlu5 receptors on the cell surface sent completely different messages than the same receptors inside the cell.
The scientists knew that most autism studies were conducted with compounds that blocked mGlu5 receptors but could not get into the cell. To determine whether blocking inside receptors would have different effects, O’Malley collaborated with Yukitoshi Izumi, MD, PhD, research professor of psychiatry, and Charles F. Zorumski, MD, the Samuel B. Guze Professor and head of the Department of Psychiatry, who study cell-based models of learning and memory.
When the scientists examined these model systems using compounds that allowed them to activate only mGlu5 receptors within cells, they found that these receptors played a bigger role in turning down the volume of brain cell communications than did the cell surface receptors.
In the last few years, scientists have found that 20 or more types of brain cell receptors located on cell surfaces also are present at high levels inside cells, O’Malley noted.
“This should be a factor we consider when we design drugs to target brain cell receptors,” she said. “Do we want to reach cell surface receptors, receptors inside the cell or both?”
(Source: news.wustl.edu)
Discovery sheds new light on marijuana’s anxiety relief effects
An international group led by Vanderbilt University researchers has found cannabinoid receptors, through which marijuana exerts its effects, in a key emotional hub in the brain involved in regulating anxiety and the flight-or-fight response.
This is the first time cannabinoid receptors have been identified in the central nucleus of the amygdala in a mouse model, they report in the current issue of the journal Neuron.
The discovery may help explain why marijuana users say they take the drug mainly to reduce anxiety, said Sachin Patel, M.D., Ph.D., the paper’s senior author and professor of Psychiatry and of Molecular Physiology and Biophysics.
Led by first author Teniel Ramikie, a graduate student in Patel’s lab, the researchers also showed for the first time how nerve cells in this part of the brain make and release their own natural “endocannabinoids.”
The study “could be highly important for understanding how cannabis exerts its behavioral effects,” Patel said. As the legalization of marijuana spreads across the country, more people — and especially young people whose brains are still developing — are being exposed to the drug.
Previous studies at Vanderbilt and elsewhere, Patel said, have suggested the following:
• The natural endocannabinoid system regulates anxiety and the response to stress by dampening excitatory signals that involve the neurotransmitter glutamate.
• Chronic stress or acute, severe emotional trauma can cause a reduction in both the production of endocannabinoids and the responsiveness of the receptors. Without their “buffering” effect, anxiety goes up.
• While marijuana’s “exogenous” cannabinoids also can reduce anxiety, chronic use of the drug down-regulates the receptors, paradoxically increasing anxiety. This can trigger “a vicious cycle” of increasing marijuana use that in some cases leads to addiction.
In the current study, the researchers used high-affinity antibodies to “label” the cannabinoid receptors so they could be seen using various microscopy techniques, including electron microscopy, which allowed very detailed visualization at individual synapses, or gaps between nerve cells.
“We know where the receptors are, we know their function, we know how these neurons make their own cannabinoids,” Patel said. “Now can we see how that system is affected by … stress and chronic (marijuana) use? It might fundamentally change our understanding of cellular communication in the amygdala.”
(Image: Shutterstock)
Researchers at The Scripps Research Institute (TSRI) and Vanderbilt University have created the most detailed 3-D picture yet of a membrane protein that is linked to learning, memory, anxiety, pain and brain disorders such as schizophrenia, Parkinson’s, Alzheimer’s and autism.
"This receptor family is an exciting new target for future medicines for treatment of brain disorders," said P. Jeffrey Conn, PhD, Lee E. Limbird Professor of Pharmacology and director of the Vanderbilt Center for Neuroscience Drug Discovery, who was a senior author of the study with Raymond Stevens, PhD, a professor in the Department of Integrative Structural and Computational Biology at TSRI. "This new understanding of how drug-like molecules engage the receptor at an atomic level promises to have a major impact on new drug discovery efforts."
The research—which focuses on the mGlu1 receptor—was reported in the March 6, 2014 issue of the journal Science.
A Family of Drug Targets
The mGlu1 receptor, which helps regulate the neurotransmitter glutamate, belongs to a superfamily of molecules known as G protein-coupled receptors (GPCRs).
