Inactivation of Erk1 and Erk2 Disrupts Cortical
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INACTIVATION OF ERK1 AND ERK2 DISRUPTS CORTICAL PROGENITOR PROLIFERATION LEADING TO ABNORMAL CYTOARCHITECTURE, CIRCUITRY AND BEHAVIOR, MODELING HUMAN NCFC AND RELATED SYNDROMES. by JOANNA PUCILOWSKA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Gary E. Landreth Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Joanna Pucilowska candidate for the PhD degree*. (signed) Robert Miller (chair of the committee) Gary Landreth Jerry Silver Stephen Maricich (date) 5/29/2012 *We also certify that written approval has been obtained for any proprietary material contained within. 2 TABLE OF CONTENTS List of figures……...……………………………………………………………….….6 ABSTRACT…...………………………………………………………………….…..9 CHAPTER 1: INTRODUCTION………………………………………………………….11 MAP KINASE Signaling Pathway………………………………………………...11 MAPK Specificity: The Right Place at the Right Time………………………..…16 ERKs and Isoform Specificity………………..…………………………………….23 ERKs in Learning and Memory………………………..………………………….26 ERKs and their FGF Ligands………………………..…………………………….28 CORTICAL DEVELOPMENT: Forebrain Regionalization and Morphogenesis………..…………………31 The Role of Cell Cycle in Corticogenesis……..…………...………………33 ERKs and the Cell Cycle Progression…………………..…………………36 Progenitor Proliferation and Neurogenesis………………..……………...39 Migration…………………..………………………………………………..45 Gliogenesis…………………………………………………………..………47 Differentiation……………………………………………………..………..48 Synaptogenesis…………………………………………………..………….50 Pathology Associated with Cortical Development……………………..…………52 Developmental Disorders of the MAP Kinase Pathway…………………..……...57 ERK and Autism……………………..……………………………………………..63 Research Goals……...………………………………………………………..……..65 3 CHAPTER 2: Disrupted ERK signaling during cortical development leads to abnormal progenitor proliferation, neuronal and network excitability and behavior, modeling human NCFC and related syndromes……………………………..…...67 Abstract…………………………………………………………………..….68 Introduction………………………………………………………..………..69 Materials and Methods………………………………………………..……71 Results…………………………………………..……………………...……82 Discussion………………………………………..………………………….95 Literature Cited……….……………………………………………..…....101 Figures…………………………………………..……………………….....106 CHAPTER 3: The Role of ERKs in Interneuron Migration………………………………..…..134 Introduction………………………………..………………………………134 Results…………………………………………..………………...………..139 Discussion……………………………………………..……...……………146 CHAPTER 4: DISCUSSION…………………………………………………….……………….149 FGF Signaling, ERKs and Cortical Development……………………....152 ERKs and Cortical Progenitor Dynamics……………………………..…157 ERKs and Cognition…………………………………...…………..……...160 Disorders of the Ras/MAPK pathway and Cognitive Deficits…….....…166 4 The contribution of MAPK/ERK signaling to Autism Spectrum Disorders associated with copy number variation of 16p11.2……………….…..…171 Potential Impact on Autism Research…………………..………………..176 Towards a Treatment for Developmental Disorders of the MAPK Pathway……………..………………………………………..…………….177 Conclusion……………………………………………………………..…..180 Literature Cited……………………………………………………..…….181 5 LIST OF FIGURES Figure 1-1 Mitogen Activated Protein Kinase cascade………………………..….12 Figure 1-2 Basic signal transduction pathway mediating ERK activation…..….15 Figure 1-3 Progression through the cell cycle is controlled by ERKs………..….39 Figure 1-4 Division modes of the expansion of the cerebral cortex in mice: Lateral and Radial………………………………………………………………….41 Figure 1-5 Disorders of the MAP Kinase Pathway………………..……………...60 Figure 2-1 ERK activity is abrogated in the dorsal telencephalon of ERK2 CKO and ERK1/2 DKOs……………………………………………………………..….106 Figure 2-2 Loss of ERK1 and ERK2 leads to layer perturbations in the adult cortex……………………………………………………..………………………...109 Figure 2-3 The frequency of neurons generated during mid-neurogenesis is altered in mutant mice lacking ERK1 and/or ERK2…………………………....111 Figure 2-4 ERK2 CKO mice show reduction in Pax6+ progenitors, mitotic radial glia and transition to intermediate progenitor cells…………………………..…113 Figure 2-5 Loss of ERK2 disrupts basal progenitor frequency and generation, resulting in premature progenitor pool depletion…………………………….....115 Figure 2-6 ERK2 CKO neural progenitors exhibit premature cell cycle exit and lengthening of the cell cycle during mid-neurogenesis………………..………...117 Figure 2-7 Loss of ERK2 alters expression of key cell cycle regulators cyclin D1 and p27Kip1 in the developing cortex………………..……………………………120 6 Figure 2-8 Cell-intrinsic electrophysiological parameters are altered in ERK2 mutant mice………………………………………………………………..………122 Figure 2-9 Network-level electrophysiological parameters are altered in Erk2 mutant mice…………………………………………………………………..