Date of Award

5-1-2024

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Dissertation/Thesis Chair

Li Niu

Committee Members

Alexander Shekhtman, Jia Sheng, Mehmet Yigit, Hua Shi

Keywords

kainate receptors, mutational analysis, RNA aptamers, SELEX

Subject Categories

Biochemistry

Abstract

Ionotropic glutamate receptors (iGluRs) play a major role in mediating the excitatoryfunctions in the central nervous system (CNS). iGluRs are ligand-gated ion channels that are activated when glutamate binds to the extracellular ligand binding domain, thereby leading to the opening of the ion channels. Three subtypes of iGluRs include kainate receptors, α-amino-3- hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, and N-methyl-D-aspartate (NMDA) receptors. Abnormal and dysregulation of these receptors have cytotoxic effects for neurons and have been implicated in various neurological diseases. For instance, elevated expression of a key AMPA receptor subunit has been linked to conditions such as ALS, where cytotoxic levels of Ca2+ ions enter the motor neurons, triggering cell death. Downregulations of iGluR surface expression has been linked to learning and mood disorders. Auxiliary proteins, such as transmembrane AMPAR regulatory proteins (TARPs) for AMPA receptors and neuropilin and tolloid-like (Neto) proteins for kainate receptors respectively, also play an important role in regulating receptor surface expression and modulating receptor activities. Developing a set of regulatory options that could control these abnormal receptor activities would be therapeutically beneficial. Small molecule compounds have been developed over the past decades to modulate iGluR activity. These compounds, however, generally have low water solubility and low selectivity to differentiate subtypes, let alone subunits. In clinical settings, the use of these compounds has shown significant sides effects but no or low therapeutic efficacy. In particular, the development of modulators for kainate receptors lags far behind in the iGluR field, as compared with the development of modulators for either AMPA or NMDA receptors. For 2 example, virtually all compounds that have developed to date have higher selectivity towards the GluK1 kainate receptor subunit. In other words, no selective modulators have been developed towards the GluK2 subunit. Yet, the GluK2 kainate receptor subunit is the most prevalent kainate receptor subunit found throughout the brain. My doctoral thesis work concerns the development of RNA-based modulators of kainate receptors. Below, I will discuss how I used two different methods in developing RNA aptamers with higher potency and higher selectivity for kainate receptors, as compared with traditional, small molecule compounds. In Chapter 1, I will describe the classic use of systematic evolution of ligands by exponential enrichment (SELEX) in developing RNA aptamers for kainate receptors. Two separate SELEX runs were performed: one with homomeric GluK2 channels and the other with heteromeric GluK2/K5 channels (kainate receptors have 5 subunits, and GluK2/K5 is a major kainate receptor complex found in vivo). Both SELEX runs included the addition of both Neto1 and Neto2 proteins to increase the chance of finding selective RNA aptamers. To further increase the chance of finding useful aptamers that may have low copy numbers, the DNA libraries from individual rounds of SELEX operation were analyzed using Next-Generation Sequencing (NGS) approach, instead of traditional Sanger sequencing technique, which limits the number of DNA clones that could be detected and analyzed. In particular, I will describe that among all the new RNA aptamers I have isolated, S-A94, a 94 nucleotide (nt)-long RNA aptamer, is a kainate receptor aptamer: whole-cell current recording showed that S-A94 selectively potentiates the closed-channel GluK1 without affecting GluK2. Through mutational analysis, I found that the minimal, but fully functional, length of this aptamer is a 52 nt RNA. 3 In Chapter 2, I will describe a new method in developing highly selective RNA aptamers, including a GluK2 subunit-selective aptamer. This work was based on a previously identified RNA aptamer by our group, termed AB9s-b, which was the first molecular agent capable of inhibiting GluK1 and GluK2 equally potently. In the sequence of AB9s-b, it was discovered that a segment of RNA sequence is responsible for kainate receptor inhibition. This provided a unique platform to mutate this known sequence so that hopefully a mutated AB9s-b would exhibit an improved selectivity such that the newly developed RNA aptamer would become GluK2-selective without any cross activity towards any other subunits of iGluRs. Two sets of AB9s-b mutants were created. The first set consisted of sequence mutants in which three-base sequence segments were changed while maintaining the original secondary structure of AB9s-b. The second set were structural mutants where the sequence was maintained while the secondary structure was changed. The function of all the mutants were determined through whole-cell current recording using HEK-293 cells that express individual iGluRs. Between these two groups of mutants, a wide variety of aptamers with varying selectivity between GluK1 and GluK2 have been created. Of note is 3sb-U9 aptamer. 3sb-U9 was one of the sequence mutants with the loop of the hairpin motif mutated from the original AB9s-b mutant. 3sb-U9 was the first modulator with single subunit selectivity in that it uniquely potentiates GluK2 without any other cross activity for any other kainate subunits or any other AMPA or NMDA receptors. No molecular modulator with such a subunit selectivity has ever been reported in the field. Furthermore, with the help from molecular docking simulations, we have gained some understanding of how the interaction of 3sb-U9 with the GluK2 vs other kainate and AMPA receptors may have endowed this aptamer to be uniquely selective. In summary, my research has been able to develop new 4 methodology and identify new RNA aptamers for regulating kainate receptors. My work has shown that developing RNA aptamers is a promising approach to address the challenge of developing subunit-selective kainate receptor modulators, which has thus far defied the field.

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