A Dissertation entitled Development of a Novel Pharmacological Model of Okadaic Acid-induced Alzheimer’s Disease in Zebrafish and its use in Drug Discovery by Daniel M Koehler Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics _________________________________________ Dr. Frederick Williams, Committee Chair _________________________________________ Dr. Zahoor Shah, Committee Member _________________________________________ Dr. Jeffrey Sarver, Committee Member _________________________________________ Dr. Caren L. Steinmiller, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2018 Copyright 2018, Daniel Maximilian Koehler This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Development of a Novel Pharmacological Model of Okadaic Acid-induced Alzheimer’s Disease in Zebrafish and its use in Drug Discovery by Daniel M Koehler Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics The University of Toledo August 2018 Alzheimer’s disease (AD) is a progressive neurodegenerative disease hallmarked by the presence of amyloid beta deposition forming plaques and neurofibrillary tangles (NFTs) that are comprised mainly of hyperphosphorylated tau protein. AD is the most common form of dementia and is currently the sixth leading cause of death in the United States. Even though AD has been heavily studied in the past several decades, no cure exists and the exact etiology of the disease is unknown and the factors that are essential for its progression are also not well understood. Many animal models of AD have been developed and have contributed to the understanding of the disease. However, these animal models are not ideal with many of the transgenic models only manifesting a portion of the disease pathology, developing motor impairments (which make cognition testing difficult), and being cost and time inefficient. In addition, the current AD animal models have yet to yield a successful drug to treat AD. Therefore, it is crucial that new animal models to study the AD pathology and to do preclinical testing of drugs are developed. The studies presented here utilized a newly developed AD model in zebrafish. A protein phosphatase inhibitor, okadaic acid iii (OKA), was dissolved in water and the zebrafish were housed in the water for a total of 9 days. This treatment resulted in the formation of many AD pathological hallmarks including cognitive decline, phosphorylation of tau, the formation of plaques, and the increase in kinase expression. The studies presented here were designed to further characterize the model and simultaneously use it as a drug screening tool. The first study showed that TDZD-8, a glycogen synthase kinase 3β (GSK3β) inhibitor, is able to treat the OKA-induced AD pathology by rescuing cognitive function, downregulating active GSK3β, downregulating pTau (Ser199), and normalizing protein phosphatase 2A activity. GSK3β has been established as a target for treating AD because of its role in phosphorylating tau. This study served as a proof of concept that the OKA-induced AD model in zebrafish can be utilized as a drug screen tool. The last studies demonstrated that a novel therapeutic, lanthionine ketimine-5-ethyl ester (LKE), is able to treat OKA insults by rescuing cognitive function and upregulating the BDNF/Akt/CREB pathway and reducing apoptosis in the zebrafish dorsal lateral pallium which is homologous to the human hippocampus. iv Acknowledgements I would like to thank Dr. Frederick Williams for being a patient advisor over the course of my dissertation. Your clear perspective and guidance throughout my troubles during my time at the University of Toledo helped me tremendously and kept me grounded, focused, and confident. Thank you for making me a better student, researcher, and person. I also would like to thank my committee members; Dr. Zahoor Shah, Dr. Jeffrey Sarver, and Dr. Caren Steinmiller. All 3 of you were a tremendous help. You were always there for me when I was seeking advice, even when it came to matters outside of research, and for that, I thank you dearly. I would like to thank my lab partner, Alexander Wisner, who has been in the lab with me during my entire Ph.D. study. You were the one who taught me everything from the beginning and always kept me even. More than a lab partner, you are a great lifelong friend. I would like to thank my family. You were honest with me and kept me strong and focused during my struggles and you celebrated my achievements in a way that inspired me. I love you all. Holly Helminski. Words cannot express the gratitude that I have for you. You have been beyond wonderful, and someone whom I will remember forever. Lastly, I would like to thank the entire Department of Pharmacology and Experimental Therapeutics at the University of Toledo for being accommodating and for allowing me this great opportunity. It has been a journey that has been difficult but has served me in becoming a better human. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables .....................................................................................................................x List of Figures .................................................................................................................... xi List of Abbreviations ........................................................................................................ xii List of Symbols ..................................................................................................................xv 1 General Introduction: Modeling Alzheimer’s Disease in Zebrafish to Aid in Drug Discovery.................................................................................................................1 1.1 Alzheimer’s Disease .........................................................................................2 1.2 Amyloid Hypothesis….. ...................................................................................2 1.3 Tau Hypothesis .................................................................................................3 1.4 Interplay between Amyloid and Tau .................................................................4 1.5 Tau Protein Kinases ..........................................................................................5 1.6 Tau Protein Phosphatases .................................................................................7 1.7 Using Okadaic Acid to Study Neurodegeneration ............................................7 1.8 Modeling Alzheimer’s Disease in Zebrafish.….. .............................................9 1.9 Utilizing Okadaic Acid in Zebrafish ...............................................................11 References…………… ....................................................………………………..13 vi 2 The GSK3β inhibitor, TDZD-8, Rescues Cognition in a Zebrafish Model of Okadaic Acid-induced Alzheimer’s Disease .........................................................28 2.1 Introduction .....................................................................................................29 2.2 Materials and Methods…... .............................................................................31 2.2.1 Animals .............................................................................................31 2.2.2 Drug Treatment .................................................................................32 2.2.3 Learning and Memory Test ...............................................................32 2.2.4 PP2A Activity Assay ........................................................................33 2.2.5 Western Blotting ...............................................................................34 2.2.6 Statistical Analysis ............................................................................34 2.3 Results…….. ...................................................................................................35 2.3.1 Treatment with TDZD-8 reduced mortality induced by OKA .........35 2.3.2 TDZD-8 rescues the OKA induced cognition impairments .............37 2.3.3 Pre-treatment cognition results .........................................................37 2.3.4 Post-treatment cognition results ........................................................38 2.3.5 OKA treated zebrafish exhibit reduced activity of PP2A .................43 2.3.6 OKA treated zebrafish exhibit reduced expression of PP2A ............45 2.3.7 TDZD-8 reduces the tau kinase, GSK3β, active: inactive expression levels in OKA treated zebrafish………… .................................................45 2.3.8 TDZD-8 reduces the expression of phosphorylated tau in OKA treated zebrafish tau kinase……… ............................................................45 2.4 Discussion. ......................................................................................................48