University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2009 Reelin Signaling Promotes Radial Glia Maturation and Neurogenesis Serene Keilani University of Central Florida Part of the Microbiology Commons, and the Molecular Biology Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Doctoral Dissertation (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Keilani, Serene, "Reelin Signaling Promotes Radial Glia Maturation and Neurogenesis" (2009). Electronic Theses and Dissertations, 2004-2019. 6143. https://stars.library.ucf.edu/etd/6143 REELIN SIGNALING PROMOTES RADIAL GLIA MATURATION AND NEUROGENESIS by SERENE KEILANI B.S. University of Jordan, 2004 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida Spring Term 2009 Major Professor: Kiminobu Sugaya © 2009 Serene Keilani ii ABSTRACT The end of neurogenesis in the human brain is marked by the transformation of the neural progenitors, the radial glial cells, into astrocytes. This event coincides with the reduction of Reelin expression, a glycoprotein that regulates neuronal migration in the cerebral cortex and cerebellum. A recent study showed that the dentate gyrus of the adult reeler mice, with homozygous mutation in the RELIN gene, have reduced neurogenesis relative to the wild type. Based on the above findings, our first hypothesis states that Reelin expression is important for the formation of radial glia and the generation of neurons from the neural progenitors. In order to investigate the role of Reelin in the process of cortical neurogenesis during development, we used human neural progenitor cells (hNPCs) that were isolated from a fetal cortex. These cells do not express Reelin. In this study, we show that Reelin addition to these hNPCs in vitro induced the formation of radial glia and increased neurogenesis significantly. Next, we investigated the mechanism by which Reelin increases the formation of radial glia and the generation of neurons. The formation of radial glia is under the control of two pathways, these are the Reelin and the Notch-1 signaling pathways. Since the level of Notch-1 activation determines if a cell would become a radial glia or an astrocyte, and since the absence of Reelin allows the transformation of a radial glia into astrocyte, we hypothesized that Reelin induces the formation of radial glia via activating Notch-1 signaling. To test this hypothesis, we investigated the effect of Reelin addition on Notch-1 activation in hNPCs. We found that Reelin addition in vitro activated Notch-1 signaling by increasing the level of Notch-1 intracellular domain (NICD). On the other hand, reducing NICD release, by inhibiting γ -secretase activity, inhibited iii the Reelin-induced radial glia, confirming that Reelin’s effect on the formation of radial glia is dependent on Notch-1 activation. Furthermore, we found that the Reelin-induced tyrosine phosphorylation of Disabled-1 (Dab-1), an adaptor protein downstream of Reelin, and the subsequent activation of Src family kinases, are essential steps for Notch-1 activation by Reelin. Finally, we found that Reelin addition increased the binding of Dab-1, recently identified as a nucleoshuttling protein, to NICD and enhanced NICD translocation to the nucleus. This resulted in the induction of BLBP expression and the subsequent formation of radial glia. Taken together, these data show that Reelin signaling, mediated by Dab-1 and Src kinase, activates Notch-1 signaling in hNPCs resulting in the induction of BLBP expression, the formation of radial glia and the generation of neurons. This work is novel because it provides that first evidence that Reelin expression is an important signal for the neuronal differentiation of the hNPCs. It also shows the crosstalk between Reelin and Notch-1 signaling, two major pathways in development and cell fate determination. The work is significant because it improves our understanding of the role of Reelin signaling in cell fate determination, differentiation and neurogenesis for the future manipulation of these processes to restore adult brain functions after brain injury or in neurodegenerative diseases. iv I dedicate this work for all my family and friends who gave me endless support and love and kept their faith in me all the way. Special thanks to Dr. Kiminobu Sugaya, my mentor for five years. I thank you for all you have taught me over the years, for your patience, and continuous support. v ACKNOWLEDGMENTS I thank my committee members, Dr. Steven Ebert, Dr. Sic L. Chan and Dr. James Turkson for all their help, feedback and support. vi TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................................ xi CHAPTER ONE: GENERAL INTRODUCTION ........................................................................ 1 Adult Neurogenesis and Radial Glial Cells ................................................................................ 1 Reelin Signaling in Radial Glial Cells ........................................................................................ 2 Notch-1 Signaling in Radial Glial Cells ..................................................................................... 6 Amyloid Precursor Protein and Neurogenesis ............................................................................ 7 Rationale and Aims ..................................................................................................................... 9 CHAPTER TWO: REELIN INDUCES A RADIAL GLIAL PHENOTYPE IN HUMAN NEURAL PROGENITOR CELLS BY ACTIVATION OF NOTCH-1 ...................................... 12 Introduction ............................................................................................................................... 12 Materials and Methods .............................................................................................................. 15 Results ....................................................................................................................................... 19 Discussion ................................................................................................................................. 37 CHAPTER THREE: REELIN ACTIVATES NOTCH-1 SIGNALING THROUGH DISABLED-1 TYROSINE PHOSPHORYLATION ................................................................... 39 Introduction ............................................................................................................................... 39 Materials and Methods .............................................................................................................. 41 vii Results ....................................................................................................................................... 48 Discussion ................................................................................................................................. 73 CHAPTER FOUR: DISABLED-1 PROMOTES γ-SECRETASE-MEDIATED PROCESSING OF AMYLOID PRECURSOR PROTEIN ................................................................................... 76 Introduction ............................................................................................................................... 76 Materials and Methods .............................................................................................................. 79 Results ....................................................................................................................................... 84 Discussion ............................................................................................................................... 101 CHAPTER FIVE: GENERAL CONCLUSION ........................................................................ 103 REFRENCES .............................................................................................................................. 108 viii LIST OF FIGURES Figure 1: A schematic diagram that illustrates the proposed role for Reelin in neurogenesis........ 5 Figure 2: A schematic diagram that demonstrates the proposed cross-talk between Reelin and Notch-1 signaling pathways. ......................................................................................................... 11 Figure 3: The formation of radial glia in hNPCs is dependent on the growth factors FGF and EGF. .............................................................................................................................................. 21 Figure 4: Reelin levels in the media of HEK293 cells that are stably transfected with Reelin. ... 23 Figure 5: Reelin treatment in vitro induces a radial glial phenotype. ........................................... 24 Figure 6: Reelin treatment of hNPCs induces Dab-1 phosphorylation on tyrosine residue 198. 26 Figure 7: Reelin treatment induces neurogenic radial glial cells in hNPCs. ................................ 28 Figure 8: Reelin