THE ROLES OF ERK1 AND ERK2 MAP KINASE IN NEURAL DEVELOPMENT AND DISEASE by IVY SAMUELS Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Gary E. Landreth Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Ivy S. Samuels _______________________________________________________________ candidate for the Ph.D. degree.* Stephen O’Gorman _______________________________________________________________ chair of the committee Gary E. Landreth _______________________________________________________________ Robert H. Miller _______________________________________________________________ Ruth E. Siegel _______________________________________________________________ 06/19/2008 * We also certify that written approval has been obtained for any proprietary material contained therein. For my teachers, past and present, who inspired me. TABLE OF CONTENTS Table of Contents………………………………………………………………….1 List of Tables………………………………………………...……………………4 List of Figures……………………………………………………………………..5 Acknowledgements……………………………………………………………..…7 Abstract…………………………………………………………………………....9 Chapter 1: Introduction..……………………………………………………….11 Mitogen-Activated Protein Kinase Signal Transduction……………………..12 Structural Deformities of the Cerebral Cortex and MR: A Historical Perspective…………………………………………………………………....17 Controlling Cortical Development: The cell cycle…………………………...19 ERK1/2 and the Cell Cycle…………………………………………………...22 Cortical Development I: The Expansion of Progenitor Cells………………...26 Cortical Development II: Neurogenesis……………………………………...28 Modes of Neuronal Generation…………………………………………..29 Cortical Malformations and Disrupted Division…………………………30 Cortical Development III: Migration………………………………………....32 Interkinetic migration during Neurogenesis……………………………...32 Cortical Malformations and Disrupted Migration………………………..34 Cortical Development IV: Differentiation of Neurons and Synaptogenesis....35 Molecular Mechanisms of Cortical Development…………………………....36 MAPK and Cell Fate Determination…………………………………………39 Disruptions in upstream elements of the MAPK signaling cascade lead to 1 anatomical and behavioral impairments in mice………………………....41 Aberrant ERK1/2 Signaling leads to Developmental Disorders…………45 ERK in Learning and Memory……………………………………………….47 Individual Roles for the ERK1 and ERK2 Isoforms………………………....49 Research Goals……………………………………………………………….54 Literature Cited……………………………………………………………….56 Chapter 2: Conditional Inactivation of ERK2 Identifies Its Key Roles in Cortical Neurogenesis and Cognitive Function………………….……………………...68 Abstract………………………………………………………………………69 Introduction…………………………………………………………………..69 Materials and Methods.………………………………………………………72 Results………………………………………………………………………..81 ERK2 is required for normal development of the cerebral cortex……….81 ERK2 inactivation alters the cellular composition of the cortex ………..83 ERK2 inactivation alters the dynamics of neurogenesis………………...86 ERK2 CKO neural progenitors generate fewer neurons in vitro…..........88 Mature ERK2 CKO mice display impaired associate learning………….89 MAPK1/ERK2 deficiency is associated with learning deficits………….90 Discussion…………………………………………………………………...92 Figures………………………………………………………………………98 Literature Cited……………………………………………………………..128 Chapter 3: ERK1 and ERK2 are required for cell cycle regulation of neural progenitor cells………………………………………………………………...132 2 Abstract……………………………………………………………………..132 Introduction…………………………………………………………………133 Materials and Methods………...……………………………………………136 Results……..………………………………………………………………..140 ERK1/2 DKO mice die embryonically, but display normal brain morphology……………………………………………………………..140 Loss of both ERK1 and ERK2 does not exacerbate the alterations in cellular composition identified in the ERK2 CKO cortex……………………....141 ERK1/2 DKO NPCs display inhibited cell cycle progression attributable to reduced in cyclin D2 and increased p27kip1 expression…...…………..142 Discussion…………………………………………………………………..144 Figures………………………………………………………………………150 Literature Cited……………………………………………………………...166 Chapter 4: Discussion…………………..……………………………………...169 Isoform specific expression regulates isoform specific function…………...169 ERK and Neurogenesis/the Cell Cycle……………………………………..174 ERK and Cognitive Function/Mental Retardation…………………………179 ERK and 22q11 Deletion Syndrome……………………………………….182 22q11 and psychopathology……………………………………………183 22q11 physical pathology………………………………………………185 ERK and Affective Function/Mood Disorders……………………………..187 Literature Cited……………………………………………………………..191 Chapter 5: Literature Cited…………………………………………………..198 3 LIST OF TABLES Table 3-1 Inactivation of ERK2 does not alter the morphology of Layer III cortical neurons………………………………………...