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F IDENTIFICATION of AMYOTROPHIC LATERAL IDENTIFICATION OF AMYOTROPHIC LATERAL SCLEROSIS DISEASE MECHANISMS BY CEREBROSPINAL FLUID PROTEOMIC PROFILING by Mahlon Angus Collins B.S. in Neuroscience/Psychology, Central Michigan University, 2006 M.S. in Experimental Psychology, Central Michigan University, 2009 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of PhD in Neurobiology University of Pittsburgh 2016 f UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Mahlon Angus Collins It was defended on April 14, 2016 and approved by Carey Balaban, PhD, Professor Michael Gold, PhD, Professor Teresa Hastings, PhD, Associate Professor Janice Robertson, PhD, Associate Professor Rebecca Seal, PhD, Assistant Professor Dissertation Advisor: Robert Bowser, PhD, Adjunct Professor ii Copyright © by Mahlon A. Collins 2016 iii IDENTIFICATION OF AMYOTROPHIC LATERAL SCLEROSIS DISEASE MECHANISMS BY CEREBROSPINAL FLUID PROTEOMIC PROFILING Mahlon Angus Collins, PhD University of Pittsburgh, 2016 Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease. Heterogeneity in clinical, genetic, and pathological features of ALS suggest the disease is a spectrum of disorders each resulting in motor neuron degeneration. Molecular profiling of ALS patients is, therefore, a useful means of characterizing and stratifying the ALS population. To this end, mass spectrometric proteomic profiling was performed on cerebrospinal fluid (CSF) from ALS, healthy control (HC), and other neurological disease (OND) subjects. This resulted in the identification of 1,712 CSF proteins, 123 of which exhibited altered relative abundance in ALS CSF. Biological processes related to these 123 proteins included synaptic activity, extracellular matrix, and inflammation. The application of feature selection and machine learning methods to these CSF proteomic profiles resulted in a classifier that used relative levels of WDR63, APLP1, SPARCL1, and CADM3 to predict independent ALS, HC, and OND samples with 83% sensitivity and 100% specificity. To aid in the validation of selected CSF proteins, a Western blot loading control method was developed and validated using a reversible, iodine-based total protein stain. This method improves the accuracy and sensitivity of the relative quantification of CSF proteins via Western blot. As RNA binding protein (RBP) pathology/dysfunction is common to several forms of ALS, the largest CSF RBP alteration, that of RNA binding motif 45 (RBM45) protein, was validated externally. The results demonstrated that RBM45 pathology is common to several forms of ALS, frontotemporal lobar degeneration (FTLD), and Alzheimer’s disease. To further understand the iv biological functions of RBM45, immunoprecipitation coupled to mass spectrometry was performed to identify RBM45 protein-protein interactions (PPIs). RBM45 PPIs and associated pathways were most strongly associated with hnRNP proteins, RNA processing, and cytoplasmic translation. RBM45 also participates in the general cellular response to stress via association with nuclear stress bodies. This association is dependent on RNA binding, is upregulated in ALS/FTLD, and is sufficient to induce the aggregation of the protein. Collectively, these results illustrate the utility of CSF proteomic profiling for characterizing mechanisms of neurological disease and provide new insights into the contributions of RNA binding protein dysregulation to ALS/FTLD. v TABLE OF CONTENTS PREFACE ................................................................................................................................... XV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 PROTEIN AGGREGATION AND INTRACELLULAR INCLUSION BODIES ..................................................................................................................................... 2 1.1.1 SOD1 ................................................................................................................. 3 1.1.2 TDP-43 .............................................................................................................. 4 1.1.3 FUS .................................................................................................................... 6 1.1.4 Neurofilament Proteins and Peripherin ........................................................ 8 1.1.5 Bunina Bodies .................................................................................................. 9 1.1.6 Other Aggregating Proteins ............................................................................ 9 1.2 INTRACELLULAR STRESS .......................................................................... 10 1.2.1 Stress Granules .............................................................................................. 10 1.2.2 Autophagy and Proteasomal Stress ............................................................. 13 1.2.3 ER Stress ........................................................................................................ 16 1.2.4 Evidence for RNA Binding Protein in the Disease Process ....................... 22 1.2.5 Altered RNA Splicing, Transport, and Translation ................................... 23 1.2.6 ADAR2 RNA Editing .................................................................................... 25 1.2.7 miRNAs........................................................................................................... 25 vi 1.2.8 C9ORF72 Hexanucleotide Repeat Expansion ............................................. 29 1.3 GLIAL AND INFLAMMATORY MECHANISMS OF DISEASE .............. 31 1.3.1 Astrocytes ....................................................................................................... 31 1.3.2 Microglia......................................................................................................... 35 1.3.3 Oligodendrocytes ........................................................................................... 38 1.3.4 Perineuronal Nets and Extracellular Matrix Dysfunction ........................ 40 1.3.5 Inflammation and Regulatory T-Cells ......................................................... 42 1.4 AXONAL TRANSPORT DEFECTS AND AXONOPATHY ....................... 45 1.4.1 Molecular Motor Proteins and the Neuronal Cytoskeleton ....................... 46 1.4.2 Anterograde Axonal Transport .................................................................... 47 1.4.3 Retrograde Axonal Transport ...................................................................... 48 1.4.4 Axonal Transport Defects in ALS ................................................................ 48 1.5 CURRENT THEORY AND OUTSTANDING CHALLENGES .................. 51 2.0 LC-MS/MS PROTEOMIC PROFILING OF AMYTROPHIC LATERAL SCLEROSIS CEREBROSPINAL FLUID ............................................................................... 56 2.1 CHAPTER SUMMARY ................................................................................... 56 2.2 INTRODUCTION ............................................................................................. 57 2.3 MATERIALS AND METHODS ...................................................................... 60 2.3.1 Subjects and CSF Collection ........................................................................ 60 2.3.2 CSF Preparation and Digestion.................................................................... 61 2.3.3 Liquid Chromatography Tandem Mass Spectrometry ............................. 62 2.3.4 Spectral Counting .......................................................................................... 63 2.3.5 Statistical Analysis of Relative Protein Abundance ................................... 63 vii 2.3.6 Ontological Enrichment Analysis................................................................. 64 2.3.7 Feature Selection, Classifier Construction, and Validation ...................... 65 2.3.8 Validation of Selected Proteins ..................................................................... 67 2.4 RESULTS ........................................................................................................... 68 2.4.1 Global CSF Analysis ...................................................................................... 68 2.4.2 Statistical Analysis of Relative Protein Abundance ................................... 73 2.4.3 Validation of Known ALS Biomarkers ....................................................... 81 2.4.4 Validation of Protein Alterations in ALS Spinal Cord Tissue .................. 83 2.4.5 Classifier Construction and Machine Learning ......................................... 87 2.5 DISCUSSION ..................................................................................................... 91 3.0 TOTAL PROTEIN IS AN EFFECTIVE LOADING CONTROL FOR CEREBROSPINAL FLUID WESTERN BLOTS ................................................................... 98 3.1 CHAPTER SUMMARY ................................................................................... 98 3.2 INTRODUCTION ............................................................................................. 99 3.3 MATERIALS AND METHODS .................................................................... 102 3.3.1 Cerebrospinal Fluid (CSF) Samples .......................................................... 102 3.3.2 Polyacrylamide Gel Electrophoresis (PAGE) and Electrophoretic Transfer ............................................................................................................................
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