Seizures are the most life-threatening, as well as disconcerting, symptoms of withdrawal in people who are alcoholics and who abruptly stop drinking. Heavy consumption of alcohol, or ethanol, the substance in beer, wine and liquor that is addictive, leads to changes in the brain. These changes allow an alcoholic to develop tolerance to ethanol. But they also trigger seizures and other symptoms of delirium tremens when alcohol consumption stops.
Now Rockefeller University scientists, in experiments with mice, have discovered a protein that regulates the seizures induced by ethanol withdrawal.
The protein, called tissue plasminogen activator, or tPA, is the same factor that dissolves the blood clots that can trigger heart attacks and strokes.
The Rockefeller scientists' finding suggests that drugs targeting tPA might prevent the seizures as well as other impairing effects of ethanol withdrawal. The results appear in the Jan.3 online issue of Proceedings of the National Academy of Sciences.
"What we have found is that tPA's interactions with certain brain receptors contribute to the development of physical dependence on ethanol," says Sidney Strickland, Ph.D., head of Rockefeller's Laboratory of Neurobiology and Genetics. "Our new findings imply that interfering with these interactions might protect against alcohol-withdrawal pathologies in the brain."
Nearly 14 million Americans abuse alcohol or are alcoholic. Without access to alcohol they go into withdrawal, which can include insomnia, tremor, muscle rigidity, hallucinations and seizures. These symptoms, often called delirium tremens, kill about 5 percent of the people who develop them.
Consuming alcohol slows down the transmission of chemical messages in the brain. Ethanol molecules sit in a receptor (called the NMDA receptor) that would normally be occupied by a stimulant — a neurotransmitter called glutamate — thus preventing glutamate from delivering its message. When a person drinks large amounts of ethanol over a long period of time, the brain compensates by making more NMDA receptors on cells.
"The increase in NMDA receptors allows the brain to function even under the depressive effect of ethanol," says Strickland. But it also leads to the symptoms of withdrawal.
"An analogy is driving a car and trying to maintain a speed of 30 miles per hour," explains Strickland. "Ingesting alcohol is like stepping on the brake. To maintain your speed, you need to press harder on the gas, which in the brain means making more NMDA receptors. Then if the brake is suddenly released, the car goes too fast. This is what happens with delirium tremens. When alcohol consumption — the brake — stops, the brain is essentially too active. The person in ethanol withdrawal feels anxious and agitated, and may have tremors or seizures."
Strickland and his colleagues knew from earlier research that tPA interacts with NMDA receptors, in particular a form of NMDA receptor with a binding site called NR2B. "tPA is better known as a clot-buster, used to treat heart attack or stroke patients," explains Strickland. "But it also functions in the central nervous system. tPA is involved in making synapses work better, to facilitate learning and memory."
To investigate further the connection between tPA and NMDA receptors in alcohol dependence, Strickland and his colleagues studied two groups of mice that were genetically identical except for the tPA gene: one group had the gene and made the protein normally; the other did not have the gene for tPA, and thus did not produce the tPA protein.
For 14 days the researchers put the mice on a well-established regimen for mimicking the development of alcohol addiction in humans. They fed all the mice a liquid diet that included vitamins and a quantity of ethanol that increased from 2.3 to 10 percent of the diet volume over the course of the study. Then, on the 15th day, they switched the mice to an alcohol-free diet.
The normal mice suffered from seizures and other symptoms of ethanol withdrawal that peaked six hours after they stopped drinking the alcohol-containing diet. The mice that lacked tPA also showed some — but much less severe — effects of ethanol withdrawal.
"The action of ethanol in the brain is complex," says Robert Pawlak, Ph.D., a postdoctoral associate in the Strickland laboratory who spearheaded the study. "It's important to stress that tPA-NMDA interactions are not solely responsible for the development of physical dependence to ethanol. Changes to other neurotransmitters and receptors could explain why the mice in our experiments that lacked tPA still developed moderate signs of ethanol withdrawal."
Strickland and colleagues also found that tPA levels in certain brain structures — the hippocampus and the amygdala — increased during the period when the mice were ingesting increasing amounts of alcohol. This confirmed the scientists' suspicion that tPA plays a role in ethanol dependence.
Next, after the mice stopped the alcohol diet, the researchers injected tPA into the brains of mice that lacked tPA. This led to an increase in seizures, confirming the link between tPA and symptoms of ethanol withdrawal.
Finally, the researchers injected ifenprodil, a drug that prevents tPA from binding specifically to the NR2B subunit of NMDA receptors, into mice undergoing ethanol withdrawal. The seizures and other symptoms abated.
"tPA sensitizes the nervous system," says Strickland. "That's good while ethanol is there, but bad once the ethanol is gone. Too much tPA has pathological effects."
Another experiment, one using antibodies to identify the molecules with which tPA was interacting, confirmed a physical bond between NR2B and tPA. The scientists also investigated further the chemistry of this interaction.
Since tPA is an enzyme that cuts apart certain proteins, "our first thought was that it was cleaving the NR2B receptor, to make it work better," says Strickland. "But our results show that it does not cleave NR2B." The researchers also determined that tPA was not activating plasminogen, which is tPA's most common physiological function.
"These findings identify a tPA-dependent pathway of neuronal activation as a potential drug target against the effects of sudden alcohol withdrawal," says Strickland. "We don't fully understand how tPA affects these processes. The next step will be to figure out in more detail its mechanism of action."