Date of Award

5-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xii, 99 pages) : color illustrations.

Dissertation/Thesis Chair

Li Niu

Committee Members

Alan Shekhtman, Alan Chen, Mehmet Yigit, Haijun Chen

Keywords

Kainic acid, Glutamic acid, Neural transmission, Neurotransmitter receptors, RNA

Subject Categories

Biochemistry | Neuroscience and Neurobiology

Abstract

Glutamate receptors act to bring about excitatory transmission in the central nervous system. The receptors are divided into two groups: ionotropic and metabotropic glutamate receptors. Ionotropic glutamate receptors are ion channels which are activated by an agonist such as glutamate or kainate. The main receptors in the ionotropic glutamate receptor family are the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors. In the central nervous system ionotropic glutamate receptors are found both pre- and postsynaptically. It has been found that most AMPA and NMDA receptors are postsynaptic receptors while the kainate receptors can be pre- or postsynaptic. Underactivity of these receptors has been implicated in many neurological disorders such as depression, learning disabilities, and Alzheimer’s disease (AD). Potentiators of these receptors can be drug candidates for these diseases. For this reason, developing potentiators which specifically target these receptors is important. The work detailed in this thesis has been done to develop an RNA aptamer which potentiates AMPA and kainate receptors. Currently, small molecule potentiators are being synthesized to potentiate AMPA receptors. Because of low solubility and low selectivity, many of these compounds have failed drug trials and have been deemed to be unsafe to use. In order to find a selective, soluble RNA aptamer which potentiates AMPA and kainate, a systematic evolution of ligands by exponential enrichment (SELEX) study was performed. This is an in-vitro evolutionary method which allows us to isolate RNA sequences (aptamers) which bind specifically to the receptor and may potentiate the receptor. My use of SELEX against GluA1 will be described in chapter 1, where I detail how I was able to isolate an RNA aptamer, AL3, which potentiates both AMPA and kainate receptors. To find the shortest active sequence for the potentiator, I truncated the AL3 aptamer. The shorter aptamer, named AL3-1, lost activity against AMPA receptors, but specifically and potently potentiates kainate receptors. This was an interesting discovery because research on kainate receptors has been slow, due to the lack of kainate receptor-specific potentiators. The shorter aptamer, AL3-1 has 69 nucleotides, while the longer AL3 has 101 nucleotides. The shorter sequence potentiates GluK1 and GluK2 more potently than the longer sequence does. Both receptors showed no activity on NMDA receptors. Additionally, this work is important because it supports the most recent work of others from this lab showing that aptamers may bind to different receptors with different moieties, and by separating these moieties of the aptamer, a different selectivity profile can be achieved. Potentiation is an increase in channel conductivity, which can be accomplished in a few different ways. The aptamer most likely potentiates the receptor by opening the channel wider to allow more ions to flow in. Both AL3 and AL3-1 increase the amplitude of channel conductivity while not affecting the steady state, or desensitization of the receptor. The mechanism of most small-molecule potentiators is either slowing desensitization or deactivation of the receptor. Drugs which potentiate in this way are seizurogenic, and many potentiators that work in this way are toxins. However in preliminary tests, small molecule potentiators have been shown to increase memory and learning. The aptamer may be a safer alternative to these small molecule drugs. In order for RNA aptamers to be functional, chemical modification must be made. I chemically modified a previously found aptamer, FN58, and my own aptamer FL3-1 with 2’-F nucleotides for A, C, and U. This is necessary because the unmodified RNA is quickly degraded in the presence of ubiquitous RNAse. The 2’ F modification replaces the 2’ OH in all A, C and U nucleotides in the sequence, rendering a more stable aptamer. It has been previously shown in this lab that the 2’-F modified nucleotides have half-lives in the order of days, rather than minutes, for unmodified nucleotides. In chapter 2, I describe a new method of increasing the transfection yield of green fluorescent protein in HEK-293 cells. In this experiment, I investigated the application of DMSO after transfection. HEK-293 cells are one of the most commonly used cell lines behind CHO cells and HeLa cells. These cells have been particularly useful in electrophysiology and neuropharmacology because they are not typical kidney cells, but contain genetic signatures of adrenal and neurologic cells. Also they are small and faithfully translate membrane proteins, such as glutamate receptors. Some previous studies have shown that brief applications of DMSO have increased protein yields in different cell lines, but the optimum concentration of DMSO and optimum exposure time seemed to be cell-line dependent. No previous studies have been done on the use of DMSO in HEK-293 cells to improve transfection yield. In my experiment, I found that a brief application of DMSO increases protein yield, and the optimum concentration of DMSO as well as the optimum exposure time for HEK-293 cells were determined. To quantify transfection efficiency, green fluorescent protein was transfected. The green cells were imaged and classified according to intensity of green, which indicates transfection yield. It was found that a 10% DMSO solution applied for 5 minutes gives approximately 1.7- fold increase in intense green cells. Also, a brief exposure is not noticeably toxic to the cells. To ensure that the DMSO does not affect channel kinetics in glutamate receptors, a whole cell recording study was performed. This study showed that the channel conductance was not significantly different between cells that were treated with DMSO and hypothermia and those which were not treated. This technique is useful in our lab because much of our research involves transfecting glutamate receptors in HEK-293 cells.

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