In the dynamic world of the central nervous system, the neurotransmitter glutamate is a key player, ceaselessly transmitting critical instructions between nerve cells. Now, UCSF researchers have identified the protein that transports the chemical signal to its launch site in nerve cells, offering a possible new target for treating such diseases as Alzheimer's disease.
The discovery opens a vast field of potential therapy, for while glutamate carries out such fundamental processes as sensory perception, learning and memory, changes in its role contribute to many brain diseases. The release of too much glutamate causes excessive excitation in the nervous system that leads to seizures, contributes to injury after stroke, the perception of pain and even the destruction of nerve cells associated with neurodegeneratives diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (Lou Gehrig's disease). A drug that blocked the glutamate transporter from loading glutamate, thus reducing the release of glutamate, could treat these illnesses.
Alternatively, increasing the amount of glutamate released from certain nerve cells could improve learning, memory skills and overall cognitive function. In this case, therapy might simply involve taking a drug that increases the expression of the gene that produces the protein transporter. The resulting increased expression of the protein would enable nerve cells to store and release more glutamate.
"We've never had a drug that inhibits the release of glutamate, but we presume it would have dramatic effects," says the senior author of the study, Robert H. Edwards, MD, UCSF professor of neurology and physiology. Glutamate, like all neurotransmitters, is a chemical message released by one nerve cell and targeted at another. Thousands of glutamate molecules are released from a single cell, prompting a response in a neighboring cell, which prompts a response in yet another cell. The millisecond relay between thousands of nerve cells causes a flurry of activity that prompts the brain to carry out all fundamental aspects of brain function.
While neurotransmitters are created in the interior of the cell, they are pumped, in large quantity, into synaptic vesicles tucked into the wall of a nerve cell's so-called "terminal," the launch pad from which chemical messages are released from the cell. The launch pad is based at the end of a cell's axon, the long fiber that extends from the cell body toward other nerve cells. The axon ultimately branches into thousands of fingers, each with its own terminal.
When a nerve cell is activated, it sends an electrical impulse down its axon to the terminal, which stimulates a vesicle within the terminal to migrate to the terminal's surrounding membrane. There, it fuses with the membrane, creating a conduit for the release of the neurotransmitters into the so-called synaptic cleft between nerve cells. The released neurotransmitters diffuse across the cleft, binding to receptors on the target cells and prompting a response in the target cell.
While some neurotransmitters, such as GABA, transmit inhibitory signals that reduce excitation and anxiety, and others, such as dopamine and serotonin, modulate the activity of neural circuits to influence mood and sleep, glutamate causes excitation, nudging the brain into high alert, enabling it to carry out the computations that underlie cognition and most other fundamental aspects of brain function.
The UCSF researchers previously discovered two families of proteins that serve as the synaptic vesicle transporters for most other key neurotransmitters.1 However, the glutamate transporter has remained elusive. Now, in the August 11 issue of Science, the University of California, San Francisco researchers report not only that they have identified the transporter, but that it is a protein that scientists have thought had a completely different role. Previously known as brain-specific inorganic phosphate transporter, the researchers have renamed the protein vesicular glutamate transporter, or VGLUT1.
"The discovery of the glutamate transporter represents a major missing component that people have sought for a long time," says Edwards. "It is one of the final things that will enable us to study the basic function of how synapses work."
The finding's importance resembles that of the discovery of glutamate receptors, the proteins that respond to glutamate on target cells, says Edwards. "Our finding represents the flip side - the release of glutamate." Given the importance of glutamate for brain function, the idea of reducing its release to treat a particular disease might seem problematic. However, studies by other investigators have previously identified a similar but distinct protein related to VTLUT1 that also exists in the adult brain, and may be particularly important in brain development. The fact that there are two forms, or "isoforms," of the glutamate transporter suggests that it may be possible to inhibit just one of the two isoforms, says Edwards.
"If we inhibited one isoform, it wouldn't necessarily undermine basic glutamate function," says Edwards, "because the other isoform might be able to carry on the day-to-day functions."
The discovery of the glutamate transporter rounds out the understanding of the mechanisms by which neurotransmitters are released, and which therefore make neurotransmission possible. While the other classical neurotransmitters, such as dopamine and serotonin, are often released at great distances from their target nerve cells, glutamate usually affects target cells immediately adjacent to the sites of release. And a key aspect of the UCSF finding is the suggestion that the transporters for these two different types of neurotransmitters may contribute to their different functions.
Many transporters function by pumping molecules such as neurotransmitters into compartments, such as synaptic vesicles. They can often generate concentrations of the molecule inside the compartments that are 100 to 10,000 times higher than outside.
The newly discovered VGLUT1 transporter also pumps glutamate into synaptic vesicles. However, the researchers made an observation that, if confirmed, suggests the transporter also has a property of another type of neurochemical conveyor - a channel. Channels, which allow a compound such as glutamate to move from a compartment with high concentrations to one with low concentrations, but not vice versa, cannot pump as large amounts of a compound into synaptic vesicles as a transporter can. However, they operate much more quickly than transporters.
The channel-like property could have evolved, says Edwards, to meet the important need of moving large amounts of glutamate quickly, for while the other neurotransmitters are involved in modulating behaviors, such as mood, and therefore don't need to act with millisecond speed, glutamate functions by rapid-fire communication between closely placed nerve cells, and could require the fast action of the channel mechanism.
"A lot of synapses have to release neurotransmitters on a very rapid time scale, and may need to refill the vesicles within a few seconds. Having this channel property could allow a vesicle to fill itself faster," says Edwards. The researchers conducted their study in rat cells. To determine whether the protein in question, at the time known as BNPI, mediated the transport of glutamate into synaptic vesicles, the researchers inserted the BNPI DNA into rat cells that normally lacked BNPI protein. The inserted DNA then directed synthesis of the protein. Using synaptic vesicles from these cells, as well as from cells lacking the BNPI DNA, the researchers then tested the ability of the two sets of vesicles to accumulate glutamate. Vesicles in the cells expressing BNPI loaded two to four times more glutamate than those from the unaffected cells.
The researchers' next step is to study the channel and transporter-like properties of VGLUT1 and its related isoform in greater detail, to see if they contributes to changes in synaptic strength that underlie learning, memory and many other aspects of cognition. To determine the protein's role in behavior, they will also knock out the genes in mice.
Researchers previously thought the VGLUT1 protein played a very different role. They thought it was a protein that functions on the plasma membrane to transport inorganic phosphate into a neuron.
###Co-authors of the study were Elizabeth E. Bellocchio, MS, a graduate student in the UCSF Program in Biological Sciences Neuroscience program; Richard J. Reimer, MD, a postdoctoral fellow in the Edwards lab and Robert T. Fremeau Jr., MD, a visiting scientist in the Edwards lab.
The study was funded by the National Institutes of Health.
* 1. One family of synaptic vesicle transporters pumps in monoamine transmitters, dopamine, serotonin, norepinephrine and acetycholine. The second family pumps in GABA, the major inhibitory neurotransmitter in the brain, and other members of the family contribute to the recycling of glutamate at the synapse.
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