Inflammation Is a Common Factor Between Central and Peripheral Neurodegeneration Brett Anthony Mcgregor
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University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2019 Inflammation Is A Common Factor Between Central And Peripheral Neurodegeneration Brett Anthony Mcgregor Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Mcgregor, Brett Anthony, "Inflammation Is A Common Factor Between Central And Peripheral Neurodegeneration" (2019). Theses and Dissertations. 2473. https://commons.und.edu/theses/2473 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. INFLAMMATION IS A COMMON FACTOR BETWEEN CENTRAL AND PERIPHERAL NEURODEGENERATION By Brett Anthony McGregor Bachelor of Science, University of North Dakota, (2014) A Dissertation Submitted to the Graduate Faculty of the University of North Dakota In partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota May 2019 Copyright 2019 Brett A. McGregor ii PERMISSION Title: Inflammation is a common factor between central and peripheral neurodegeneration Department: Biomedical Sciences Degree: Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work, or in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Brett A. McGregor May, 2019 iv TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………...……..…viii LIST OF TABLES………………………………………………………………………..x ACKNOWLEDGEMENTS…………………………………………………..………...xii ABSTRACT…………………………………………………………….……………….xv CHAPTER I. INTRODUCTION………………………………………………………..1 Preface…………………………………………………………...1 Neuroinflammation……………………………………………...2 Cellular Response to Inflammation in the CNS……...3 Parkinson’s Disease…………………………………………....5 Treatment and Proposed Causes……………….....…6 Animal Models of Parkinson’s Disease…………....…9 Peripheral Inflammation...…….………………………...…….11 Diabetic Peripheral neuropathy.……………………………...13 Animal Models of Diabetic Peripheral Neuropathy...15 Dissertation Research Objection………………………...…..18 II. CONSERVED TRANSCRIPTIONAL SIGNATURES IN HUMAN AND MURINE DIABETIC PERIPHERAL NEUROPATHY...……...19 Abstract………………………………………………………....19 Introduction……………………………………………………..20 Research Design and Methods…………………………...….21 Microarray Data………………………………………...21 Study Design……………………………………………22 Transcriptome Profiling…………………………….….23 Diabetes- and Age-Comparisons of DEGs Sets…….25 v Transcriptional Network Comparison……………......25 Functional Enrichment Analysis……………………...26 Construction of Merged Human-Mouse Conserved Transcriptional Network and Network Centrality Analysis…………………………………………………26 Results…………………………………………….………...….28 Identification of Changes in Gene Expression……...28 Transcriptional Network Analysis …………………....34 Merged Transcriptional Network and Centrality Analysis…………………………………………………41 Discussion……………………………………………………...46 III. CONSERVED GENE EXPRESSION CHANGES AND DYSREGULATED PATHWAYS IN COMPLICATION-PRONE TISSUES OF STREPTOZOTOCIN-INDUCED DIABETIC MICE…53 Abstract………………………………………………………....53 Introduction……………………………………………………..54 Methods ………………………………………………………...56 Sample Collection and Processing…………………..56 Differential Expression and Pathway Enrichment....60 Network Clustering…………………………………….60 Results ………………………………………………………….61 Changes in Gene Expression……………………...…61 Enriched Canonical Pathways………………………..62 Network Analysis of Concordant Gene Pathways…..67 Discussion………………………………………………………71 IV. ALPHA-SYNUCLEIN-INDUCED METHYLATION AND GENE EXPRESSION CHANGES IN MICROGLIA………………...………78 Abstract………………………………………………………....78 Introduction……………………………………………………..80 vi Materials and Methods………………………………………...84 Study Design……………………………………………84 Animals………………………………………………….84 Animal Use……………………………………………...86 Microglia Isolation……………………………………...86 Dual DNA/RNA Extraction and Isolation……………..87 Global Methylation Assay……………………………..87 Methylation Processing using Reduced Representation Bisulfite Sequencing (RRBS)……...88 RNA-Sequencing Processing………………………...89 Enrichment and Network Analysis…………………...90 Results ………………………………………………………….91 α-synuclein-Induced Methylation Changes in Microglia………………………………………………...92 α-synuclein-Induced Gene Expression Changes in Microglia……………………………………………….106 Correlation between Identified DMGs and DEGs….119 Discussion …………………………………………………….121 V. DISCUSSION ………………………………………………………...128 Future Directions ……………………………………………..133 Limitations of the Work Presented in this Dissertation…….136 Summary Conclusions…………………………………….