GPCRs sit in the cell membrane and sense various molecules outside the cell, including odors, hormones, neurotransmitters and light. After binding these molecules, GPCRs trigger a specific response inside the cell. More than one-third of therapeutic drugs target GPCRs—including allergy and heart medications, drugs that target the central nervous system and anti-depressants.
The Stevens lab’s work has revolved around determining the structure and function of GPCRs. GPCRs are not well understood and many fundamental breakthroughs are now occurring due to the understanding of GPCRs as complex machines, carefully regulated by cholesterol and sodium.
When the Stevens group decided to pursue the structure of mGlu1 and other key members of the mGlu family, it was natural the scientists reached out to the researchers at Vanderbilt. “They are the best in the world at understanding mGlu receptors,” said Stevens. “By collaborating with experts in specific receptor subfamilies, we can reach our goal of understanding the human GPCR superfamily and how GPCRs control human cell signaling.”
Colleen Niswender, PhD, director of Molecular Pharmacology and research associate professor of Pharmacology at the Vanderbilt Center for Neuroscience Drug Discovery, also thought the collaboration made sense. “This work leveraged the unique strengths of the Vanderbilt and Scripps teams in applying structural biology, molecular modeling, allosteric modulator pharmacology and structure-activity relationships to validate the receptor structure,” she said.
The Challenge of the Unknown
mGlu1 was a particularly challenging research topic.
In general, GPCRs are exceedingly flimsy, fragile proteins when not anchored within their native cell membranes. Coaxing them to line up to form crystals, so that their structures can be determined through X-ray crystallography, has been a formidable challenge. And the mGlu1 receptor is particularly tricky as, in addition to the domain spanning the membrane, it has a large domain extending into the extracellular space. Moreover, two copies of this multidomain receptor associating in a dimer are needed to transmit glutamate’s signal across the membrane.
The task was made more difficult because there was no template for mGlu1 from closely related GPCR proteins to guide the researchers.
“mGlu1 belongs to class C GPCRs, of which no structure has been solved before,” said TSRI graduate student Chong Wang, a first author of the new study with TSRI graduate student Huixian Wu. “This made the project much harder. We could not use other GPCRs as a template to design constructs for expression and stabilization or to help interpret diffraction data. The structure was so different that old school methods in novel protein structure determination had to be used.”
Surprising Results
The team decided to try to determine the structure of mGlu1 bound to novel “allosteric modulators” of mGlu1 contributed by the Vanderbilt group. Allosteric modulators bind to a site far away from the binding site of the natural activator (in this case, presumably the glutamate molecule), but change the shape of the molecule enough to affect receptor function. In the case of allosteric drug candidates, the hope is that the compounds affect the receptor function in a desirable, therapeutic way.
"Allosteric modulators are promising drug candidates as they can ‘fine-tune’ GPCR function,” said Karen Gregory, a former postdoctoral fellow at Vanderbilt University, now at Monash Institute of Pharmaceutical Sciences. “However, without a good idea of how drug-like compounds interact with the receptor to adjust the strength of the signal, discovery efforts are challenging."
The team proceeded to apply a combination of techniques, including X-ray crystallography, structure-activity relationships, mutagenesis and full-length dimer modeling. At the end of the study, they had achieved a high-resolution image of mGlu1 in complex with one of the drug candidates, as well as a deeper understanding of the receptor’s function and pharmacology.
The findings show that mGlu1 possesses structural features both similar to and distinct from those seen in other GPCR classes, but in ways that would have been impossible to predict in advance.
“Most surprising is that the entrance to a binding pocket in the transmembrane domain is almost completely covered by loops, restricting access for the binding of allosteric modulators,” said Vsevolod “Seva” Katritch, assistant professor of molecular biology at TSRI and a co-author of the paper. “This is very important for understanding action of the allosteric modulator drugs and may partially explain difficulties in screening for such drugs.
“The mGlu1 receptor structure now provides a solid platform for much more reliable modeling of closely related receptors,” he continued, “some of which are equally important in drug discovery.”