Structure, Function and Pharmacology of Neurotransmitter Reuptake Systems

National Institute Of Mental Health

Investigators

Linked publications, trials & patents

Abstract

Drugs which act on neurotransmitter transport systems are among the most widely used treatments for neuropsychiatric disorders, including major depression and attention-deficit disorders. These transporters are also a target for drugs of abuse such as methamphetamine (METH), MDMA (ecstasy) and cocaine. My laboratory has used cell biological, biochemical, physiological, and transcriptomic approaches to understand the function, regulation, and effects of drugs on neurotransmitter transporters. We have continued to explore the presynaptic actions of amphetamines by further examining the signaling events that they elicit within dopamine neurons. When amphetamine enters a dopamine neuron, it activates the small GTPases, RhoA and Rac1 and triggers internalization of the dopamine transporter (DAT) and a neuronal glutamate transporter (EAAT3). Intriguingly, studies from other groups have shown that the gene encoding EAAT3 is associated with several psychiatric disorders including obsessive-compulsive disorder, autism, and in schizophrenia. Additional work examining the effects of reduced global EAAT3 gene expression has confirmed a role for the transporter in both amphetamine-mediated and basal ganglia dependent repetitive behaviors. We have shown that the removal of transporters from the cell surface results in the acute potentiation of both glutamate and dopamine signaling. We identified the trace amine receptor, TAAR1, as an intracellular receptor target that mediates the initial actions of AMPHs, trace amines, and other drugs, and showed that TAAR1 signals through two G-proteins, Gs and G13, to activate Protein Kinase A (PKA) and the small GTPase, RhoA, respectively. In experiments on transgenic mouse lines lacking the TAAR1 receptor we have shown that the intracellular effects of amphetamine, including both the activation of PKA and RhoA, depend absolutely upon the presence of TAAR1. We have continued to explore the role of TAAR1 as an intracellular target for amphetamines and have shown that similar intracellular events regulate the trafficking of transport proteins in serotonin and norepinephrine neurons. We have recently expanded our efforts to consider the mechanisms that regulate EAAT3 trafficking to and from the cell surface after amphetamine treatment, and how these events affect the clearance of glutamate. Using transgenic mouse and rat lines we have now shown that deletion of the EAAT3 gene selectively in dopamine neurons blocks the increased locomotor activity seen after AMPH or METH administration, underscoring the importance of EAAT3 and the neurotransmitter glutamate in the behavioral actions of AMPHs. To better examine the proteins that regulate and shape the itinerary of EAAT3 as it is internalized from the neuronal cell surface after AMPH administration, we used a proximity labeling technique that fuses an enzyme (a biotin ligase) to EAAT3 and enables labeling of protein complexes that associate with EAAT3 in different subcellular compartments. The proteins that are tagged with biotin by the ligase can be purified and their sequences determined by mass spectrometry. Over a hundred proteins have been identified, many are components of the membrane protein trafficking machinery and regulatory pathways. Current efforts focus on defining the complexes, confirming the interactions, and identifying the subcellular compartments where the regulatory events take place. While our work has focused on the presynaptic effects of amphetamine-related compounds on TAAR1 signaling, efforts by drug companies have led to the identification of several new TAAR1 ligands that may prove therapeutically useful in psychiatric disorders. One novel TAAR1 agonist from Sunovion, SEP-363856 (Ulotaront), was identified in a phenotypic behavioral screen for antipsychotics and received breakthrough therapy status from the FDA for the treatment of schizophrenia. It proved promising in Phase 2 clinical trials, although Phase 3 results in humans to date have been inconclusive. Unlike current antipsychotics that mainly target D2 dopamine receptors, SEP-363856 is an agonist at TAAR1 and serotonin 5-HT1A receptors, making it a new potential therapeutic class for schizophrenia. We have examined the effects acute administration of SEP-363856 on intracellular signaling and found that it also activates the same signaling pathways that are activated by other TAAR1 agonists, such as amphetamine and MDMA, but does so in a broader range of neuronal cell types. Because we have learned that AMPHs activate TAAR1-dependent signaling pathways within dopamine neurons, we wondered whether these signals could also activate the expression of specific genes within the nucleus of the neuron. In initial experiments, we used transcriptomic analyses of RNA isolated from single nuclei from midbrain or from actively translating ribosomes in DA neurons to identify groups of genes that are upregulated or downregulated by the acute administration of AMPH or METH. Unexpectedly, we found that acute methamphetamine exposure leads to robust upregulation of cholesterol metabolism genes in dopaminergic neurons of the ventral tegmental area and substantia nigra—a response that was largely absent in other midbrain cell types. Further analysis using a ribosome tagging strategy combined with RNA sequencing revealed that like METH, AMPH also induced similar changes, whereas methylphenidate (Ritalin), another drug used to treat ADHD did not, highlighting drug-specific transcriptional responses. In addition, we found that dopaminergic neurons are highly enriched for genes in the cholesterol synthesis pathway, including the rate-limiting enzyme for cholesterol synthesis and target for statin drugs, HMG CoA reductase, the LDL receptor, and the key cholesterol pathway regulators, Srebf2 and Insig1, challenging the prevailing view that neurons rely mainly on astrocyte-derived cholesterol. These results imply dynamic changes in cholesterol metabolism dopaminergic neurons in response to AMPHs and uncover the potential importance of cholesterol homeostasis for dopaminergic neuron function.