Researchers Unlock Clues To SSRI Mechanism

In groundbreaking research published this spring, Nobel Prize–winner Paul Greengard, Ph.D., and his colleagues at Rockefeller University have detailed for the first time the cellular effects of fluoxetine on neurons thought to be involved in the development of depression.

The new reports arose directly from the line of research for which Greengard, a professor of psychiatry and pharmacology and director of the Laboratory of Molecular and Cellular Neuroscience at Rockefeller, was awarded the 2000 Nobel Prize in Medicine. Greengard shared that prize with psychiatrist Eric Kandel, M.D., of Columbia University and pharmacologist Arvid Carlsson, M.D., of the University of Göteborg in Sweden (Psychiatric News, November 3, 2000).

Greengard was recognized by the Nobel committee for his line of work, which began in the mid-1960s, on defining the role of dopamine and other neurotransmitters in the central nervous system. It was Greengard who developed the now universally accepted model of cell membrane receptors and their binding by various neurotransmitters to cause various cellular effects. Greengard’s research detailed the addition or removal of phosphate chemical groups associated with what he called at the time “key proteins.” The process is now known as phosphorylation and dephosphorylation. With the addition or subtraction of phosphate groups, the structure and therefore the shape of a key protein changes and, consequently, so does the protein’s function.

The new research, presented as two reports in the March 5 Proceedings of the National Academies of Science, details the interaction of the serotonin reuptake inhibitor fluoxetine and an intracellular protein, DARPP-32, which is abundant inside neurons in the brain’s prefrontal cortex and striatum.

The research was funded through grants from the National Institutes of Health and the National Institute of Mental Health. Greengard’s team worked collaboratively with researchers from Eli Lilly and Company, maker of the Prozac brand of fluoxetine.

Defining a Mechanism

“We had shown earlier that [phosphorylation of] DARPP-32 is regulated by dopamine,” Greengard told Psychiatric News. “And since this area of the brain is rich in serotonergic innervation, we wondered if serotonin might also regulate the state of phosphorylation of DARPP-32 in a manner similar to what we had shown with dopamine and, subsequently, the antipsychotic medications.”

In the two companion papers, Greengard’s group showed exactly that. In a combination of studies using slices of mouse brain as well as whole animals, the researchers showed that serotonin regulates the phosphorylation of DARPP-32 at three specific sites on the very large protein, adding phosphate groups to two of the sites, and removing phosphate at the third specific site on the protein. This combination of changes in DARPP-32 directly leads to a decrease in the activity of a key intracellular enzyme, protein-phosphatase-1 (PP-1).

‘Far-Reaching Effects’

“This has extremely far-reaching effects,” Greengard explained. “PP-1 removes phosphates from receptors for neurotransmitters and ion channels and from what are called transcription factors that regulate protein synthesis within the neuron.”

Simply put, Greengard continued, decreasing the activity of PP-1 would lead to changes in how neurotransmitters bind or are blocked from binding to receptors, changes in movements of ions like sodium, potassium, and calcium into and out of the nerve cell, and changes in how much or how little of certain proteins the neuron produces.

Greengard’s group then tested what response ensued when they increased serotonin availability. They increased serotonin through a number of mechanisms, including adding serotonin agonists as well as adding fluoxetine, a known reuptake inhibitor. In each case, they observed the characteristic changes in phosphorylation and a decrease in the activity of PP-1, confirming that DARPP-32 is essential not only for dopaminergic but also for serotonergic neurotransmission.

The protocols used in the brain-slice experiments were then replicated in whole animals to test for the presence of the changes in phosphorylation and activity of PP-1. In addition, the researchers tested the effects of the serotonin agonists, including an agent known to increase the release of serotonin from the pre-synaptic terminal and 5-HTP, the metabolic precursor of serotonin. Again they saw the characteristic changes. The tests were then repeated, with low doses of clozapine, the atypical antipsychotic drug, which at the low dose used has a strong attraction to serotonin receptors but little attraction for dopamine receptors. The researchers expected to see little change in phosphorylation or activity of PP-1 with the serotonin pathway blocked by the drug. Again, the results pointed in the same direction.

To test whether their target protein, DARPP-32, was actually responsible for the changes noted, the tests were repeated in additional mice, comparing a control group of animals with a second group in which the gene for DARPP-32 had been removed. As the team hypothesized, the “knockout” mice did not show the characteristic changes in phosphorylation or activity of PP-1.

Finally, the team studied the effects of the serotonin-agonist fluoxetine and clozapine on two standard models of mouse behavior, the locomotor activity test, and the tail-suspension test for antidepressant efficacy. As they expected, boosting the serotonin pathway led to a change in mouse behavior analogous to an “antidepressant effect,” while clozapine strongly counteracted the effect.

“This line of work has really opened up a tremendous field [of research],” Greengard said.

By working out the details of the pathway by which fluoxetine exerts behavioral effects, Greengard explained, the team has been able to shed light on many biochemical processes in the brain that can now be associated with depression. In effect, by discovering how the antidepressant drug exerts its effects in the brain, the team has opened the door to discovering the physiological mechanisms of depression.

“The major implication here is that by working out this pathway,” Greengard told Psychiatric News, “this provides new [biochemical] targets for new drug development.”

With more research, Greengard continued, new antidepressant drugs could be targeted to increase efficacy while narrowing and minimizing side effects.

Although the team’s work published in these two reports centered on the effects of fluoxetine in particular, Greengard is confident that the results have broad applicability.

“I am certain that [all SSRIs] work through this pathway. There may be small differences between [other SSRI] drugs, but we are working on that now, and with other [non-SSRI] antidepressants, to see whether they do the same thing.”

“DARPP-32 Mediates Serotonergic Neurotransmission in the Forebrain” is posted on the Web at www.pnas.org/cgi/doi/10.1073/pnas.052712699. “Involvement of Striatal and Extrastriatal DARPP-32 in Biochemical and Behavioral Effects of Fluoxetine (Prozac)” is posted at www.pnas.org/cgi/doi/10.1073/pnas.052712799. ▪

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PNAS 2002 99 3182