The contribution of N-terminally modified amyloid beta to the etiology of Alzheimer’s disease Dissertation for the award of the degree “Doctor of Philosophy” Division of Mathematics and Natural Sciences of the Georg-August-Universita¨t Go¨ttingen submitted by Jessica L. Wittnam from Billings, MT, USA Go¨ttingen, 2012 PhD thesis committee: Prof. Dr. Thomas A. Bayer (Reviewer) Department for Psychiatry Division of Molecular Psychiatry Georg August University Go¨ttingen Prof. Dr. Andr´e Fischer (Reviewer) Laboratory of Aging and Cognitive Diseases European Neuroscience Institute Go¨ttingen Prof. Dr. Dr. Hannelore Ehrenreich Division of Clinical Neurosciences Max Planck Institute of Experimental Medicine Go¨ttingen Date of the oral examination: May 21, 2012 Declaration: I hereby declare that the PhD thesis entitled “The contribution of N- terminally modified amyloid beta to the etiology of Alzheimer’s disease”was written independently and with no other sources and aids than quoted. Jessica Wittnam Go¨ttingen, 2012 Contents Contents i 1 Introduction 3 1.1 Clinical presentation of Alzheimer’s disease . . . . . . . . . . . . . . . . . . 4 1.1.1 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.1.2 Risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.1.3 Disease progression . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.1.4 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.1.5 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2 Neuropathology of Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . 7 1.2.1 Amyloid deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2.2 Neurofibrillary tangles . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.2.3 Brain atrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.2.4 Microglia and astrocyte activation . . . . . . . . . . . . . . . . . . . 10 1.3 The amyloid precursor protein . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.3.1 Non-amyloidogenic processing of the amyloid precursor protein . . . 12 1.3.2 Amyloidogenic processing of the amyloid precursor protein . . . . . 13 1.4 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.5 The amyloid hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.5.1 Soluble amyloid hypothesis . . . . . . . . . . . . . . . . . . . . . . . 17 1.5.2 Intraneuronal amyloid hypothesis . . . . . . . . . . . . . . . . . . . 19 1.6 Amyloid beta variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 1.7 Pyroglutamate-modified amyloid beta . . . . . . . . . . . . . . . . . . . . . 22 1.7.1 Generation of pyroglutamate-modified amyloid beta . . . . . . . . . 23 1.7.2 Biochemical properties of pyroglutamate-modified amyloid beta . . 25 1.7.3 Toxicity of pyroglutamate-modified amyloid beta . . . . . . . . . . 25 1.8 Amyloid beta 4-42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 1.9 Transgenic mouse models of Alzheimer’s disease . . . . . . . . . . . . . . . 27 1.9.1 5XFAD mouse model . . . . . . . . . . . . . . . . . . . . . . . . . . 29 i Contents 1.10 Project objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 1.10.1 ProjectI:GenerationandcharacterizationoftheTBA42mousemodel 32 1.10.2 ProjectII:Exploringthepyroglutamate-modifiedamyloidbetaseed- ing hypothesis using the FAD42 mouse model . . . . . . . . . . . . 33 1.10.3 Project III: Generation and characterization of the TBA83 mouse model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2 Materials and Methods 35 2.1 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.1.1 Housing and general considerations . . . . . . . . . . . . . . . . . . 35 2.1.2 TBA42 transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.1.3 5XFAD transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . 36 2.1.4 FAD42 transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.1.5 TBA83 transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.1.6 Tissue preparation for biochemistry . . . . . . . . . . . . . . . . . . 37 2.1.7 Tissue preparation for immunohistochemistry . . . . . . . . . . . . 37 2.2 Behavioral analysis of mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2.1 Clasping test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2.2 Balance beam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.2.3 String suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.2.4 Inverted grip hang . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.2.5 Y-maze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 2.2.6 Cross maze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 2.2.7 Elevated plus maze . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.2.8 Open field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.2.9 Morris water maze . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.3 Molecular biology and biochemistry . . . . . . . . . . . . . . . . . . . . . . 43 2.3.1 Media preparation for Escherichia coli culturing . . . . . . . . . . 43 2.3.2 TBA1 plasmid mutagenesis . . . . . . . . . . . . . . . . . . . . . . 44 2.3.3 Escherichia coli transformation . . . . . . . . . . . . . . . . . . . . 44 2.3.4 Plasmid purification . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.3.5 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.3.6 Isolation and purification of TBA8 transgene . . . . . . . . . . . . 46 2.3.7 Pronuclear injection of TBA8 transgene . . . . . . . . . . . . . . . 47 2.3.8 DNA isolation for genotyping of transgenic mice . . . . . . . . . . . 47 2.3.9 RNA isolation from mouse brain . . . . . . . . . . . . . . . . . . . . 48 2.3.10 Nucleic acid concentration measurement . . . . . . . . . . . . . . . 48 2.3.11 Reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . 48 2.3.12 Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 2.3.13 Quantitative real-time polymerase chain reaction (qPCR) . . . . . . 49 2.3.14 Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . 51 ii Contents 2.3.15 DNA electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2.3.16 Protein isolation from mouse brain . . . . . . . . . . . . . . . . . . 55 2.3.17 Protein concentration determination . . . . . . . . . . . . . . . . . 56 2.3.18 Enzyme-linked immunosorbent assay (ELISA) . . . . . . . . . . . . 56 2.3.19 Glutaminyl cyclase enzyme activity measurement . . . . . . . . . . 57 2.4 Mass spectrometric analysis of mouse brain . . . . . . . . . . . . . . . . . . 57 2.5 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2.6 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 2.6.1 Paraffin embedding of mouse brain and spinal cord . . . . . . . . . 59 2.6.2 3,3’-Diaminobenzidine (DAB) immunohistochemistry . . . . . . . . 59 2.6.3 Combined DAB/HistoGreen immunohistochemistry . . . . . . . . . 61 2.6.4 Fluorescent immunohistochemistry . . . . . . . . . . . . . . . . . . 61 2.6.5 Microscopy and image preparation . . . . . . . . . . . . . . . . . . 61 2.6.6 Plaque load analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 62 2.7 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3 Results 63 3.1 Project I: Generation and characterization of the TBA42 mouse model . . 63 3.1.1 Selection of the TBA42 mouse line . . . . . . . . . . . . . . . . . . 63 3.1.2 Amyloid beta accumulation in TBA42 mice . . . . . . . . . . . . . 65 3.1.3 Gliosis in TBA42 mice . . . . . . . . . . . . . . . . . . . . . . . . . 66 3.1.4 Intracellular localization of amyloid beta in TBA42 mice . . . . . . 69 3.1.5 General physical assessment of TBA42 mice . . . . . . . . . . . . . 70 3.1.6 Motor function in TBA42 mice . . . . . . . . . . . . . . . . . . . . 71 3.1.7 Working memory in TBA42 mice . . . . . . . . . . . . . . . . . . . 72 3.1.8 Anxiety and exploratory behavior in TBA42 mice . . . . . . . . . . 72 3.2 Project II: Exploring the pyroglutamate-modified amyloid beta seeding hy- pothesis using the FAD42 mouse model . . . . . . . . . . . . . . . . . . . . 75 3.2.1 Behavioral analysis of FAD42 mice . . . . . . . . . . . . . . . . . . 75 3.2.2 Immunoprecipitation and mass spectrometric (IP/MS) characteri- zation of wild-type, TBA42, 5XFAD and FAD42 mouse brain . . . 76 3.2.3 Localization of cortical amyloid beta in 5XFAD and TBA42 mice . 78 3.2.4 Analysis of cortical plaque load in 5XFAD and FAD42 mice . . . . 78 3.2.5 MeasurementofsolubleandinsolubleamyloidbetainTBA42,5XFAD and FAD42 mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 3.2.6 Glutaminylcyclaseactivityinwild-type,TBA42,5XFADandFAD42 mouse brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 3.3 Project III: Generation and characterization of the TBA83 mouse model . 83 3.3.1 Selection of the TBA83 mouse line . . . . . . . . . . . . . . . . . . 83 3.3.2 Amyloid beta accumulation in TBA83 mice . . . . . . . . . . . . . 85 3.3.3 Gliosis in TBA83 mice . . . . . . . . . . . . . . . . . . . . . . . . . 85 iii Contents 3.3.4 General physical assessment and motor function in TBA83 mice . . 87 3.3.5 Working memory in TBA83 mice . . . . . . . . . . . . . . . . . . . 88 3.3.6 Anxiety and exploratory behavior in TBA83 mice . . . . . . . . . . 88 3.3.7 Spatial reference memory in TBA83 mice . . . . . . . . . . . . . . . 89 4 Discussion 92 4.1 Project I: Generation and characterization of the TBA42 mouse model . . 92 4.1.1 TBA42 micedevelopintraneuronalandsparse extracellular amyloid deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.1.2 Intraneuronal amyloid beta found in the endosomal/ lysosomal sys- tem in TBA42 mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.1.3 Age-dependent increase in gliosis in TBA42 mice . . . . . . . . . . 96 4.1.4 Age-dependent motor deficits and weight loss in TBA42 mice . . . . 97 4.1.5 Age-dependent working memory deficits in TBA42 mice . . . . . . 98 4.1.6 Decreased anxiety and altered exploratory activity in TBA42 mice . 100 4.1.7 Conclusions of Project I . . . . . . . . . . . . . . . . . . . . . . . . 101 4.2 Project II: Exploring the pyroglutamate-modified amyloid beta seeding hy- pothesis using the FAD42 mouse model . . . . . . . . . . . . . . . . . . . . 102 4.2.1 Enhanced behavioral deficits in FAD42 mice . . . . . . . . . . . . . 102 4.2.2 Minor alterations in the amyloid beta profile of FAD42 mice as determined by IP/MS . . . . . . . . . . . . . . . . . . . . . . . . . 