Year of Award

2020

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Neuroscience

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Michael P. Kavanaugh

Commitee Members

Richard Bridges, Kasper Hansen, Nathan Insel, Travis Hughes, Leonid Kalachev

Keywords

ASCT1, asymptotic analysis, D-serine, EAAT, glutamate, NMDAR

Abstract

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Glutamate release and reuptake control both tonic activation of glutamate receptors and phasic signaling during synaptic transmission. Here we study different aspects of the system. The first three chapters focus on the most abundant glutamate transporters found in the brain, excitatory amino acid transporters 1-3 (EAAT1- 3/SLC1A3,2,1). The last chapter is devoted to a paralogous neutral amino acid transporter (ASCT1/SLC1A4) and its role in transport of D-serine, a coagonist of the N-methyl-Daspartate (NMDA) glutamate receptor.

In the first chapter, we discuss the existing discrepancy in estimates of EAAT1-3 turnover rates in the research literature. We talk about the general approach used for the data analysis and its pitfalls. We use a boundary function method to construct a reduced model that describes the kinetics of EAAT1-3, and we use it to derive an independent and statistically reliable estimate of turnover rates (15, 24, and 2 glutamate molecules per second for EAAT1-3, respectively). We discuss and compare our results to the ones existing in the literature. We create a 3-D diffusion model of synaptic transmission using these turnover rates, as a proof of concept. We show that the simulation is consistent with previously reported time-course of glutamate in the synaptic cleft.

The second chapter is devoted to studying the efficacy of the transporters and their ability to create a significant diffusion gradient near the membrane surface. We continue the discussion from the previous chapter about the importance of an ambient glutamate level. There is a 100-fold discrepancy in the glutamate concentration estimates between two different methodologies in the literature. Electrophysiological studies are consistent with 25-87 nM glutamate concentration, while microdialysis studies suggest >2 μM glutamate concentration. We address this issue by constructing another mathematical model that provides a potential explanation for such discrepant results.

In the third chapter, we focus on a novel photosensitive EAAT blocker, an azobenzene derivative of TFB-TBOA named azo-TFB-TBOA (ATT). The drug rapidly shifts between its cis- and trans- conformations upon the stimulation with 350nm or 450nm wavelength light, respectively. The drug's affinity for each EAAT subtype is also changed with its conformational structure.

In the fourth chapter, we focus on D-serine homeostasis, an endogenous NMDA receptor coagonist. We investigate ASCT1 transporter (also known as SLC1A4) and propose it to be the prime source of extracellular D-serine in the brain. We also study a new ASCT1/SLC1A4 blocker, 3-biphenyl-4-hydroxyproline (BiPro), as potentially valuable research and therapeutic tool.

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© Copyright 2020 Denis Mikhailovich Shchepakin