Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Gonzalo Torres

Committee Members

Itzhak Mano

Susana Mingote

Nesha Burghardt

Gary Mawe

Subject Categories

Biology | Molecular and Cellular Neuroscience | Neuroscience and Neurobiology | Substance Abuse and Addiction

Keywords

Serotonin, monoamines, psychostimulants, amphetamine, efflux, monoamines transporters

Abstract

Serotonin is a vital neurotransmitter and hormone with significant roles in almost every organ system. In the central nervous system, serotonin mediates physiological functions that in turn guide behavior and mood. Here, serotonin is released from serotonergic neurons and exerts its effects through serotonin receptors. Regulation of serotonin neurotransmission is important for the maintenance of its physiological functions; thus, extracellular serotonin must be sequestered to limit the intensity and duration of serotonin transmission. Disproportionate transmission is strongly linked with neurological and psychiatric ailments.

Extracellular serotonin levels are primarily mediated by the serotonin transporter (SERT), a critically important plasma membrane protein that is highly regulated. Serotonergic signaling is terminated in a process known as uptake (i.e., recycling serotonin from the extracellular space to the presynaptic neuron). Due to its dynamic nature, SERT may also act as a secondary mode for serotonin release through a process known as efflux. As neurotransmitter release traditionally occurs due to calcium-dependent exocytotic vesicular release from the presynaptic cell, the mechanism underlying neurotransmitter release by transporter protein remains poorly understood. Much of the current knowledge regarding serotonin efflux through SERT is due to the actions of amphetamines, important therapeutics but also potent drugs of abuse. Amphetamines are exogenous SERT substrates which promote the efflux of serotonin. Several second messenger systems including proteins, kinases, and phospholipids interact with SERT. These interactions often lead to functional changes in SERT activity and impair the actions of amphetamines. These signaling mechanisms are often heavily involved in the signaling cascade of G protein coupled receptors (GPCRS).

In order to gain a deeper understanding of SERT mediated serotonin efflux, we sought to investigate a new interaction between SERT and the Gbg subunits of GPCRs. After confirming an interaction between Gbg and SERT through co-immunoprecipitation and GST-pulldown assays, we went to determine if this interaction leads to a functional change in SERT activity in both a heterologous and ex vivo brain slice model. Our results indicate that activation of Gbg results in serotonin release in heterologous cells overexpressing SERT and potentiates the effects of amphetamine while inhibition of Gbg drastically reduced the effects of amphetamine in rodent brain tissue. Based on these data, we propose that there is an endogenous GPCR that, when activated, may promote endogenous 5HT release through SERT. Overall, this work has both profound effects on not only basic science but clinical improvements for the treatment of 5HT-ergic disorders.

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