Differential expression of neuregulin-1 isoforms and downregulation of erbin are associated with Erb B2 receptor activation in diabetic peripheral neuropathy By Pan Pan Submitted to the graduate degree program in Pharmacology and Toxicology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ______________________________ Rick T. Dobrowsky, Ph.D., Chairperson _______________________________ Jeff Staudinger, Ph.D. _______________________________ Alexander Moise, Ph.D. _______________________________ Honglian Shi. Ph.D. ________________________________ Brian Ackley, Ph.D. Date Defended: April 18th The Dissertation Committee for Pan Pan certifies that this is the approved version of the following dissertation: Differential expression of neuregulin-1 isoforms and downregulation of erbin are associated with Erb B2 receptor activation in diabetic peripheral neuropathy By Pan Pan _______________________________ Rick T. Dobrowsky, Ph.D., Chairperson th Date approved: April 18 ii Table of Contents Table of Contents ......................................................................................................................... iii Abstract ......................................................................................................................................... vi Acknowledgements ..................................................................................................................... vii List of Tables and Figures ........................................................................................................... ix List of Abbreviations .................................................................................................................. xii Chapter 1: Introduction ............................................................................................................... 1 1.1. Overview of Diabetes Mellitus ........................................................................................ 1 1.1.1. Diabetes Mellitus .......................................................................................................... 1 1.1.2. Diagnostic Criteria of Diabetes Mellitus ...................................................................... 2 1.1.3. Classification and Clinical Features of Diabetes Mellitus............................................ 4 Type 1 Diabetes Mellitus ......................................................................................................... 5 Type 2 Diabetes Mellitus ......................................................................................................... 8 Gestational Diabetes and Other Sub-types ........................................................................... 11 1.1.4. The Action of Insulin .................................................................................................. 12 1.1.5. Mitochondrial Dysfunction in β-cell Death ................................................................ 14 1.2. Diabetic Complications .................................................................................................. 17 1.2.1. Diabetic Retinopathy .................................................................................................. 18 1.2.2. Diabetic Nephropathy ................................................................................................. 21 1.2.3. Cardiovascular Disease............................................................................................... 24 1.3. Diabetic Neuropathy ...................................................................................................... 25 1.3.1. Classification and Clinical Features ........................................................................... 25 Diabetic Autonomic Neuropathy (DAN) ............................................................................... 26 Diabetic Sensorimotor Polyneuropathy (DPN) ..................................................................... 27 1.3.2. Animal Models of Diabetic Peripheral Neuropathy ................................................... 30 Type 1 Diabetic Models ......................................................................................................... 30 Type 2 Diabetic Models ......................................................................................................... 32 1.3.3. Pathology, Current Management and Treatments ...................................................... 32 Advanced Glycation End-products Pathway ......................................................................... 33 Polyol Pathway ...................................................................................................................... 34 iii Protein Kinase C Pathway .................................................................................................... 35 Inflammation Pathway........................................................................................................... 36 Oxidative Stress and Mitochondrial Dysfunction .................................................................. 37 1.4. Growth Factors in Peripheral Nervous System .............................................................. 39 1.4.1. Neurotrophin Family .................................................................................................. 40 Nerve Growth Factor ............................................................................................................ 41 Brain-derived Neurotrophic Factor and Neurotrophin-3 ..................................................... 43 1.4.2. Insulin-like Growth Factor Family ............................................................................. 44 1.5. Neuregulin-Erb B Signaling in Peripheral Nervous System .......................................... 46 1.5.1. Ligand Neuregulin-1 Family and Erb B Receptors .................................................... 46 1.5.2. Neuregulin-Erb B Signaling in Peripheral Nerve Development ................................ 49 1.5.3. Neuregulin-Erb B Signaling in Nerve Injury ............................................................. 53 1.5.4. Adapter Protein Erb B2-Interacting Protein ............................................................... 54 1.6. Dissertation Hypothesis .................................................................................................. 55 Chapter 2: Materials and Methods ........................................................................................... 57 2.1. Materials ......................................................................................................................... 57 2.1.1. Animals ....................................................................................................................... 57 2.1.2. Antibodies ................................................................................................................... 57 2.1.3. Peptide Competition Assay ........................................................................................ 58 2.1.4. Reagents...................................................................................................................... 59 2.2. Methods .......................................................................................................................... 60 2.2.1. Induction of Diabetes and Measurement of Fasting Blood Glucose (FBG) ............... 60 2.2.2. Measurement of Glycated Hemoglobin ...................................................................... 60 2.2.3. Assessments of Sensory Sensitivity ........................................................................... 60 Thermal Sensitivity ................................................................................................................ 60 Mechanical Sensitivity ........................................................................................................... 61 2.2.4. Nerve Conduction Velocity ........................................................................................ 61 Motor Nerve Conduction Velocity ......................................................................................... 62 Sensory Nerve Conduction Velocity ...................................................................................... 63 2.2.5. Erlotinib Administration ............................................................................................. 64 2.2.6. Immunohistochemistry of Footpad Tissue ................................................................. 64 iv 2.2.7. Immunoblot Analysis ................................................................................................. 65 2.2.8. Erbin-shRNA Lentivirus Preparation and Infection ................................................... 66 2.2.9. Preparation of Myelinated Mouse DRG/Schwann Cell Co-cultures .......................... 67 2.2.10. Preparation of Schwann Cell Cultures........................................................................ 67 2.2.11. Measurement of Mitochondrial Respiration ............................................................... 68 2.2.12. Mitochondrial Fraction ............................................................................................... 69 2.2.13. Statistical Analysis ..................................................................................................... 69 Chapter 3: Dysfunction of Neuregulin-Erb B2 Ligand-Receptor Signaling Pathway in Diabetic Peripheral Neuropathy ...............................................................................................