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EXCITOTOXIC MECHANISMS IN HUNTINGTON'S DISEASE by MIT LIBRARIES JU N 09 1992 Andrew Freese SCHERING Submitted to the Division of Health Sciences and Technology in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY November, 1991 Copyright (c) 1991, Massachusetts Institute of Technology Signature of Autthor__ - _ , ,- r,~- ~ ,_r1__ , ,,...C Division of Health Sciences and Technology November, 1991 Certified by G.E. Schneider, Thesis Sponsor J.B. Martin, Thesis Advisor M. DiFiglia, Thesis Advisor A Accepted by by_MASCUET _ _ __ _ Chairman,___ _ _ _____,T MASSACHUSETTS INSTNTE Chairman, Department Committee OF TECHNIOI OGY J N',0 7 1992 LIBRARIES "-.r. _ . ^ 1-. a.1 1f. "This doctoral thesis has been examined by a Committee of the Division of Health Sciences and Technology as follows:" Professor William Thilly Chairman Professor Gerald Schneider Thesis Snsor Professor Joseph Martin ..... _ f .. -- Thesis Advisor J..__ Professor Marian DiFiglia Thesis Advisor - - Professor Helmut Zarbl -C---~----'~ it / Professor Edward Bird U- - - - - I - 1dl I' I I Abstract Acting at the N-methyl-D-aspartate (NMDA) excitatory amino acid receptor, a variety of endogenously synthesized excitotoxic compounds, including glutamate and quinolinic acid (QUIN), may play a role in the pathogenesis of a number of neurodegenerative diseases, including Huntington's Disease. When injected into the corpus striatum of animals, QUIN produces a neurochemical pattern of neuronal degeneration strikingly similar to that seen in the striatum of a Huntington's Disease victim. The goal of this Ph.D. thesis is to identify mechanisms of neuronal degeneration in the striatum induced by excitotoxins using both an in vitro tissue culture system and in vivo systemic manipulations. In primary striatal cultures derived from neonatal rats, examination of the mechanisms of excitotoxicity focused on studies determining the relationships among activation of excitatory amino acid receptors (especially the NMDA receptor), subsequent ion fluxes into the cell, modulation of toxicity by outside factors and second messenger systems, and the induction of cell death. In addition, examination of the neurochemical correlates of cell death and pharmacological approaches towards reducing excitoxicity in these cultures was performed. Using in vivo systemic manipulations, the dramatic dependence of QUIN on its biosynthetic precursor, tryptophan, was determined. In turn, this observation was the basis of an attempt to perturb physiological controls of this biosynthetic pathway to develop a systemic model for Huntington's Disease. However, it was shown that brain QUIN biosynthesis downregulates in the presence of chronic elevations of its precursor. In contrast, brain levels of the excitatory amino acid receptor antagonist, kynurenic acid (KYA), another metabolite of tryptophan, remain elevated, suggesting that precursor-based therapy for Huntington's Disease might be achieved. In turn, the neuroprotective effect of precursor therapy was demonstrated. Implications of these results for the therapy and understanding of Huntington's Disease are discussed. Thesis Advisors: Professors Joseph B. Martin and Marian DiFiglia Massachusetts General Hospital Thesis Sponsor: Professor Gerald E. Schneider Massachusetts Institute of Technology 2 Acknowledgements I want to thank all of the members of my thesis committee for their advice and encouragement. In particular, I thank my thesis advisors, Marian DiFiglia and Joe Martin, for letting me join their productive and noble research efforts towards finding a cause and cure of Huntington's Disease, and my thesis sponsor, Professor Gerald Schneider, for constructively assessing and supporting the progress of my research work. I am indebted to the Chairman of my thesis committee, Bill Thilly, who has become a mentor and friend. I greatly appreciate the input and guidance provided by the additional members of my thesis committee, including Professors Helmut Zarbl and Edward Bird. I am indebted to Professors Richard Kitz and Roger Mark, who co-directed the HST Program during my graduate years, for their faith in me. I also thank my many colleagues, friends, and fellow graduate students including: Matthew During, Kenton Swartz, Stephen Finn, Walter Koroshetz, Melvyn Heyes, Rosy Roberts, John Steichen, and Alfred Geller for putting up with me during my graduate school career. A special note of thanks