DURHAM, N.C. -- Researchers at the Howard Hughes Medical Institute at Duke
University Medical Center have deleted the gene for a crucial molecular
component of a mouse's nervous system and created an animal that, in essence,
mimics a person constantly high on illicit drugs.
Marc Caron, a Hughes investigator and professor of cell biology at Duke, said initial studies with the mice already have yielded surprising insights that challenge conventional theories about drug addiction and Parkinson's disease and may provide the first realistic model for testing new treatments for psychiatric disorders.
"We were astonished that a single genetic deletion would have such a profound effect on both biology and behavior," Caron said. "We believe this mouse will provide an ideal model to study addictive behavior and psychiatric disease."
Caron and his colleagues report their findings in the Feb. 15 issue of the journal Nature. Bruno Giros, the study's lead author, Mohamed Jaber, and Sara Jones of the department of cell biology; and R. Mark Wightman, of the department of neurobiology and chemistry at the University of North Carolina at Chapel Hill, also contributed to the research. The study was funded in part by the National Institutes of Health and an unrestricted neuroscience award from Bristol Myers Squibb.
Caron said in an interview that while it may seem a stretch for one genetically engineered mouse to help answer fundamental questions about the mechanism of addictive drugs, Parkinson's disease, and psychiatric disorders, all are linked by a common problem: a malfunction in the body's regulation of dopamine, an essential messenger of the nervous system.
Neurotransmitters such as dopamine, serotonin and adrenaline are chemical messengers that neurons release to their neighbors to signal them to fire nerve impulses or initiate metabolic changes. Because neurotransmitters are so critical to the smooth functioning of the nervous system, the body has evolved a precise system for regulating them.
This neurotransmission system involves two main elements: receptors and transporters. Receptors are the molecular "locks" on nerve cells that receive a neurotransmitter "key," causing a neuron to fire a nerve impulse. Transporters are "pumps" on the surface of the transmitting neuron that recycle neurotransmitters back to the nerve cell, to prepare for the next burst.
The neurotransmitter dopamine is stored in tiny hollow spheres, called vesicles, within synaptic "bulbs," which are tiny buds on the nerve cell's surface. When a nerve impulse reaches a bulb, the transmitting neuron releases a flood of dopamine into the narrow space between neurons, called the synapse. When the dopamine reaches the receiving neuron, it binds to specific dopamine receptors, thereby triggering a response in that neuron. Almost immediately, the dopamine transporter scavenges excess dopamine from the synapse to terminate the signal.
It is this dopamine transporter protein that Caron and his colleagues knocked out by disrupting the gene that encodes its production in mice. The resulting mice have no functional dopamine transporter, which means dopamine signals flood the brain and can't be shut off. Depending on where in the brain such overstimulation occurs, it may cause the temporary "high" perceived by cocaine or amphetamine addicts or the permanent and severe delusions experienced by schizophrenics. On the other hand, the lack of dopamine in the brain's motor areas produces the symptoms of Parkinson's disease.
"Virtually every addicting substance appears to modify the dopamine system, implying it may have a central role in addiction," said Alan I. Leshner, director of the National Institute on Drug Abuse (NIDA). "This finding provides a fundamentally new scientific tool in the arsenal to understand and ultimately break the addictive power of drugs."
Caron said the research "points to neutrotransmitter transporters as the most important determinant of the strength and duration of cellular communications in the nervous system."
Dr. Ralph Snyderman, chancellor for health affairs at Duke, said, "These findings represent a superb example of the way in which basic research in academic medical centers leads to practical benefit. This discovery may well change our understanding of the nature of addiction and lead to new treatments for psychiatric disorders and Parkinson's disease. It once again shows how our nation's commitment to research is needed to provide practical solutions for common health care problems."
Insights into the mechanism of amphetamine drugs derived from studying the "knockout" mice should yield new strategies for treating amphetamine addiction, Caron said.
"Much of what we know about dopamine's function comes from studies of drugs that tamper with the dopamine system," he said. Cocaine, for example, temporarily blocks the transporter from outside the cell, so the dopamine remains at high concentrations in the synapse and continues to stimulate adjacent neurons, producing cocaine's characteristic "high."
Amphetamines, by contrast, are believed to enter the neuron via both the transporter and directly through the cell membrane, where they burst dopamine vesicles inside the cell and promote the release of dopamine.
