Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2001 Astrocytes: a driving force in brain signaling and encephalopathies Aleksandar Jeremic Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Neuroscience and Neurobiology Commons, and the Neurosciences Commons Recommended Citation Jeremic, Aleksandar, "Astrocytes: a driving force in brain signaling and encephalopathies " (2001). Retrospective Theses and Dissertations. 648. https://lib.dr.iastate.edu/rtd/648 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Astrocytes: A driving force in brain signaling and encephalopathies by Aleksandar Jeremic A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Co-majors: Veterinary Anatomy; Neuroscience Program of Study Committee: Srdija Jeftinija, Co-major Professor Anumantha Kanthasamy, Co-major Professor Lloyd Anderson Walter Hsu Mary Helen Greer Donald Sakaguchi Iowa State University Ames, Iowa 2001 UMI Number: 3034192 UMI UMI Microform 3034192 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 u Graduate College Iowa State University This is to certify that the doctoral dissertation of Aleksandar Jeremic has met the dissertation requirements of Iowa State University Signature was redacted for privacy. C<vmaj Signature was redacted for privacy. o-major Profess Signature was redacted for privacy. For the G -r jor Progra Signature was redacted for privacy. For he Co-maj r P iii To my wife Jelena and Luka & Mina iv TABLE OF CONTESTS DEDICATION iii ABBREVIATION TABLE vi ABSTRACT viii CHAPTER 1. GENERAL INTRODUCTION 1 Thesis Organization 1 Literature Review 2 CHAPTER 2. ATP STIMULATES CALCIUM-DEPENDENT GLUTAMATE RELEASE FROM CULTURED ASTROCYTES 15 Abstract 15 Introduction 16 Materials and Methods 18 Results 21 Discussion 45 Acknowledgment 48 References 48 CHAPTER 3. CANNABINOID-INDUCED ASTROTOXICITY 55 Abstract 55 Introduction 56 Materials and Methods 57 Results 61 Discussion 76 Acknowledgment 82 References 83 V CHAPTER 4. ASTROCYTE ACTIVATION AND APOPTOSIS INDUCED BY gpl20 AND SDF-la ARE MEDIATED BY CXCR4 RECEPTOR 91 Abstract 91 Introduction 92 Materials and Methods 94 Results 97 Discussion 125 Acknowledgment 130 References 130 CHAPTER 5. GENERAL CONCLUSIONS 136 General Discussion 136 Summary 144 REFERENCES 149 ACKNOWLEDGMENTS 167 vi ABBREVIATION TABLE a-MEM-a-minimum essential medium ATP- adenosine 5'tri-phosphate Ach-acetylcholine Asp-aspartate AO-acridine orange B APT A-AM-1,2-bis- ethane-N,N,N\N'-tetracetic acid-acetoxymethyl ester Ca2+-calcium [Ca2+]j -intracellular calcium concentration CI -chloride CNS-central nervous system DAG-1-Oleoyl-2-acetyl-sn-glycerol D-AP5-D(-)-2-Amino-5-phosphonovaleric acid EAA-excitatory amino acids EGTA-ethylene glycol-bis-N,N,N' ,N* -tetraacetic acid GFAP-glial fibrillary acidic protein Glu-glutamate HCO3 -bicarbonate Hepes-(N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) IP3-inositol 1,4,5-trisphosphate K+-potassium Vil MTT-[3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide Na+-sodium NPPB-5-nitro-2(3-phenylpropylamino)-benzoate PCMPS- phenylsulfonic acid PDC-L-/razis-pyrolidine-2,4-dicarboxylate PI-propidium iodide PNS- peripheral nervous system PTX-pertussis toxin RNase-ribonuclease ROS-reactive oxygen species SDF-la- stromal cell-derived factor 1 a THC-A9-tetrahydrocannabinol WIN-Win 55,212-2 viii ABSTRACT For decades, astrocytes, the most numerous cell of the central nervous system, were considered to be passive supporters of nerve cell architecture and function, lacking excitability and having no role in signal integration. However, recent findings have progressively challenged that view, highlighting the active participation of astrocytes in synaptic transmission and information processing. This dissertation presents evidence for the major contribution of astrocytes in the modulation of neuronal excitability that is in large part mediated by the excitatory neurotransmitter glutamate. Neuroligands, ATP and SDF-la released glutamate from astrocytes that stimulated adjacent neurons by the NMDA-receptor activation. Furthermore, rapid bi-directional signaling loops between the neurons and glia have been revealed in experiments performed on co-cultures. In addition to its fundamental physiological importance, this dissertation postulates a new role for astrocytes in brain pathology. Cell culture experiments revealed a direct toxic effect of neurotoxins, gpl20, an HIV-1envelope glycoprotein, and cannabinoids on astrocytes. A propensity of astrocytes to undergo apoptosis and to release excitotoxic glutamate in response to insult elicited by gpl20 and cannabinoids suggests that, along with neurons, astroglia are an important site of action for neurotoxins in brain. Potential mechanisms of cannabinoid toxicity include formation of ceramide and free radicals and activation of several primary and secondary effector enzymes. An indirect contribution of astrocytes to the gpl20 and cannabinoid-induced neuron injury is implied with this study. Potential mechanisms include disturbances of glia function and their ability to support ix neurons and the astrocytes-mediated excitotoxins formation in the brain. This part of the dissertation gave well-deserved respect to the issue of cannabinoid and gpl20 astrotoxicity because a better understanding of the pathogenesis of astrocytes in encephalopathies may have important therapeutic implications in the future. 1 CHAPTER 1. GENERAL INTRODUCTION The mammalian nervous system is composed of more than 100 billion neurons surrounded by glial cells. Glial cells outnumber the neurons 10-50:1. A generally accepted view is that glial cells are nothing more than passively subservient to neurons. The development and advances in monitors of intracellular ion concentration open a new field of cellular physiological investigation. With these technical advances, studies show that glial cells are much more complex entities than had been appreciated. The focus of this dissertation is twofold: first, the study of the effect of neuroligands, ATP and SDF-la, on astrocytes in order to further our understanding of astrocytes in brain function and second, the study of the effect of neurotoxins on astrocytes and the mechanism of astrotoxicity. Thesis organization This dissertation is composed of three journal papers. Each journal paper compromises a single chapter of the dissertation. The papers are preceded by a general introduction chapter containing a literature review, and followed by a general conclusion chapter consisting of a general discussion section and summary. References from all chapters are listed in a comprehensive listing at the end of the dissertation. 2 Literature Review Neuroglia Glial cells were first described in 1846 by German pathologist Rudolf Virchow who named them neuroglia (Greek, meaning nerve glue). In the vertebrates, glia can be divided into four major categories. In the peripheral nervous system (PNS) are the Schwann cells, and in the central nervous system (CNS) are the microglia, oligodendrocytes and astrocytes. Anatomists have divided normal astrocytes in the mature brain into two broad categories: fibrous astrocytes, which contains abundant intermediate filaments and are predominant in white matter, and protoplasmic astrocytes, which contain fewer filaments and are predominant in gray matter. The glial filaments are composed of glial fibrillary acidic protein (GFAP), a specific histochemical marker of astroglia. A third class known as reactive astrocytes appears following injury