California State University, San Bernardino CSUSB ScholarWorks Electronic Theses, Projects, and Dissertations Office of aduateGr Studies 6-2017 AN ISOGENIC STEM CELL MODEL OF ALZHEIMER'S DISEASE: DIRECT EXPRESSION OF AMYLOID-BETA Teresa Marie Ubina California State University - San Bernardino Follow this and additional works at: https://scholarworks.lib.csusb.edu/etd Part of the Nervous System Diseases Commons Recommended Citation Ubina, Teresa Marie, "AN ISOGENIC STEM CELL MODEL OF ALZHEIMER'S DISEASE: DIRECT EXPRESSION OF AMYLOID-BETA" (2017). Electronic Theses, Projects, and Dissertations. 523. https://scholarworks.lib.csusb.edu/etd/523 This Thesis is brought to you for free and open access by the Office of aduateGr Studies at CSUSB ScholarWorks. It has been accepted for inclusion in Electronic Theses, Projects, and Dissertations by an authorized administrator of CSUSB ScholarWorks. For more information, please contact [email protected]. AN ISOGENIC STEM CELL MODEL OF ALZHEIMER'S DISEASE: DIRECT EXPRESSION OF AMYLOID-BETA A Thesis Presented to the Faculty of California State University, San Bernardino In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by Teresa Marie Ubina June 2017 AN ISOGENIC STEM CELL MODEL OF ALZHEIMER’S DISEASE: DIRECT EXPRESSION OF AMYLOID-BETA A Thesis Presented to the Faculty of California State University, San Bernardino by Teresa Marie Ubina June 2017 Approved by: Nicole Bournias-Vardiabasis, Committee Chair Paul Salvaterra, Committee Member Jeffrey Thompson, Committee Member © 2017 Teresa Marie Ubina ABSTRACT Alzheimer’s disease (AD), identified over 100 years ago and intensively studied since the 1970s, has no effective treatments or mechanistic understanding of the underlying neurodegenerative process. Most investigators believe accumulation or aggregation of amyloid beta (A) proteins plays a causative role. Aβ peptides (~39-43 residues) are generated by proteolysis of the transmembrane protein APP. One reason we know so little about AD is an incomplete understanding of the cellular mechanisms responsible for A proteotoxicity. Human ES and iPSC models of AD are recent additions to many other models used to investigate these mechanisms. AD, however is a chronic progressive condition of old age and cultured neurons may not live long enough to model what goes wrong in neurons from AD patients. In my research, I used hESCs which directly express A peptides thus avoiding the time it takes to process APP. One App allele in H9 hESCs was previously edited using TALEN. A homologous recombination cassette coding directly for a secretory form of either A1-42 or A1-40 and containing a stop codon, was inserted into the first exon of App upstream of the normal translational start site. I used multiple independently isolated clones of edited cells with 3 genotypes: App/App (unedited), App/Aβ1-40 and App/Aβ1-42. Expression of A from edited alleles was confirmed by qRT-PCR using primers specific for the edit. I first sought to establish if editing changed any aspects of neuronal differentiation in culture. All 3 genotypes have similar embryoid body (EB) development, and similar numbers iii and sizes of neuronal clusters (NC) up to 34 days after EB dissociation and neural differentiation. Immunostaining of neuronal markers, NeuN and DCX (doublecortin), likewise revealed no difference among edited and unedited cells, suggesting that the edits do not affect the ability of my stem cells to differentiate into neurons. I next measured accumulation of aggregated A using an aggregate specific antibody, 7A1a. Data at 34-days post EB dissociation indicates NCs in the Aβ1-42 edited cells accumulate significantly more aggregates relative to either unedited or A-40 edited lines, a result consistent with the increased ability for A1-42 to form aggregates. Aβ aggregates also appear to be concentrated around fragmented nuclei within neuronal clusters suggesting that intracellular accumulation may play a key role in proteotoxicity. Additionally, I observed a significant decrease in the number of synapsin1 puncta, a marker of synapses, another feature of AD. I documented a nearly 3-fold greater neuronal cell death in both the Aβ1-40 and Aβ1-42 neurons at 70 days after differentiation. RNA sequencing data also shows independently isolated clones group together and show differential expression of genes related to memory and neuronal cell death. The early presence of Aβ aggregation and subsequent cell death is in line with the chronic and progressive nature of AD and this is the first known model to exhibit a neurodegenerative phenotype. These isogenic cell lines thus appear to be useful to screen for therapeutics that may prevent or slow A1-42 dependent