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Measuring D-Amino Acids in the Central Nervous System Using Capillary Electrophoresis

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Measuring D-Amino Acids in the Central Nervous System Using Capillary Electrophoresis MEASURING D-AMINO ACIDS IN THE CENTRAL NERVOUS SYSTEM USING CAPILLARY ELECTROPHORESIS BY TING SHI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2012 Urbana, Illinois Doctoral Committee: Professor Jonathan V. Sweedler, Chair Professor Ryan C. Bailey Professor Rhanor Gillette Professor Wilfred A. van der Donk Abstract Understanding what molecules are used by neurons and glia for cell-to-cell signaling is important as this knowledge provides insights into neuronal network function within the brain. Even after decades of research, novel signaling molecules remain to be uncovered in the nervous system. Since their discovery in the central nervous system, D- amino acids (D-AAs) have drawn intense research interest because several of them are involved in signal transmission processes and brain development. However, a lack of appropriate analytical tools has prevented more refined studies to elucidate the roles of D-AAs in intercellular signaling. Here new and improved analytical platforms are described with the aim of sampling the complex neuronal microenvironment, accomplishing chiral separations and quantifying low abundant D-AAs reliably. Among D-AAs found across the metazoan, two D-AAs are studied extensively, namely D-serine (D-Ser) and D-aspartate (D-Asp). D-Ser can be synthesized endogenously by serine racemase which converts L-Ser to D-Ser. D-Ser can affect the activity of N-methyl-D-aspartate (NMDA) receptor, which is an important receptor involved in learning and memory formation. D-Asp levels correlate with embryonic development stages and D-Asp concentrations drop to much lower levels in adult animals. Through the past ten years of studying D-Asp within Sweedler group, we have determined its neuronal localization, biosynthesis, transport, and demonstrated several of the physiological responses it elicits within Aplysia californica (A. californica). Capillary electrophoresis (CE) is an ideal method to characterize D-AAs in the central nervous system due to the compatibility of CE with small size sample and the low detection limits it provides when coupled with laser-induced fluorescence (LIF) as a ii detection method. Through a variety of CE measurements, two main questions are answered in this thesis. First, is D-Asp released from A. californica neurons and is it released in a stimulation-dependent manner? Second, is D-Ser released from glia via a vesicular release pathway? The first question was answered by selectively extracting and quantitatively measuring the extremely diluted D-Asp from the releasates of A. californica ganglia. Our results indicate a stimulation-dependent release of endogenous D-Asp, which is the last criterion needed to be satisfied to show that D-Asp meets the requirements of a traditional neurotransmitter within A. californica. The second question was addressed by quantifying the amino acid contents in intact and leaked glial vesicles. Various CE methods were incorporated to validate the quantitation results. These analytical measurements clearly show the enrichment of D-Ser, along with another signaling molecule L-glutamate (L-Glu), inside glial vesicles. Together with the results of biochemical assays from our collaborator, our data demonstrate D-Ser being stored in glial vesicles and released from glia via vesicular release pathway. iii To my parents and my fiancé iv Acknowledgements I would like to thank my advisor and mentor, Professor Jonathan V. Sweedler, for his support and invaluable guidance over the last five years. It has truly been a privilege and honor of mine to be one of his students. He is one of the most intelligent, passionate, knowledgeable and considerate scientists I have ever encountered. I have learned far more important values than knowledge from him, which is by no means short. I have learnt in my childhood an old Chinese axiom, that a day as a mentor constitutes a lifetime of parenthood. Jonathan had me really believe in it. I am also deeply thankful to the other members of my committee, Dr. Wilfred A. van der Donk, Dr. Ryan C. Bailey and Dr. Rhanor Gillette for their helps throughout the course of my graduate studies. The knowledge they have taught me in graduate courses, as well as many insightful suggestions on my research have been of incalculable value in this process. I have had the wonderful opportunity to work with a number of amazing collaborators on very interesting projects. I especially would like to thank Professor Jean-Pierre Mothet from Universite Aix-Marseille for letting me have the opportunity to participate in such an international collaborated project that involves many top scientists in different fields. I appreciate the collaboration with Dr. Magalie Martineau, the former student of Professor Mothet, who is very knowledgeable and intelligent. Besides off- campus collaboration, I also had the opportunity to work closely with Dr. Ann M Knolhoff and Nobutoshi Ota in Sweedler lab on a variety of challenging separation and measurement tasks. The dedication and talents of all my collaborators have contributed tremendously to the success of my Ph.D. project. v Over the years in Sweedler lab, I was really fortunate to be surrounded by brilliant colleagues who have been always willing to lend a hand when needed. In particular, I would like to give my special thanks to Dr. Fang Xie who provided initial orientation when I joined the group and is a very good friend throughout and after my graduate career. I am also very grateful to Dr. Stanislav Rubakhin and Dr. Elena Romanova, who spent a lot of time training me on dissection and provided samples for me. Additionally, my thanks would go to my past and present incredible officemates at Beckman Institute 2620, where I spent almost all my time in the past five years. I have greatly enjoyed working with every single member in Sweedler group and I feel proud to be one of this remarkable team. Finally, my whole-hearted thanks go to my loving family. My fiancé, Dr. Zhou Li, has made my Ph.D. journey not only possible, but also enjoyable by always giving his unconditional love, continuous encouragement and inexhaustible support. Without him by my side, I would not have come so far. I would like to sincerely thank my parents, Jianhua Luo and Xudong Shi, for always believing in me, being proud of me and encouraging me whenever, wherever I needed. vi Table of Contents List of Figures ..................................................................................................................... x List of Tables ................................................................................................................... xiii Chapter 1 Introduction .................................................................................................... 1 1.1 Research summary .................................................................................... 1 1.2 Neurochemistry and signaling molecules ................................................. 2 1.3 Capillary electrophoresis ........................................................................... 3 1.4 Overview of thesis ..................................................................................... 4 1.5 References ................................................................................................. 7 Chapter 2 Measuring Endogenous D-Amino Acids in Brains ...................................... 12 2.1 Introduction ............................................................................................ 12 2.2 Separation based techniques.................................................................... 14 2.3 Non-separation based techniques ............................................................ 26 2.4 Clinical and pharmaceutical applications ................................................ 32 2.5 Conclusions ............................................................................................ 34 2.6 References ............................................................................................... 36 2.7 Tables and figures ................................................................................... 51 Chapter 3 Investigating D-Aspartate Release in the Aplysia Californica Central Nervous System ................................................................................................ 60 3.1 Introduction ............................................................................................. 61 vii 3.2 Experimental procedures ......................................................................... 63 3.3 Results ..................................................................................................... 67 3.4 Discussion ............................................................................................... 72 3.5 Conclusions ............................................................................................. 73 3.6 References ............................................................................................... 74 3.7 Tables and figures ................................................................................... 81 Chapter 4 Measuring D-Serine in Astrocytic Synaptic-like Vesicles Using Capillary Electrophoresis ................................................................................................. 92 4.1 Introduction ............................................................................................
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