The findings, reported by researchers at Rockefeller University with collaboration from three pharmaceutical and biotech companies, provides, for the first time, a cellular model detailing how this crucial protein, known as DARPP-32, interacts with multiple neurotransmitter systems to produce behavior.
The scientists demonstrate that DARPP-32 acts like the thin neck in an hourglass, through which all signals taken into a nerve cell must pass and be processed, producing a wide variety of biochemical reactions. In this case, three different drugs of abuse, LSD, PCP ("angel dust") and amphetamine, work on three different neurotransmitters, serotonin, glutamate, and dopamine, respectively. All three drugs, which are classified as psychotomimetics or psychostimulants, are processed within the DARPP-32 hourglass neck through the same pathway, thus producing very similar physiological symptoms.
"For the first time, we can explain through a molecular model why these drugs all produce the same kind of behavioral symptoms," says the study's first author, Per Svenningsson, M.D., Ph.D., a research assistant professor in the Laboratory of Molecular and Cellular Neuroscience, headed by Paul Greengard, Ph.D.
Clinically, the study does not suggest that DARPP-32 is the root cause of schizophrenia, but it does provide new avenues in which to treat the disease, says Greengard, Vincent Astor Professor at Rockefeller and the study's principal investigator.
By experimentally blocking the function of one of the 205 amino acids that make up DARPP-32, the research team was able to abolish the effects of the drugs, all of which have long been known to produce schizophrenia-like behavior in both mice and humans.
"This is remarkable because it shows that a single amino acid on a single protein, by being altered, can abolish the effects of these psychotomimetic drugs on behavior," says Greengard, who shared the 2000 Nobel Prize in Medicine or Physiology for his work on neurotransmitters and DARPP-32. "The research certainly indicates new targets for the development of antipsychotic drugs."
The study also answers a long-standing debate in psychiatry as to which neurotransmitter is primarily responsible for schizophrenia, says Greengard, because researchers have known that drugs like LSD, PCP and amphetamines, which act on different transmitters, create the same psychoses as seen in schizophrenia.
"It turns out everyone was right, because each of these drugs work on a common pathway regulated by DARPP-32," says Greengard.
Previous research by Svenningsson and Greengard also has demonstrated that DARPP-32 regulates the actions of medications such as Prozac, to treat depression, as well as drugs of abuse such as cocaine, opiates and nicotine.
"We have begun to believe that DARPP-32 is really a master molecule that integrates information coming in from all parts of the brain and is involved in mediating and regulating the actions of many, many neurotransmitters," says Greengard.
The investigators knew that, like many such proteins, DARPP-32 can be activated by the addition of a phosphate molecule (a process called "phosphorylation") or by removal of a phosphate molecule ("dephosphorylation") on specific amino acid sites.
In the findings reported in Science, the Rockefeller team found that DARPP-32 was phosphorylated or dephosphorylated at three sites by the studied psychotomimetics, in a pattern that worked together to inhibit an enzyme downstream of DARPP-32 called protein phosphatase-1 (PP-1). PP-1 helps regulate its own series of biochemical reactions that lead to physiological responses.
In order to understand the precise functional importance of these three phosphorylation sites, the scientists created a series of "knockin" mice, in which each of these sites on the DARPP-32 protein were mutated. The behavioral responsively to LSD, PCP and amphetamine were thereafter compared between these mutant mice and normal mice. It turned out that single mutations in the amino acid sequence of DARPP-32 virtually abolished the behavioral actions of the psychotomimetics.
Schizophrenia-like symptoms such as repetitive movements and sensory perception defects induced by the psychotomimetics were strongly attenuated in two of the three different mutant mouse lines, implicating a critical involvement of two distinct, but interacting, phosphorylation sites of DARPP-32 in the actions of LSD, PCP and amphetamine, says Svenningsson.
Ongoing research is aimed at further understanding how DARPP-32 can process a wide variety of neurotransmitters that affect behavior, he says. "This master molecule seems to be involved in many behaviors, including those related to mood and the way we perceive the world," says Svenningsson.
Co-authors of the study, funded by the National Institutes of Health, include researchers from Rockefeller University (Robert Carruthers and Ilan Rachleff), Eli Lilly and Company (Eleni Tzavara, David McKinzie, George Nomikos), Lexicon Genetics, Inc. (Sigrid Wattler and Michael Nehls) and Intra-Cellular Therapies (Allen Fienberg). Fienberg also is affiliated with Rockefeller University.
Journal
Science