15 June 2011. Two recent studies turn up new clues for the regulation of anxiety in some obscure places. One study, published in Nature on 19 May 2011, identifies an extracellular matrix protease called neuropsin as a key player in converting stressful events into anxious behavior in mice. Led by Robert Pawlak of the University of Leicester, U.K., this study outlines a path in which stress spurs neuropsin to cleave EphB2 receptors on amygdala neurons, which then triggers a cascade of events involving NMDA receptors and Fkbp5 gene transcription. The second study, published in PNAS on 17 May 2011, explores the somewhat mysterious trace amine-associated receptor 1 (TAAR1). A team led by Marius Hoener of F. Hoffmann-La Roche in Basel, Switzerland, reports the discovery of a selective agonist for TAAR1, which not only has anxiolytic effects in mice, but also mimics antipsychotic drugs in various hyperlocomotion tests used for screening drugs for schizophrenia.
Both studies offer a glimpse into the multiple and complicated mechanisms behind anxiety, which is a common comorbidity for people with schizophrenia, and touch on other clues related to the disorder. The first study centers on how neuropsin cleaves EphB2, a receptor tyrosine kinase that associates with NMDA receptors, whose function may be compromised in schizophrenia (see SRF hypothesis). Other threads of evidence link a ligand for EphB2, called ephrin B2, to schizophrenia, including a genetic association study (see SZGene entry), and a recent report identifying ephrin B2 as participating in the control of neuronal migration during development via the reelin (see SZGene entry) pathway (Sentürk et al., 2011).
The second study explores the selective activation of TAAR1, a receptor for trace amines. Trace amines are byproducts of amino acid production and are found in the brain at levels so low as to seem irrelevant. But trace amines garnered more respect in 2001, when researchers discovered a family of receptors for them, including TAAR1 (Borowsky et al., 2001), which localizes to dopamine and serotonin-containing neurons in the brain. Abnormal levels of trace amines have been reported for schizophrenia and other disorders, and have been proposed to play a role in regulating dopamine and serotonin systems (Burchett and Hicks, 2006).
Stressful steps to anxiety
To explore the possibility of stress-related signaling at the extracellular matrix-neuron interface, first author Benjamin Attwood and his colleagues in Leicester began with a demonstration that neuropsin selectively cleaves the extracellular portion of EphB2 in cultured cells. To address whether this cleavage was impacted by emotional "stress," the researchers restrained the mice, which activates the amygdala, and measured a 50 percent increase in neuropsin levels, and a twofold increase in EphB2 receptors in the neuronal membranes afterwards. Co-immunoprecipitation studies indicated that cleaved EphB2 receptors dissociated from the NR1 subunit of the NMDA receptors, with which they normally cluster. This dissociation did not transpire in knockout mice lacking neuropsin, but it could be rescued by injecting the amygdala of these animals with neuropsin.
The researchers also found that this neuropsin-dependent EphB2 cleavage boosted transcription of Fkbp5, a gene whose activation is implicated in turning stressful events into post-traumatic stress disorder (Segman et al., 2005). When stressed, mice lacking neuropsin had a smaller induction of Fkbp5 and less anxiety than wild-type animals, as measured by entries into open arms of an elevated-plus maze. These unflappable mice could be turned into apprehensive ones by injecting neuropsin into their amygdalae before stressing them. Other manipulations supported a role for other steps in the neuropsin pathway: wild-type mice displayed less anxious behavior after an episode of stress when EphB2 receptors were blocked with an antibody, or when Fkbp5 gene expression was decreased in the amygdala.
A new TAAR1-get
TAAR1 is a G protein-coupled receptor residing in the dopamine and serotonin-containing cells of the brain and, like other amine receptors, it activates cAMP production inside the cell. TAAR1 knockout mice are hypersensitive to amphetamine, showing elevated locomotion and increased release of dopamine, serotonin, and noradrenaline relative to wild-type mice (Wolinsky et al., 2007). This suggests that TAAR1 normally keeps monoaminergic signaling in check.
But studying the consequences of TAAR1 activation directly with any precision has been stymied by the lack of a selective agonist. This is where first author Florent Revel of Hoffmann-La Roche and colleagues step in, with an introduction to and characterization of a new selective TAAR1 agonist, called RO166017. This agonist had high affinity for and potency at mouse, rat, monkey, and human versions of TAAR1, spurring cAMP production to levels similar to those reached by endogenous trace amines. Testing for binding to 123 other proteins found that the agonist preferred to bind to TAAR1 in every case, usually with a greater than 100-fold selectivity, making the compound a clean way to activate the TAAR1 receptor.
In mouse brain slices, the agonist clearly modulated the firing rates of other aminergic neurons. It decreased the firing rates of dopamine neurons in the ventral tegmental area (VTA) and of serotonin neurons in the dorsal raphe nucleus (DRN), but not in the locus ceruleus (LC), which is devoid of TAAR1 receptors. Importantly, RO166017-induced responses weren't apparent in knockout animals lacking the TAAR1 receptor, and they were blocked by a TAAR1 antagonist, called EPPTB, in wild-type mice—which both support the selectivity of the agonist's action.
Behaviorally, the RO166017 agonist had anxiolytic and antipsychotic-like effects in mice. When given orally, the agonist prevented stress-induced hyperthermia in wild-type mice in a dose-dependent manner, but not in TAAR1 knockouts. The agonist also inhibited the hyperlocomotion induced by cocaine in wild-type mice (but not in TAAR1 knockouts), achieving a level of inhibition similar to the antipsychotic olanzapine. In a more specific test, the agonist stemmed the hyperlocomotion due to elevated levels of synaptic dopamine that occur in mice lacking the dopamine transporter (DAT) that pumps dopamine back into the neuron. The agonist could also temper hyperlocomotion triggered by inhibition of the NMDA receptor—a model for schizophrenia.
These results put TAAR1 forward as an alternative route to modulating monoaminergic systems in the brain—one that might have fewer side effects than the direct modifications to these systems made by current antipsychotic drugs. Together, the two studies provide a reminder that there is still a lot to learn about the mechanisms behind anxiety—a situation that can lead to new and unexpected therapeutic targets.—Michele Solis.
Attwood BK, Bourgognon JM, Patel S, Mucha M, Schiavon E, Skrzypiec AE, Young KW, Shiosaka S, Korostynski M, Piechota M, Przewlocki R, Pawlak R. Neuropsin cleaves EphB2 in the amygdala to control anxiety. Nature. 2011 May 19;473: 372-5. Abstract
Revel FG, Moreau JL, Gainetdinov RR, Bradaia A, Sotnikova TD, Mory R, Durkin S, Zbinden KG, Norcross R, Meyer CA, Metzler V, Chaboz S, Ozmen L, Trube G, Pouzet B, Bettler B, Caron MG, Wettstein JG, Hoener MC. TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity. Proc Natl Acad Sci U S A. 2011 May 17;108: 8485-90. Abstract