21 November 2003. Dopaminergic, serotonergic, and glutamatergic: three CNS neurotransmitter pathways that are distinct. Or are they? A paper in this week's Science reveals that all three pathways eventually lead to one protein, the dopamine- and cAMP-regulated protein of 32 kilodaltons, commonly known as DARPP-32.
DARPP-32 is an inhibitor of protein phosphastase-1 (PP1), one of the few phosphatases in the mammalian cell, and which has been implicated in the pathogenesis of neurodegenerative diseases, particularly Parkinson's disease (see Alzheimer Research Forum related news story). Now, the current finding provides an explanation for why three distinct types of drugs—dopaminergic agonists, serotonergic agonists, and glutamatergic antagonists—can all induce, in animals, a psychotomimetic state resembling schizophrenia.
Paul Greengard and colleagues at the Rockefeller University, New York, investigated the effect of knocking out the DARPP-32 gene in mice. First author Per Svenningsson and colleagues found that D-amphetamine, D-lysergic acid diethylamide (LSD), and phencyclidine (PCP), representatives of each drug class, respectively, all cause a loss of sensorimotor gating in wild-type mice but not in DARPP-32 knockouts. This suggests that the phosphatase inhibitor mediates the psychotomimetic actions of these compounds. Sensorimotor gating, or the processing and filtering of stimuli and information, is also compromised in non-medicated sufferers of schizophrenia.
To investigate how the three drugs may converge on DARPP-32, Svenningsson determined the phosphorylation status of the protein following administration of the drugs. The inhibitor is known to be phosphorylated at several sites. Modified at threonine 34, it becomes a potent inhibitor of PP1; at serine 97, it is rendered more susceptible to phosphorylation of T34 by protein kinase A (PKA), while with serine 130 phosphorylated, it becomes resistant to protein phosphatase 2B, which can dephosphorylate T34. Thus, phosphorylation at these three sites leads to more potent PP1 inhibition. In contrast, phosphorylation of threonine 75 attenuates PP1 inhibition because it converts DARPP-32 into an inhibitor of PKA.
When Svenningsson and colleagues treated tissue samples from frontal cortex and striatum with any of the three drugs, phosphorylation of T34 and S130 was significantly increased. Amphetamine had the strongest effect, almost tripling the T34-Pi content, while PCP increased it by about 50 percent.
To rule out some nonspecific cell-wide increase in phosphorylation in response to the drugs, the authors examined downstream targets of DARPP-32, including CREB and GSK-3β, and proteins thought to be regulated independently, including STAT-3 and CaMKII. While the three drugs caused phosphorylation of CREB and GSK-3β, STAT-3 and CaMKII were unaffected.
Svenningsson next used threonine-alanine mutants to confirm the relationship between DARPP-32, its downstream targets, and sensorimotor function. Compared to wild-type tissues, in those from T34A and S130A transgenic mice, phosphorylation of CREB and GSK-3β was significantly reduced in response to the three drugs. Furthermore, expression of c-fos, which is stimulated by activated CREB and GSK-3β, was suppressed in these mice. In addition, in transgenic animals the over twofold increase in repetitive movements caused by the psychotomimetics was significantly reduced. In contrast, T75A mutations had no effect on these movements or on DARPP-related phosphorylation activity.
Overall, the study suggests that it is through DARPP-32, and its target PP1 that these agonists and antagonists exert their psychotomimetic effects. Finding the substrates of PP1, the authors suggest, may lead to new insights into the behavioral effects of the drugs.—Tom Fagan.
Svenningsson P, Tzavara ET, Carruthers R, Rachleff I, Wattler S, Nehls M, McKinzie DL, Fienberg AA, Nomikos GG, Greengard P. Diverse psychotomimetics act through a common signaling pathway. Science. 2003 Nov 21;302(5649):1412-5.