Defense Date

2022

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Sammanda Ramamoorthy

Abstract

Amphetamine (AMPH) has been used clinically and recreationally around the world for years. A 2021 study reports a 67.5% rise in AMPH use from 2010-2017 across all fifty states. Study also shows that 30% of patients with AMPH-induced psychosis end up with primary psychosis over time. AMPH works by inhibiting monoamine (Norepinephrine (NE), Serotonin (5-HT), and Dopamine (DA) reuptake followed by triggering transporter mediated monoamine release and consequently increases monoamine concentrations in extracellular synaptic space. AMPH also works as a substrate for monoamine transporters, enters the neuron, and once its inside the neuron, it will prevent the storage of monoamines in vesicles by inhibiting vesicular monoamine transporter 2 (VMAT2). AMPH also regulates monoamine transporter surface expression by affecting transporters trafficking. Creating this neurochemical imbalance corrupts the normality of central nervous system function and results in reduced monoamines in vesicles. Because of the neurochemical imbalance AMPH causes, there has been no drug to fully treat and remain effective on abstinence. Recreational use of AMPH and Methamphetamine (METH) can cause addiction which can eventually lead to death. Since monoamine transporter is a phosphoprotein, various kinases such as protein kinase C (PKC) downregulates the transporters. An in vitro study was done with mutated T258A and S259A motif in norepinephrine transporter (NET) sequence to see the double mutant (DM) transporter attenuating the effects of AMPH. The results showed low AMPH-mediated downregulation of DM as well as resistance to AMPH-induced endocytosis of NET. In rats, same motif was injected into intra-accumbal region by using membrane permeable TAT-NET peptide and the results indicate that TAT-peptide containing NET-T258/S259 motif blocked AMPH-mediated NET inhibition. Similarly, AMPH-induced conditioned place preference (CPP)/ reinstatement and locomotor sensitization was attenuated by NET-T258/S259. Since human DAT and NET have similar sequence homology especially in intracellular loop 2 (ICL2), and AMPH affects all three transporters by the same mechanism, we explored the AMPH-mediated regulation of DAT by mutating the phosphorylation sites threonine 261 and serine 262 to non-phosphorylatable alanine in hDAT. We examined the mechanisms involved in AMPH-induced DAT regulation in human embryonic kidney cells (HEK-293) expressing wild-type (WT)-hDAT or double mutant (DM)-hDAT. Our uptake experiment reveals that treatment of AMPH on cells carrying WT-hDAT results in decrease in hDAT-mediated DA uptake while DM-hDAT carrying cells blunted the effects of AMPH. The kinetic analysis of DA uptake in HEK-293 cells transfected with hDAT showed that AMPH treatment resulted in a significant decrease in the maximal velocity (Vmax) in WT-hDAT and a non-significant change in the case of DM, while not affecting the affinity (Km) of DA for DAT. Our surface biotinylation studies showed decrease cell surface density of hDAT in AMPH-treated cells expressing WT-hDAT but not DM-hDAT. Therefore, our findings with previous results demonstrate that T261 and S262 are involved in AMPH-mediated DAT downregulation.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-12-2022

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