is extended to Gloria Sessler, a wonderful author, lady, friend and neighbor, who always lent an attentive ear. 3 My stepmother, Kit Bick Freese, has been a great support during this entire process, listening to my ideas and providing sage advice. I wish that I could thank my parents, Ernst and Elisabeth, in person for their inspiration and love, but their lives were prematurely ended by cancer--cancers that may well have been caused by their pioneering work with mutagens. Their dedication to science, truth and family has been the cornerstone of my research work and my life. Finally, I thank wholeheartedly my wife, Marcia, for her love and encouragement; without her support this thesis would not have been completed. 4 Dedication This thesis is dedicated to Luise Bautz, my grandmother, whose clear and soft blue eyes are still filled with love. 5 Table of Contents Page Abstract 2 Acknowledgements 3 Dedication 5 Table of Contents 6 List of Figures 7 List of Abbreviations 10 Chapters: 1. Introduction 13 2. Characterization and Mechanism of 33 Excitotoxicity in Striatal Cultures 3. Molecular Modulation of Excitotoxicity 92 4. Quinolinic Acid: In Vivo Metabolism 122 5. Kynurenic Acid and the Kynurenine 165 Pathway Summary 188 References 191 6 List of Figures Figure 1.1 Tryptophan Metabolism Figure 2.1 GABA Neuron and Age Dependent Changes in Striatal Neurons Figure 2.2 Neuropeptide Y Release into Culture Medium Figure 2.3 Striatal Cultures Figure 2.4 Toxicity of Glutamate Figure 2.5 Time Course of Glutamate Toxicity Figure 2.6 Neuronal Surface Area Changes During Glutamate Exposure Figure 2.7 Concentration and Age Dependence of Glutamate Toxicity Figure 2.8 Comparison of Glutamate Toxicity Between PND 0 and PND 6 Rats Figure 2.9 Effect of Extracellular Calcium Figure 2.10 Calcium Dependence of Toxicity Figure 2.11 Magnesium Attenuation of Toxicity Figure 2.12 Quinolinic Acid Toxicity Figure 2.13 APV Blockade of Toxicity Figure 2.14 Toxicity of Kainic Acid Figure 2.15 Quantitation of GABA Cells Figure 2.16 Quantitation of Enkephalin Cells 7 Figure 2.17 Age-Dependent Changes in Electrophysiology (<8 DIC) Figure 2.18 Age-Dependent Changes in Electrophysiology (>8 DIC) Figure 2.19 Electrophysiology of Survivor Neurons Figure 3.1 Effect of bFGF on Glutamate Toxicity Figure 3.2 Effect of bFGF on Quinolinate Toxicity Figure 3.3 Protective Effect of bFGF Figure 3.4 Effect of bFGF on Kainate Toxicity Figure 3.5 Time Course of bFGF Protective Effect Figure 3.6 Neuronophagia Figure 3.7 TPA Protective Effect Figure 4.1 ECF Quinolinate: Effect of Acute Tryptophan Loading Figure 4.2 Tissue Quinolinate: Effect of Acute Tryptophan Feeding Figure 4.3 Tissue Quinolinate: Effect of Chronic Tryptophan Feeding Figure 4.4 Tryptophan Plasma Levels Figure 4.5 Downregulation of Quinolinate Biosynthesis Figure 4.6 Neurochemical Markers of Toxicity Following Tryptophan Feeding: Striatum Figure 4.7 Neurochemical Markers of Toxicity Following Tryptophan Feeding: Cortex 8 Figure 4.8 Neurochemical Markers of Toxicity Following Tryptophan Feeding: Hippocampus Figure 4.9 Neurochemical Markers of Toxicity In Utero Exposure to Tryptophan: Striatum Figure 4.10 Neurochemical Markers of Toxicity In Utero Exposure to Tryptophan: Cortex Figure 4.11 Neurochemical Markers of Toxicity In Utero Exposure to Tryptophan: Hippocampus Figure 5.1 Kynurenine Pathway Figure 5.2 Kynurenic Acid Levels Following Acute Tryptophan Feeding Figure 5.3 Kynurenic Acid Levels Following Chronic Tryptophan Feeding Figure 5.4 Tryptophan and ECF Kynurenic Acid Figure 5.5 Neuroprotective Effect of Tryptophan Feeding: Neurochemical Markers 9 Abbreviations Ab Antibody aFGF Acidic Fibroblast Growth Factor AMPA Amino-3-hydroxy-5-methyl-4- isoxazoleproprionic acid Anova Analysis of Variance ANOVA Anaylsis of Variance AP4 2-amino-4-phosphobutyric acid APV 2-amino-5-phosphonovaleric acid bFGF Basic Fibroblast Growth Factor CSF Cerebrospinal Fluid DAB Diaminobenzidine DAG Diacylglycerol DIC Days in culture DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethylsulfoxide EC50 Effective Concentration (50% effect) FGF Fibroblast Growth Factor GABA Gamma amino butyric acid GAD Glutamic acid decarboxylase GAGP Goat anti-guinea pig GAR Goat anti-rabbit GC-MS Gas Chromatography-Mass Spectrometry Glu Glutamate 10 3-HAO 3-Hydroxyanthranilate Oxygenase HD Huntington's Disease HPLC High Pressure Liquid Chromatography IgG Immunoglobulin IP3 Inositol trisphosphate KA Kainic Acid Km Michaelis-Menten constant