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Spinophilin-Dependent Regulation of The SPINOPHILIN-DEPENDENT REGULATION OF THE PHOSPHORYLATION, PROTEIN INTERACTIONS, AND FUNCTION OF THE GLUN2B SUBUNIT OF THE NMDAR AND ITS IMPLICATIONS IN NEURONAL CELL DEATH by Asma Beiraghi Salek A Dissertation Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Biology at IUPUI Indianapolis, Indiana December 2020 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. AJ Baucum II, Chair Department of Biology Dr. Theodore Cummins Department of Biology Dr. Nicolas Berbari Department of Biology Dr. Yvonne Lai Department of Psychological and Brain Sciences Dr. Andy Hudmon Department of Medicinal Chemistry and Molecular Pharmacology Dr. Jason Meyer Department of Medical and Molecular Genetics Approved by: Dr. Theodore Cummins 2 �ﻮ � ﻮ �ای ﻣﺎ� و �ﺪر و �ای روز� �ﺨﺎ�� ��ﻖ و �ﻤﺎی� ﮫﺎی �ﯽ ��ﻎ و �ﺨﺎ�� � ﻮدﺷﺎن �� �� � ﺧﺎ�ﻢ �ﺸﺎش ﻌ�ﻢ ��� م �ﻪ ﺑﺎ ��ﺮ و ا�تﯿﺎق و �ایت � ﮑﺎو�ﻢ �ای ﻋ�ﻮم ز �ﯽ را �ورا�ﺪ �� و د��ﺮ �ع�� و�ﻦ ��ﺖ �ﻪ ﺑﺎ �ﺮا�ت و دا�ﺶ و �ﮔﺎ�ﯽ �ﻌ� ﻪ ��ﻘﺎت ���و ا�ﺼﺎب را � ذ��ﻢ �ا��و�ﺖ 3 ا�ﺮار ازل را � �ﻮ دا�ﯽ و � �ﻦ � و�ﻦ ﻞ ��ﻤﺎ � �ﻮ �ﻮا�ﯽ و � �ﻦ �� �سﺖ �ز �ﺲ �ده ��ﻮی �ﻦ و � ﻮ �ﻮن �ده �ا��ﺪ � �ﻮ ﻣﺎ�ﯽ و � �ﻦ ﺣ��ﻢ ��� �ﯿﺎم 4 If you keep knocking on a door long enough, sooner or later someone will answer you. 5 ACKNOWLEDGMENTS I would like to acknowledge Dr. A.J. Baucum, my supervisor, for believing in me, for his help, advice, patience, guidance, and full support during my years in his lab. Also, Drs. Nicolas Berbari, Theodore Cummins, Yvonne Lai, Andy Hudmon, and Jason Meyer for serving on my advisory committee and providing much-needed feedback and constructive criticism. I would also like to acknowledge the Biology Department at IUPUI for giving me the opportunity to further my education in such a positive environment. Sue Merrell, Shari Dowell, Laura Flak, Angela Longfellow, Julie Driscol and Anita Sale for their care and help along the way. I would like to acknowledge Drs Mike Edler and Sarah Ohlemacher for their help and advice during all these years. Thank you, Cameron, Kaitlyn and Darryl. I have learned so much from you. Thank you for your help and support and for creating a fun, positive and interactive environment in the lab. And thank you for setting the bar so high with being hardworking and caring at the same time. I would also like to give my very special thanks to Indiana CTSI, Eli Lilly and Company and Stark Neurosciences research institute for believing in me and giving me the opportunity to focus on my research by selecting and generously supporting me as a Eli Lilly/Stark Neurosciences predoctoral fellow. Thank you to Dr. Lisa Jones and the Indiana School of Medicine Proteomics Core facility for mass spectrometry-based sequencing. These studies were supported by NIH grant (K01NS073700/ R21/R33DA041876) IUPUI School of Science and Department of Biology to AJB and partly by the Stark Neurosciences Research Institute, the Indiana Alzheimer Disease Center, Eli Lilly and Company, and by the Indiana Clinical and Translational Sciences Institute to ABS, funded in part by grant # UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 6 TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ 11 LIST OF FIGURES ...................................................................................................................... 12 LIST OF ABBREVIATIONS ....................................................................................................... 14 ABSTRACT .................................................................................................................................. 16 INTRODUCTION .............................................................................................. 17 Hippocampus, Hippocampal Function, And Behavior ..................................................... 17 NMDA Receptor Subunits and Function .......................................................................... 19 The Balance Between Protein Kinases and Phosphatases ................................................ 21 NMDA Receptor Phosphorylation .................................................................................... 23 Spinophilin Function and Localization ............................................................................. 25 Hypothesis......................................................................................................................... 29 SPINOPHILIN REGULATES PHOSPHORYLATION AND INTERACTIONS OF THE GLUN2B SUBUNIT OF THE N-METHYL-D-ASPARTATE RECEPTOR ............... 30 Abstract ............................................................................................................................. 30 Introduction ....................................................................................................................... 30 Materials and Methods ...................................................................................................... 32 Materials .................................................................................................................... 32 Mutagenesis ............................................................................................................... 33 Mammalian Protein Expression ................................................................................. 33 Mice ........................................................................................................................... 34 Tissue homogenization .............................................................................................. 35 IPs .............................................................................................................................. 35 Immunoblotting ......................................................................................................... 36 Mass Spectrometry .................................................................................................... 37 Statistical Analyses .................................................................................................... 38 Randomization, Preregistration, and Blinding ........................................................ 39 Results ............................................................................................................................... 39 Spinophilin Associates With GluN2B Subunit Of NMDARs ................................... 39 7 Overexpression Of The Catalytic Subunit Of PKA (PKAc) In HEK293 Cells Increases Spinophilin And GluN2B Interaction .................................................................................... 41 Activation Of Endogenous PKA Increases The Interaction Between Spinophilin And The GluN2B Subunit ............................................................................................................. 42 Spinophilin-GluN2B Interaction Domains ................................................................ 43 Spinophilin Decreases The Association Of PP1 With GluN2BTail ............................ 46 Spinophilin Rescues PP1-Dependent Dephosphorylation Of Ser-1284 On GluN2BTail…… ..................................................................................................................... 47 Spinophilin KO Mice Have Enhanced GluN2B-PP1 Interaction And Decreased Ser- 1284 Phosphorylation In P28 Mouse Hippocampus ............................................................. 51 Spinophilin KO Modulates The P28 GluN2B Interactome ....................................... 54 Discussion ......................................................................................................................... 57 Mechanisms Regulating The Spinophilin-GluN2B Interaction ................................ 57 Spinophilin Attenuates PP1 Targeting To GluN2B ................................................... 58 Spinophilin Enhances GluN2B Phosphorylation At Ser-1284 .................................. 58 Loss Of Spinophilin Alters GluN2B Phosphorylation And The GluN2B Interactome in vivo ................................................................................................................................... 61 Summary ........................................................................................................................... 62 SPINOPHILIN-DEPENDENT REGULATION OF GLUN2B CONTAINING NMDA RECEPTORS AND GLUTAMATE TOXICITY ........................................................... 63 Abstract ............................................................................................................................. 63 Introduction ....................................................................................................................... 63 Materials and Methods ...................................................................................................... 65 Reagents ..................................................................................................................... 65 Animals ...................................................................................................................... 67 Mutagenesis ............................................................................................................... 67 Mammalian
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