…137 VI. REFERENCES ……………………………………………………….140 vii LIST OF FIGURES Figure Page 1. Model and Network DEG Comparison Workflow…………...…………….…….24 2. DEG patterns in study 1 DPN models…………………………….……………..32 3. Shared transcriptional networks between murine and human datasets identified by TALE, a graph matching software……………………………………..……...36 4. Highly connected DEGs across TALE networks……………………..………….39 5. IPA Enriched Pathway Clustering……………………………………….………..45 6. Metabolic Phenotype db/+ mice testing following STZ treatments………....…57 7. Neuropathy Phenotype db/+ mice testing following STZ treatments……….....58 8. Nephropathy Phenotype db/+ mice testing following STZ treatments…..…….59 9. Gene Overlap Common between Complication-Prone Tissue……………..….63 10. Enriched Pathways Common Between Complication-Prone Tissue……….…65 11. Clustering of Enriched Concordant Pathways……………………………….….68 12. Principal Component Analysis of Four Tissues in STZ Treated Mice and Wild Type Controls……………………………………………………………...……….72 13. Study 3 Workflow and Description……………………………………………..…85 14. Global DNA Methylation ELISA Results…………………………………………86 15. Genomic Regions of 3-Month DMCs……………………………………………..98 16. Enrichment Network from 3-Month DMCs……………………………………….99 17. Genomic Regions of 13-month DMCs………………………………………….104 viii 18. Enrichment Network from 13-Month DMCs…………………………………....105 19. Enrichment Network from 3-Month DEGs…………………………………...…110 20. GO Enrichment in 13-Month DEGs……………………………………………..113 21. KEGG Enrichment in 13-Month DEGs………………………………………….115 22. Reactome Pathway Enrichment in 13-Month DEGs…………………………..116 23. Enrichment Network from 13-Month DEGs…………………………………….117 24. Correlation Summary of Genes Both Differentially Methylated and Expressed………………………………………………………………………..120 25. Summary Figure………………………………………………………………….139 ix LIST OF TABLES Tables Page 1. Metabolic and neuropathy phenotypic measures of microarray datasets…….30 2. Study 1 Dataset Summary………………………………………………………...31 3. Most frequently dysregulated canonical pathways in study 1 datasets identified by Ingenuity Pathway Analysis (IPA)………………………….……………….…33 4. Summary of the microarray datasets…………………………………………….35 5. The most commonly dysregulated differentially expressed genes among the shared transcriptional networks………………………………………………..…37 6. Disrupted Pathways Based on TALE DEGs……………………………………..40 7. Centrality Analysis Gene Results…………………………………………………43 8. Top 20 IPA Canonical Pathways Based on the Most Central Genes………….44 9. Gene Overlap Common between Complication-Prone Tissue………………...64 10. Enriched Pathways Common Between Complication-Prone Tissue………….66 11. Concordant Gene IPA results from Complication-Prone Tissues……………..70 12. Sample Alignment Summary……………………………………………………...94 13. Top 20 DMCs at 3 Months…………………………………………………………96 14. Bottom 20 DMCs at 3 Months…………………………………………………..…97 15. Top 20 DMCs at 13 Months……………………………………………………...102 16. Bottom 20 DMCs at 13 Months……………………………………………….…103 17. 20 most up regulated DEGs at 3 Months……………………………………….107 x 18. 20 most down regulated DEGs at 3 Months……………………………………108 19. 20 most up regulated DEGs at 13 Months……………………………………...111 20. 20 most down regulated DEGs at 13 Months…………………………………..112 21. DEGs shared between 3 and 13-Month Groups……………………………….118 xi ACKNOWLEDGEMENTS First, I would like to thank Dr. Junguk Hur and Dr. James Porter for the opportunity to study in their laboratories, for funding this research, and for their extensive mentorship over the years of graduate school. Dr. Hur generated the core ideas behind the diabetic peripheral neuropathy studies conducted and provided me an opportunity to learn the ins and outs of bioinformatics analysis. Dr. Porter provided me with invaluable guidance through my undergraduate education, which continued into my graduate education, as he taught me how to conduct research in a laboratory environment. Both of my mentors have been very understanding of my many mistakes and have provided countless opportunities for me to test a variety of ideas throughout graduate school. I thank Dr. Saobo Lei, Dr. Keith Henry, Dr. Roxanne Vaughan, and Dr. Peter Meberg for being a part of