Ben-Gurion University of the Negev Faculty of Engineering Sciences The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering The Impact of PLEKHM2 Mutation on Neuronal Differentiation and Impaired Autophagy Thesis Submitter in Partial Fulfillment of the Requirements for the M.Sc. Degree By Hadas Ben-Zvi Supervised by Prof. Smadar Cohen, Dr. Rivka Ofir and Dr. Ginat Narkis October 2020 Ben-Gurion University of the Negev Faculty of Engineering Sciences The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering The Impact of PLEKHM2 Mutation on Neuronal Differentiation and Impaired Autophagy Thesis Submitter in Partial Fulfillment of the Requirements for the M.Sc. Degree By Hadas Ben-Zvi Supervised by Prof. Smadar Cohen, Dr. Rivka Ofir and Dr. Ginat Narkis Author:………………………… Date:……………..24.9.2020 Supervisor:…………………….. Date:……………..24.9.2020 Supervisor:…………………….. Date:……………..29.9.2020 Supervisor:…………………….. Date:…29.9.2020………….. Chairman of Graduate Studies Committee:………… Date:…………….30.9.20 . October 2020 I Abstract Mutated PLEKHM2 (mPLEKHM2) causes dilated cardiomyopathy with left ventricular noncompaction (DCM-LVNC) resulting in a premature death of mPLEKHM2 individuals due to heart failure. PLEKHM2 (also known as SKIP) is a factor in autophagy, a master regulator in cell homeostasis, decomposes pathogens, proteins and other cellular compartments. Autophagy is mainly carried out by the lysosome that contains enzymes for degradation, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 is a part of a protein complex which allows lysosomal movement toward the cell periphery, therefore as anticipated, mPLEKHM2 patient fibroblasts exhibited perinuclear localization of the lysosomes. This could be one of the reasons for the autophagic dysregulation discovered in patient fibroblasts cells, resulting with a severe disease DCM-LVNC. Autophagic dysregulation was observed in the context of neurodegenerative diseases. Many factors could stimulate neuronal cell death in neurodegenerative disorders such as cellular stressors, bioenergy failure, misfolded protein accumulation and impaired autophagy. Thus, modulation of autophagy holds considerable potential as a therapeutic approach to these diseases. Previously, induced pluripotent stem cells (iPSCs) were generated from mPLEKHM2 patient and healthy fibroblasts in our laboratory (BGU Regenerative Medicine and Stem Cell -RMSC). Our hypothesis is that neurons derived from iPSCs with mPLEKHM2 will present impaired functions as compared to normal iPSCs-derived neurons. In our study, a protocol of directed neural differentiation has been employed and optimized upon two healthy and two patient (mutated) iPSC lines including characterizing neurons in the culture. Both control and patient lines showed similar pattern of differentiation into immature motor neuron cells; the cultures were composed of 64% Tuj1+ cells, 28% GFAP+ cells and 6% isl1+ cells. An assay designed to identify autophagosome accumulation at different stages of the differentiation showed that patient lines had a trend of reduced generation of autophagic bodies during D6 and D18. Neuron functionality was examined using microelectrode array (MEA). Patient cells presented maximal activity during D30 to D33 of differentiation, with irregular and II more frequent firing rate as compare to healthy cells. Our results suggest that patient neuronal cell cultures were unable to maintain homeostasis properly as result of impaired autophagy flux. Keywords: Neurons, Motor Neurons, Autophagy, PLEKHM2, DCM-LVNC, Cardiomyopathy, Neurodegeneration, Lysosomes, Autophagosomes, iPSCs, Disease Model. III Acknowledgments This work was performed under the supervision of Prof. Smadar Cohen, Dr. Rivka Ofir and Dr. Ginat Narkis in the Department of Biotechnology, Ben-Gurion University of the Negev. I would like to thank all three for many hours spent debating our study and future progress, excellent guidance, encouragement and support. A very special thanks to Dr. Gad Vatine, whose door was always open, sharing his abundant knowledge of neuroscience whole heartedly. Also, to Tatiana Rabinski for taking me under her wing, like any student, teaching and assisting me with great patience and respect. Last but not least, my boyfriend Ran, who supported me through this study with endless love, by my side come what may. IV Table of Contents Abstract....................................................................................................................................... I Keywords................................................................................................................................... II Acknowledgments.................................................................................................................... III Table of Contents ......................................................................................................................IV List of abbreviations ................................................................................................................ VI List of Figures ........................................................................................................................VIII List of Tables ............................................................................................................................IX 1. Introduction .......................................................................................................................... 1 1.1. PLEKHM2 Mutation Causes Dilated Cardiomyopathy (DCM) with Left Ventricular Noncompaction (LVNC)………………………………………………………………1 1.2. Induced Pluripotent Stem Cells (iPSCs) as a DCM-LVNC Disease Model………..….2 1.2.1. DCM-iPSCs………………………………………………………………………3 1.3. Autophagy…………………………………………………………………………….5 1.3.1. Autophagosome………………………………………………………………….5 1.3.2. Lysosome………………………………………………………………………...6 1.3.3. DCM Fibroblasts and Autophagy………………………………………………...7 1.4. Neurodegenerative Diseases…………………………………………………………..8 1.5. Neural Disease Models………………………………………………………………..9 1.5.1. Neural Subtypes in Scientific Research…………………………………………10 1.5.2. Motor Neuron Differentiation…………………………………………………...10 1.5.3. Autophagy and Neurodegeneration…………………………………………..…11 1.5.3.1. Stress Induction……………………………………………………….12 2. Research Hypothesis and Objectives...................................................................................14 2.1. Research Hypothesis…………………………………………………………………14 2.2. Objectives……………………………………………………………………………15 3. Materials and Methods ........................................................................................................16 V 3.1. Materials .................................................................................................................... 16 3.2. Cell Culture..................................................................................................................16 3.3. Monolayer Differentiation............................................................................................16 3.4. Karyotype Analysis......................................................................................................17 3.5. Mycoplasma Contamination Examination...................................................................17 3.6. Flow Cytometry Quantification....................................................................................18 3.7. Autophagy Flux Detection............................................................................................18 3.8. Microelectrode Array (MEA) Assay.............................................................................18 3.9. Western Blot.................................................................................................................19 3.10. Immunocytochemistry.......................................................................................19 3.11. Metabolism Assay.............................................................................................20 3.12. Statistical Analysis............................................................................................20 4. Results ............................................................................................................................... 21 4.1. Characterization of DCM-iPSC ..................................................................................21 4.2. MN Differentiation......................................................................................................22 4.3. Stress Induced Autophagy Flux...................................................................................25 4.3.1. Flow Cytometry....................................................................................................26 4.3.2. Western Blot.........................................................................................................28 4.4. Functional Tests...........................................................................................................29 4.4.1. Cellular Metabolism..............................................................................................29 4.4.2. iPSCs-derived MN Activity..................................................................................32 5. Discussion ..........................................................................................................................33 5.1. Mutation in PLEKHM2 Does Not Impact Neural Differentiation…………………33 5.2. Stress Induction of mPLEKHM2 Cells Results