neurodegeneration and a tool to investigate A-dependent mechanisms with relevance to AD. iv ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Nicole Bournias-Vardiabasis, who helped me discover my love for the rigors of research and has always offered me opportunities, support, and fantastic advice not just in academics but also in life. I would also like to thank Dr. Paul Salvaterra for always challenging me to do better and for helping me to become a better scientist. I would like to thank the California Institute for Regenerative Medicine’s Bridges program which granted me a generous stipend as well as funds for me to work at the Beckman Research Institute at City of Hope. I am thankful for the members of the Salvaterra lab, Martha Magallanes and Dr. Saumya Srivastava, for all of their help during my research. I would also like to thank Dr. Beer and everyone at the CSUSB Office of Student Research who have supported me in my research and made it possible for me to attend several conferences as well as expand my knowledge. Finally, I am thankful to my family and my husband who have always believed in me, pushed me to be better, and continue to support me throughout all my pursuits in life. v TABLE OF CONTENTS ABSTRACT .......................................................................................................... iii ACKNOWLEDGEMENTS ..................................................................................... v LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ...............................................................................................ix CHAPTER ONE: ALZHEIMER’S IS A CHRONIC, PROGRESSIVE, AND COMPLEX DISEASE Background ................................................................................................ 1 Neuropathology ......................................................................................... 2 Proteolytic Production of Aβ....................................................................... 3 Where is Aβ Produced? ............................................................................. 5 Tau Tangle Formation and Its Pathological Appearance in AD ................. 6 Vesicular Accumulation in AD .................................................................... 7 The Genetics of AD ................................................................................... 7 Current Models .......................................................................................... 9 Constructing a New Human ES Cell Based Model .................................. 12 CHAPTER TWO: NEURONAL DEVELOPMENT App and Aβ Insert Expression .................................................................. 14 Alzheimer’s is Not a Developmental Disease .......................................... 17 CHAPTER THREE: ALZHEIMER’S-LIKE PHENOTYPES Aβ Aggregation ........................................................................................ 22 Synapse Decrease .................................................................................. 24 Neurodegeneration .................................................................................. 26 Changes in Gene Expression and Pathway Analysis .............................. 28 vi CHAPTER FOUR: SUMMARY AND DISCUSSION ........................................... 36 CHAPTER FIVE: MATERIALS AND METHODS Cell Culture and Maintenance .................................................................. 40 Quantitative Real-Time PCR .................................................................... 43 Immunocytochemistry .............................................................................. 44 Image Analysis ........................................................................................ 46 Brightfield Imaging ................................................................................... 47 Statistical Analysis ................................................................................... 47 APPENDIX A: INSERTED CASSETTE SEQUENCES USING TALEN .............. 48 APPENDIX B: RNA SEQUENCING DATA ......................................................... 51 REFERENCES ................................................................................................... 57 vii LIST OF TABLES Table 1. Quantitative Real-Time PCR Primers ................................................... 44 Table 2. Antibodies Used for Immunocytochemistry ........................................... 45 viii LIST OF FIGURES Figure 1. Aβ Formation via the Amyloidogenic Pathway. ..................................... 4 Figure 2. Expression of Aβ1-40/42 Inserts at Different Developmental Stages. ..... 15 Figure 3. Expression of App and Aβ in Neurons of All Three Genotypes.