103 4.2.3 Alteredlevelsofpyroglutamate-modifiedamyloidbetaandincreased plaque pathology in FAD42 mice . . . . . . . . . . . . . . . . . . . 104 4.2.4 Elevated glutaminyl cyclase activity in FAD42 mice . . . . . . . . . 106 4.2.5 Conclusions of Project II . . . . . . . . . . . . . . . . . . . . . . . . 107 4.3 Project III: Generation and characterization of the TBA83 mouse model . 108 4.3.1 Age-dependent gliosis and amyloid beta accumulation in TBA83 mice108 4.3.2 Selective deficits in motor function and spatial working memory in TBA83 mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 4.3.3 Conclusions of Project III . . . . . . . . . . . . . . . . . . . . . . . 111 5 Summary 112 Bibliography 114 List of Symbols and Abbreviations 137 List of Figures 141 List of Tables 143 iv Acknowledgments My doctoral work would not have been possible without the encouragement and guid- ance of Prof. Dr. Thomas Bayer. I truly appreciate the patience and understanding he displayed regarding the challenges I encountered during my project. I thank Prof. Dr. Andr´e Fischer and Prof. Dr. Dr. Hannelore Ehrenreich for agreeing to serve as members of my PhD committee. Their support and advice were indispensable. I also express my gratitude toward PD Dr. Oliver Wirths for his help and willingness to share his extensive knowledge of all things scientific. I am deeply grateful to Daniela Reich-Erkelenz, Prof. Dr. Stefan Treue and all the other members of the CSN board for being so kind and supportive during my term as CSN student representative. Working with them added a wonderful dimension to my PhD. A huge thanks to Kirsten P¨ohlker, Christina Bach and the other members of the GGNB office. Despite their workload, they were always able to offer assistance. To an oft-stressed PhD student, that small gesture is a lifesaver. Additional acknowledgments go to the former and current members of the Bayer lab: Ditte Christensen, Marie-Caroline Cotel, Andrea Marcello, Vivek Venkataramani, Katha- rina Dietrich, and Yvonne Bouter. Together they set the stage for my work and will keep it running once I leave. I thank Anika Saul for her pluck and general insightfulness. Her vivid personality made all those conference trips memorable. I am forever indebted to Nadine Ilse, Nina Karbe and the other members of the Klinikum animal facility. Without them, my work would have literally gone extinct. My sincerest thanks go to Petra Tucholla for always providing support, technical and otherwise. I will not forget her amazing ability to discern when I needed a kind word or some emergency chocolate. I appreciate the opportunity that all the rotation students of the Bayer lab have given me to teach and learn at the same time. 1 I will always be eternally grateful to Sadeem Jawhar and Antje Hillmann. They have been the best of friends at the worst of times, and my PhD would not have been the same in their absence. I am thankful to Sadeem for her willingness to share her scientific acumen, culture and generally fabulous sense of style. I hope I have proven to her that I am more than my picture implies. Antje has been one of the few people to understand and appreciate my idiosyncrasies. Without her, I would still be mired in the German bureaucracy. May these wonderful ladies and I remain close in the coming years. I also thank my parents for supporting and respecting me during my time in Germany. Theirabilitytoacceptmyunconventionalplanssaysvolumesaboutwhatwonderfulpeople they are. Finally, to Ivan Rajkovic: The love, kindness and unselfish support you have shown me throughout the years is beyond measure. Who knows where I would be without you. 2 Chapter 1 Introduction “You have to begin to lose your memory, if only in bits and pieces, to re- alize that memory is what makes our lives. Life without memory is no life at all, just as an intelligence without the possibility of expression is not really an intelligence. Our memory is our coherence, our reason, our feeling, even our action. Without it, we are nothing.” - Luis Bun˜uel Auguste Deter likely represents the most famous embodiment of this quotation by Spanish filmmaker Luis Bun˜uel. Over 100 years ago, Auguste’s failing memory and erratic behavior made her the patient of Dr. Alois Alzheimer at the Institution for the Mentally Ill and for Epileptics in Frankfurt, Germany. Alzheimer recalled Auguste as a woman confused by her surroundings and devoid of comprehension. Despite repeated questioning, she only seemed capable of providing one coherent description of her state, “It is like I have lost myself ”(Alzheimer, 1907). Following Auguste’s death in 1906, her brain was given to Alzheimer for postmortem analysis. He then described the histopathological features now commonly associated with Alzheimer’s disease (AD): neuron loss, extracellular plaques and intracellular tangles. Ultimately, Alzheimer’s case report on Auguste Deter gave this “lost woman”a place in history as the first AD patient. Millions of people have since been diagnosed with AD, making it the most common form of dementia. No cure for AD presently exists. As a result, AD has become a pressing medical, social and economic issue. 3