4D Mapping of Network-Specific Pathological Propagation in Alzheimer's Disease by Rebecca Gail Canter B.S. The Johns Hopkins University (2009) Submitted to the Department of Brain and Cognitive Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2016 2016 Massachusetts Institute of Technology. All rights reserved. Signature of Author Signature redacted Department of Brain and Cognitive Sciences June, 2013 Certified By Signature redacted Li-Huei Tsai, Ph.D., D.V.M. Director, The Picower Institute for Learning and Memory Picower Professor of Neuroscience Thesis Supervisor Accepted By Signature redacted Matthew A. Wilson, Ph.D. Sherman Fairchild Professor of Neuroscience Direc of Graduate Education for Brain and Cognitive Sciences MASSACHUSETTS INSTITUTE OF TECHNOLOGY 1 DEC 20 2016 LIBRARIES ARCHIVES 77 Massachusetts Avenue Cambridge, MA 02139 MITLibraries http://Iibraries.mit.edu/ask DISCLAIMER NOTICE Due to the condition of the original material, there are unavoidable flaws in this reproduction. We have made every effort possible to provide you with the best copy available. Thank you. The images contained in this document are of the ,best quality available. 2 4D Mapping of Network-Specific Pathological Propagation in Alzheimer's Disease By Rebecca Gail Canter Submitted to the Department of Brain and Cognitive Sciences on June 13, 2016 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Neuroscience Abstract Alzheimer's disease (AD) causes a devastating loss of memory and cognition for which there is no cure. Without effective treatments that slow or reverse the course of the disease, the rapidly aging population will require astronomical investment from society to care for the increasing numbers of AD patients. Additionally, the financial and emotional burden on families of affected individuals will be profound. Traditional approaches to the study of AD use either biochemical assays to probe cellular pathophysiology or non-invasive imaging platforms to investigate brain- wide network alterations. Though decades of research using these tools have advanced the field significantly, our increased understanding of AD has not led to successful interventions. One reason for this impediment may be that the tools used in neither approach can achieve the spatial and temporal precision necessary to study the consequences of molecular insults across the brain over time. In this thesis, I capitalize on recent advances in tissue processing technologies to gain a network-level perspective on the molecular and cellular progression of AD. First, I present optimized methods for in situ proteomic phenotyping of large-volume tissue specimens. Then, I use the techniques to map amyloid-beta (AP) aggregates at the whole-brain scale across disease stages in a mouse model of AD. The spatially-unbiased, temporally-precise map demonstrates hierarchical susceptibility of increasingly large, memory-related brain networks to Ap deposition. Importantly, the 4D nature of the map reveals that subcortical nodes and white matter tracts of the Papez memory circuit exhibit unique, early vulnerability to AP aggregates. Finally, using large-volume labeling approaches, I confirm the molecular findings by showing disease- specific AP aggregation in human samples from the early hub regions. Together, this data unites desperate observations of network-level deficits and identifies critical locations of early AP deposition in the brain. By linking molecular and network observations, I begin to provide biological explanations for the clinical manifestation of AD. This perspective can guide earlier patient identification and refine experimental approaches to developing cognitively efficacious treatments. These discoveries emphasize the necessity of multi-level investigations in neuroscience research and highlight the potential impacts of tools that enable researchers to bridge the gap. Thesis supervisor: Li-Huei Tsai, Ph.D., D.V.M. Title: Director, The Picower Institute for Learning and Memory Picower Professor of Neuroscience 3 "Then leaf subsides to leaf. -Robert Frost 4 TABLE OF CONTENTS ACK NO W LEDG EM ENTS ........................................................................................................................ 7 PREFACE .................................................................................................................................................... 9 THE COSTS OF M ENTAL ILLNESS ........................................................................................................... 9 TRADITIONAL APPROACHES TO AD RESEARCH .................................................................................. 10 A CIRCUIT AND NETW ORK PERSPECTIVE OF AD.................................................................................... 11 ADDRESSING THE M ULTIFACETED ETIOLOGY OF AD ......................................................................... 12 THESIS OVERVIEW .................................................................................................................................. 13 CHAPTER 1: INTRODUCTION ............................................................................................................ 15 PREFACE .................................................................................................................................................. 15 INTRODUCTION........................................................................................................................................ 16 AD THROUGH THE AMYLOID HYPOTHESIS LENS..................................................................................... 17 M ULTIPLE FACTORS INDUCE NETWORK DYSFUNCTION IN AD ........................................................... 22 PROSPECTIVE .......................................................................................................................................... 33 CHAPTER 2: ISWITCH LABELING FOR IN SITU PROTEOMIC INVESTIGATION OF LARGE VO LUM E TISSUE SPECIM ENS ........................................................................................ 39 ABSTRACT ............................................................................................................................................... 39 INTRODUCTION........................................................................................................................................ 40 RESULTS.................................................................................................................................................. 42 DISCUSSION............................................................................................................................................. 49 M ETHODS ................................................................................................................................................ 52 ACKNOW LEDGEMENTS AND CONTRIBUTIONS:.................................................................................... 55 CHAPTER 3: 4D MAPPING OF CIRCUIT-SPECIFIC ALZHEIMER'S PATHOLOGY REVEALS EARLY SUBCORTICAL VULNERABILITY TO AB DEPOSITION.......................63 ABSTRACT ............................................................................................................................................... 63 INTRODUCTION........................................................................................................................................ 64 RESULTS..................................................................................................................................................65 DISCUSSION............................................................................................................................................. 73 M ETHODS ................................................................................................................................................ 76 ACKNOW LEDGEMENTS AND CONTRIBUTIONS:.................................................................................... 80 CO NCLUSIO NS AND FUTURE DIRECTIONS............................................................................... 93 APPENDIX A: PROTOCOL FOR ISWITCH LABELING THICK TISSUE SECTIONS......97 APPENDIX B: PROTOCOL FOR ISWITCH LABELING INTACT TISSUE SAMPLES...........101 APPENDIX C: PROTOCOL FOR ISWITCH LABELING HUMAN AUTOPSY SPECIMENS.. 103 APPENDIX D: RECOMMENDATIONS TO THE NIA-AA 2015 AD SUMMIT............................ 107 REFERENCES........................................................................................................................................113 5 6 Acknowledgements This thesis culminates years of work, not only my own and my colleagues within the laboratories at MIT, but also on behalf of my teachers, mentors, friends, and family who have supported me in getting to this point. Without them, I would not be the researcher, thinker, or person that I am today, and I am eternally grateful for their contributions to this work. First and foremost I must acknowledge Dr. Li-Huei Tsai, my advisor and mentor and without whom I would not have been able to write this thesis on multiple levels. I wish I could acknowledge each way in which Li-Huei has significantly impacted my life, but it would take too many pages to list each of the things I have learned from her mentorship, advice, and example. To Li-Huei - thank you for taking me in when I thought there was nothing left