PROTEOMIC CLUES TO THE PATHOGENESIS OF ALEXANDER DISEASE by DANIEL L. FLINT MICHAEL BRENNER, COMMITTEE CHAIR STEPHEN BARNES HARALD SONTHEIMER ELIZABETH SZTUL SCOTT WILSON A DISSERTATION Submitted to the Graduate faculty of The University of Alabama at Birmingham in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, AL 2012 Copyright by Daniel L. Flint 2012 ii PROTEOMIC CLUES TO THE PATHOGENESIS OF ALEXANDER DISEASE DANIEL L. FLINT NEUROBIOLOGY ABSTRACT Since the initial report identifying mutations in GFAP as the primary genetic de- fect in the astrogliopathy Alexander Disease (AxD) much progress has been made in elucidating the molecular details underpinning the pathogenic role of mutant GFAP in astrocytes. In particular, data collected from mouse, fly, and cell culture models of AxD have isolated several prominent cellular changes that are associated with mutant GFAP expression including oxidative stress, proteasome inhibition, reduced expression of the astrocyte glutamate transporter GLT-1, and loss of neurons; these models have also successfully recapitulated the pathological hallmark of AxD—protein aggregates known as Rosenthal fibers (RFs). To date, however, much less attention has been focused on collecting data from AxD patient brain. As such, the current work sought to confirm and extend the previous findings from AxD models systems to patient neocortex by performing a proteomic study on patient and control neocortical tissue using gel- enhanced liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). The datasets generated by this approach confirmed many of the previously reported findings from AxD model systems; in addition, numerous novel features of AxD neocortex were described such as decreased abundance of the enzyme, glutamine synthetase, and signs of oligodendrocyte cell loss. Other findings of interest included the identification of atrogin-1 as a novel RF-associated protein and as a candidate AxD biomarker; the latter of which relates to the fact that this report is the first to document atrogin-1 at the protein iii level in the brain of any species. Finally, this study uncovered evidence using bioinformatics that cellular pathways related to integrins and the actin cytoskeleton are activated in AxD and suggested a role for RhoA/ROCK signaling in mediating this response. As small molecule inhibitors of ROCK (e.g. Fasudil) are currently approved for use in humans, these findings may suggest a therapeutic role for these compounds in the clinical management of AxD. Keywords: Alexander Disease, GFAP, astrocyte, glutamate-glutamine cycle, RhoA/ROCK, atrogin-1 iv DEDICATION And one of the weird things about science is that the truth always wins, and that makes it different from many other subjects. Whereas with history, economics, and philosophy people can argue for generations, and this is one of the things which is least understood by the public and the media because the media always thinks that—as said in politics—they have to bring together two opposing views. But in science there are not two opposing views. Nature always tells you what is right, and that is final. —Max Perutz v TABLE OF CONTENTS Page ABSTRACT......................................................................................................................iii DEDICATION..................................................................................................................v LIST OF TABLES ............................................................................................................viii LIST OF FIGURES ..........................................................................................................xi LIST OF ABBREVIATIONS ...........................................................................................xv INTRODUCTION ............................................................................................................1 History...................................................................................................................1 Clinical Presentation .............................................................................................1 Pathology...............................................................................................................4 Diagnosis ...............................................................................................................7 GFAP Mutations ...................................................................................................8 Mechanism of Disease ..........................................................................................12 Cellular Functions .....................................................................................12 Model Organisms ......................................................................................15 Treatment ..............................................................................................................18 Goals of Dissertation Research.............................................................................19 ALEXANDER DISEASE ................................................................................................22 SPLICE SITE, FRAMESHIFT AND CHIMERIC GFAP MUTATIONS IN ALEXANDER DISEASE ................................................................................................66 IDENTIFICATION OF NOVEL PROTEINS ASSOCIATED WITH ALEXANDER DISEASE ................................................................................................103 CONCLUSION................................................................................................................353 Genetics.................................................................................................................354 Proteomics and Proposed Model ..........................................................................355 Future Directions ...................................................................................................366 vi GENERAL LIST OF REFERENCES ..............................................................................370 APPENDIX A. RELEVANT FIGURES..................................................................................384 B. RELEVANT TABLES ...................................................................................404 C. IACUC AND IRB APPROVAL FORMS .....................................................429 vii LIST OF TABLES Tables Page ALEXANDER DISEASE 1 Characteristics of Alexander disease patients recently tested for GFAP mutations ..............................................................................................62 2 Summary of characteristics for Alexander disease patients with GFAP mutations............................................................................................64 3 Evidence for disease causality of new novel mutations ........................................65 SPLICE SITE, FRAMESHIFT AND CHIMERIC GFAP MUTATIONS IN ALEXANDER DISEASE 1 Patients with GFAP mutations identified in this study.........................................102 IDENTIFICATION OF NOVEL PROTEINS ASSOCIATED WITH ALEXANDER DISEASE 1 Clinical details associated with tissues used in the current work.........................317 2 Distribution of total tissue protein by protein fraction ..........................................318 3 Proteins increased in abundance in the RF fraction from AxD case 613 and control 105 (P ≤ 0.05)............................................................319 4 Comparison of the SpCs for different cytoskeletal proteins in protein fractions generated by different methods employed in the current study ...............................................................................................321 5 Proteins found to be more abundant in the cytoskeletal fraction from AxD case 613 when compared to control 105 by 2D-DIGE .......................322 6 Proteins increased in abundance in the cytoskeletal fraction dataset from AxD case 613 and control 105 (P ≤ 0.05).......................................323 viii 7 Proteins decreased in abundance in the cytoskeletal fraction dataset from AxD case 613 and control 105 (P ≤ 0.05).......................................325 8 Proteins increased in abundance in the soluble fraction from AxD case 613 and control 105 (P ≤ 0.05) ...................................................327 9 Proteins decreased in abundance in the soluble fraction from AxD case 613 and control 105 (P ≤ 0.05) ...................................................329 10 List of proteins showing apparent redistribution between the soluble fraction and the cytoskeletal fraction from AxD case 613 and control 105 .............................................................................331 11 List of manually compiled synaptic proteins from the cytoskeletal and soluble fraction datasets from AxD case 613 and control 105.....................................................................................................332 12 Proteins increased in abundance in the total protein fraction (stringent dataset) from AxD case 613 and control 105 (P ≤ 0.05) .....................334 13 Proteins decreased in abundance in the total protein fraction (stringent dataset) from AxD case 613 and control 105 (P ≤ 0.05) .....................337 14 Abundance of select groups of proteins in the total protein fraction (stringent dataset) .................................................................................................338 15 Bioinformatics analysis of the total protein fraction (stringent dataset) using the KEGG module in DAVID.......................................339 16 Bioinformatics analysis of the total protein fraction (stringent dataset) using PANTHER gene expression tool #1.............................340 17 Bioinformatics analysis of the total protein fraction (stringent and normal dataset) using PANTHER gene expression tool #2........................................................................................341 18 Manual compilation of proteins from the total protein fraction (normal and stringent datasets) associated with integrin and actin cytoskeleton signaling pathways..................................................................342 19 Manual compilation of proteins from the total protein fraction (normal and stringent datasets) associated with the Nrf2-mediated oxidative stress response pathway................................................343 ix 20 Manual compilation of proteins from the total protein fraction (normal and stringent datasets) associated with reactive and malignant astrocytes .............................................................................................344 21 Manual compilation of proteins of interest from the total protein fraction (normal and stringent datasets) due to proposed molecular function .................................................................................345 22 Relative abundance ratios for select proteins of interest in the total protein fraction (stringent dataset) calculated using eXtracted Ion Chromatogram peak area ratios .....................................................347 23 Comparison of differentially abundant cytoskeletal proteins across all three AxD and control tissue groups ....................................................348 24 List of manually compiled mitochondrial proteins from the cytoskeletal and soluble fraction datasets from AxD case 613 and control 105.....................................................................................................349 25 List of antibodies used in the current work ..........................................................351 26 List of antibody combinations used in the current work ......................................352 x