"What happens when amphetamines break dopamine vesicles has been a matter of some speculation, since it has never been directly tested,"said Jones, a post-doctoral fellow.
These studies provide the first direct demonstration that the dopamine transporter is absolutely required for amphetamine's releasing action on dopamine. Amphetamine appears to reverse the dopamine transporter pump so instead of pumping dopamine into the cell, it pumps dopamine out of the cell, Caron said.
In the knockout mice with no dopamine transporters, amphetamines can still get into the neuron through the cell membrane and disrupt dopamine vesicles. However, without transporters, dopamine cannot get out of the cell into the synapse.
"Our results show the transporter is absolutely essential for amphetamine dependent transport of free dopamine into the synaptic space, to the exclusion of any other mechanism," Caron said. "In essence, no functional transporter would mean no amphetamine high. This information should provide new understanding of the mechanism of amphetamine drugs and new strategies for treating amphetamine addiction."
Besides dopamine's direct role in producing a drug-induced high, said Caron, recent studies have shown that dopamine plays a central role in the brain's reward centers. These dopamine-activated pleasure centers are key to the addictive reinforcing pattern, even though addictive substances such as alcohol, nicotine, and cocaine may exert their influence on many different areas of the brain, Caron said.
Additional background: Insights into Parkinson's disease
The research with the knockout mice could also provide new insights into the management of Parkinson's disease, said Caron. The mouse research may point the way toward drugs that maintain the higher levels of dopamine in the brains of Parkinson's sufferers.
In Parkinson's disease, dopamine-producing neurons in the brain's motor control center, called the substantia nigra, slowly deteriorate and die. Thus, Parkinson's disease begins with small tremors and progresses to a total inability to initiate movement. Currently incurable, Parkinson's disease affects almost a million Americans and 50,000 new cases are diagnosed each year.
Drug therapies for Parkinson's disease have focused on replenishing the body's diminished supply of dopamine.
"Nobody has ever considered the dopamine transporter as a target, in part because its crucial role in maintaining dopamine levels had never been fully appreciated," said Jaber, a post-doctoral fellow.
Indeed, research with the knockout mouse shows that conserving existing dopamine may be more effective.
"When we measured the amount of dopamine being released in the knockout mice, we were astounded to find they make only 5 percent to 10 percent the normal amount of dopamine, about the same as Parkinson's patients," said Caron. "Yet we thought the dopamine levels would be much higher than normal because without the transporter protein, there would be no way to remove excess dopamine from the synapse."
To resolve this apparent paradox, Jones and Wightman measured how long dopamine stays in the synapse after it is released. Their measurements showed that in a normal mouse, the dopamine is scavenged by the transporter protein in less than one second. However, in the knockout mice with no dopamine transporter protein, the dopamine stays in the synapse at least 100 times longer. Because dopamine is staying in the synapse longer, it prolongs the neurotransmission like cocaine or amphetamine would, even though there is very little of it.
The researchers realized this very dopamine-conservation strategy could be used to treat Parkinson's patients. Because Parkinson's patients make very little dopamine, the trick is to keep what little dopamine they have in the synapse longer, significantly increasing neurotransmission, Caron said. This could be achieved with drugs that selectively block the transporter protein. Such a strategy may prove to be more potent than standard treatments. Giving more dopamine to Parkinson's patients, as is done now, just keeps the active transporters busy longer, he said.
"Blocking the transporter with therapeutic drugs should greatly benefit Parkinson's patients," Caron said. "We believe these findings could have profound implications for the treatment of Parkinson's disease."
Knockout mice and schizophrenia
The researchers believe the mice may accelerate the pace of research into
schizophrenia by providing the first realistic animal model to test new
Researchers have known for many years that schizophrenics behave as if they have too much dopamine in the brain's limbic system, which controls emotion and behavior. Conversely, not enough dopamine contributes to clinical depression. Although therapeutic drugs for schizophrenia block dopamine receptors, they have limited effectiveness and produce side effects in many patients.
"Years of research by many investigators have helped determine the cellular components that regulate dopamine levels in the synapse," said Giros, now a researcher at INSERM in Paris. "We are now in the position to ask questions about what role each of the components play and what happens when one crucial component is missing or malfunctioning."