……124 Figure 2-10 Loss of ERK2 leads to behavioral alterations and cognitive impairment in adult mice…………………………………………………….…...126 Supplementary Figure 2-1 The cortical size and body weight is reduced in the ERK2 CKO and DKO mice………………………………………………..…..…128 Supplementary Figure 2-2 Apical and basal mitosis are reduced in the CKO and DKO mice during mid-neurogenesis…………………………………..…………130 Supplementary Figure 2-3 Cell death in not significantly altered in the developing dorsal telencephalon of CKO mice……………………..…………...133 Figure 3-1 Do laminar alterations in the dorsal telencephalon caused by ERK1/2 deletion affect the migration and final positioning of the sub-pallially derived interneurons?............................................................................................................138 Figure 3-2 The migration pattern but not the total number of SST+ interneurons is altered in the ERK2 deficient dorsal cortex at E13.5……………………...….141 Figure 3-3 The migration pattern but not the total number of SST+ interneurons is altered in the ERK2 deficient dorsal cortex at E16.5……………...………….143 Figure 3-4 Loss of ERK2 from dorsal telencephalon results in an increase in SST+ interneurons in dorsal but not lateral postnatal cortex……………...…..145 Figure 3-5 Loss of ERK2 from dorsal telencephalon results in a decrease in Calretinin+ interneurons in the cortex of P10 mice……………………….……146 7 Figure 4-1 Cell cycle progression during cortical development……………......151 Figure 4-2 The Wnt and Notch developmental pathways that are important in corticogenesis are not altered in ERK deficient mice…………………..……….156 Figure 4-3 ERK deficient mice exhibit changes in hippocampal volume and severely reduced Tbr2+ progenitor population in the dentate gyrus……...…..164 Figure 4-4 Mutations in upstream and downstream elements of the MAPK/ERK pathway lead to NCFC and other developmental syndromes………..……...…167 Figure 4-5 General schematic illustrating changes in cortical development in ERK deficient mice………………………………………………………………..170 Figure 4-6 The cell cycle dependent mechanism of ERK action during cortical development……………………………………………………………………….171 8 The Role of ERKs in Cortical Development ABSTRACT by JOANNA PUCILOWSKA Genetic disorders arising from copy number variations in the ERK1 and ERK2 MAP kinases and mutations in their upstream regulators result in Neuro- cardio-facial cutaneous (NCFC) syndromes that are a significant genetic cause of mental retardation in humans. Furthermore, these disorders are associated with developmental abnormalities, cognitive deficits and psychiatric disease. Deletions or duplications of the human ERK genes provide the strongest genetic linkage to autism spectrum disorders and to distal 22q11 syndrome. Our findings demonstrate that deletion of one or both ERK isoforms at the beginning of neurogenesis disrupts apical and basal progenitor generation and proliferation, altering cortical cytoarchitecture of the adult brain in a gene-dose dependent manner. The changes in proliferation dynamics were due to ERK-dependent regulation of the potent cell cycle progression regulators, cyclin D1 and p27Kip1. Loss of ERK signaling in dorsal telencephalic progenitors resulted in altered levels of cyclin D1 and p27Kip1 leading to premature elongation of the cell cycle, which favors neurogenic over self-renewing divisions. The precocious neurogenesis caused premature progenitor pool depletion, subsequently altering the number and distribution of cortical pyramidal neurons in the postnatal cortex. Importantly, loss of ERK2 alters the intrinsic excitability of cortical 9 neurons and contributes to global perturbations in network activity. Together, these molecular and physiological changes observed in the ERK-deficient mice may contribute to severe anxiety and cognitive deficits observed in these mice. This study describes in detail the functional mechanisms through which ERKs act to regulate cortical development, providing a novel insight into their important role in normal brain function as well as why deregulation of this pathway may contribute to the pathology associated with autism and related cognitive disorders. 10 INTRODUCTION: All eukaryotic cells possess multiple mitogen-activated protein kinase (MAPK) pathways, which coordinately regulate diverse cellular functions. The mammalian family of MAPKs is divided into five distinct subfamilies, based on sequence homology, activity and substrate specificity. The MAPK family includes the Extra cellular signal Regulated Kinases (ERK1 and ERK2, also known as p44 and p42, respectively), the stress activated protein kinases p38MAPKs (α, β and γ) and c- Jun NH2-terminal kinases (JNK1, 2 and 3), as well as ERKs 3, 4