3,4‐methylenedioxymethamphetamine (MDMA) stimulates activation of TAAR1 and subsequent neurotransmitter transporter internalization in serotonin neurons
Authors: Underhill Suzanne , Amara Susan 
Published: May 2022

The use of 3,4‐methylenedioxymethamphetamine (MDMA) has recently gained interest as a potential therapeutic tool for the treatment of post‐traumatic stress disorder (PTSD). However, the mechanism of action of this drug has not yet been fully elucidated. Here we demonstrate that MDMA acts by targeting the trace amine associated receptor 1 (TAAR1), an intracellular G‐protein coupled receptor (GPCR) expressed throughout the brain and in peripheral tissues. In previous studies, we have shown that a closely‐related compound, amphetamine (AMPH), enters neurons through the dopamine and norepinephrine transporters (DAT and NET) and activates TAAR1. This activates the small GTPase RhoA that in turn increases internalization of the DAT and NET. We have also found that a neuronal glutamate transporter, EAAT3, is also internalized. These actions result in an increase in extracellular DA, NE and glutamate concentrations. In this study, we show that MDMA enters serotonin neurons through the 5‐HT transporter (SERT) and activates a similar signaling cascade.We isolated brain tissue encompassing the Raphe nucleus from E15 mice and prepared primary cultures of serotonin (5‐HT) neurons. 5‐HT neurons were identified by immunocytochemistry for 5‐HT. Functional SERT activity was confirmed by citalopram sensitive 3H‐5‐HT uptake. Pre‐treatment of these cultures for thirty minutes with 1 mM MDMA resulted in a decrease of SERT transport activity. This loss of function was blocked by an inhibitor of RhoA, indicating that MDMA regulates SERT through a Rho‐dependent signaling pathway.Using total internal reflection fluorescence (TIRF) microscopy in HEK293 cells we confirmed that the loss in SERT activity produced by MDMA was due to internalization of the SERT. The application of 1 mM MDMA led to a decrease in GFP‐tagged SERT at the cell membrane. Similarly, GFP‐tagged EAAT3 was also internalized in response to MDMA. Parallel studies in HEK293 cells in which the TAAR1 gene was deleted by CRISPR‐Cas9 showed no effects of MDMA treatment on either SERT or EAAT3 trafficking and confirmed that TAAR1 is an obligate target for the actions of MDMA,To examine the effects of MDMA on endogenous SERT and EAAT3 in situ, we prepared acute brain slices from wildtype and TAAR1 knockout mice. Slices containing the Raphe nucleus were treated with either vehicle control or MDMA (1 mM) and subsequently treated with a cell‐impermeable biotin reagent that would label all surface proteins. By western blot analysis, we observed internalization of SERT and EAAT3 in response to the MDMA treatment. However, neither transporter exhibited any change in cell‐surface expression in response to MDMA in the TAAR1 knockout tissue.Our data indicate that in serotonergic neurons MDMA potentiates the action of both serotonin and glutamate by activating TAAR1 signaling and triggering SERT and EAAT3 internalization. These observations point to new mechanisms to target for the development of drugs to treat PTSD.