………………………………..127 Table 3-2 Inactivation of ERK2 does not alter the morphology of Layer V cortical neurons………………………………………………………………………….127 4 LIST OF FIGURES Figure 1-1 MAPK Signal Transduction…………………………………………13 Figure 1-2 General cycle cycle regulation……………………………………….20 Figure 1-3 Temporal development of the murine cerebral cortex...……………..21 Figure 1-4 ERK1/2 regulates cell cycle entry and progression………………….23 Figure 1-5 Modes of Cellular Generation in the Developing Cerebral Cortex….30 Figure 1-6 ERK1/2 Activity Regulates Cell Fate Determination………………..40 Figure 1-7 Mutations in elements of the MAPK Signaling Cascade lead to MR and CFC Syndromes………………………………..……………………………46 Figure 2-1 Generation of ERK2 Conditional Knockout mice…………………...99 Figure 2-2 ERK2 CKO mice display reduced cortical thickness………………101 Figure 2-3 Loss of ERK2 expression and activity in ERK2 CKO cortices…….103 Figure 2-4 Inactivation of ERK2 in neural progenitor cells results in generation of fewer cortical neurons…………………………………………………..……105 Figure 2-5 Inactivation of ERK2 in neural progenitor cells results in the presence of more astrocytes within the cerebral cortex…………………………107 Figure 2-6 ERK2 CKO mice display changes in the dynamics of NPC proliferation……………………………………………………………………..109 Figure 2-7 ERK2 CKO NPCs exhibit reduced neuronal generation…………....111 Figure 2-8 ERK2 CKO cortical progenitors generate more astrocytes in the presence of gliogenic stimuli…………………………………………………...113 Figure 2-9 Male ERK2 CKO mice have deficits in associative learning……....115 5 Figure 2-10 ERK2 expression is abolished in NPCs beginning at E13.5.………117 Figure 2-11 ERK2 expression is reduced in the hippocampus, but not the amygdala……………………………………………………………….………..119 Figure 2-12 The ERK2 CKO cortex does not exhibit apoptosis……….……….121 Figure 2-13 The number of Pax6 and Tbr2 immunoreactive cells at E14.5 is not altered by conditional inactivation of ERK2…………………………………....123 Figure 2-14 Patients with deletions of distal 22q11 have reduced ERK2 levels..125 Figure 3-1 Generation of the ERK1/2 Double Knockout Mouse………………151 Figure 3-2 ERK1/2 Double knockout mice die embryonically but display normal brain morphology...……………………………………………………………..153 Figure 3-3 Loss of ERK1/2 expression and activity in DKO cortices………….155 Figure 3-4 Inactivation of ERK1 and ERK2 in neural progenitor cells results in the generation of fewer cortical neurons…………………………………..……157 Figure 3-5 DKO NPCs exhibit reduced neuronal generation…………………..159 Figure 3-6 DKO neural progenitor cells exhibit reduced proliferation throughout neurogenesis………………………………………………………………….…161 Figure 3-7 DKO mice exhibit reductions in number of mitotic Tbr2+ intermediate progenitor cells………………………………………………………………….163 Figure 3-8 Decreased proliferation of DKO NPCs is mediated by reductions in cyclin D2 and increased p27 expression……………...………………………...165 Figure 4-1 Mutations in elements of the MAPK Signaling Cascade lead to MR and CFC Syndromes…………...……………………………………………….179 6 ACKNOWLEDGEMENTS To Dr. Gary Landreth. Without his unwavering belief in my ideas, his enthusiasm for science, and his ability to follow the biology, this dissertation would not have been possible. His patience, understanding and caring have nurtured my life and my career. To my thesis committee, Dr. Robert Miller, Dr. Stephen O’Gorman, Dr. Ruth Siegel and Dr. Karl Herrup, their ideas and advice were extremely helpful. To the Landreth Lab. It has been said that this is a special place to work. It is so much more, because it is a home and a family. I can’t thank them enough for their advice, challenges and support in all endeavors. I would especially like to express my gratitude to Colleen Karlo for her initial and continuous contributions to this project, her endless support, and her contagious laughter. To the Alzheimer Laboratory. I didn’t study AD, but they didn’t care. I would like to thank them for all their advice, help and the fun times. To my parents, who recognized and nurtured my potential and encouraged me to become the first doctor in our family. And to Vicki and Mike. The love and support of my family inspires me everyday. 7 To Randall, who is not only an amazing scientist, but also my best friend and cheerleader. I am most appreciative of his support, advice, and love. And to Zander, Malone and Callie, my munchkins. To my immediate, extended, and “Cleveland” families and friends. They fill my life with happiness and were always interested, supportive and encouraging. I would specifically like to thank Jessica Lerch, Brandy Wilkinson, Barbara Schwartz, the Kossiver’s, and the York’s for helping to make Cleveland a wonderful place to work and live. 8 The Roles