GDE3 FUNCTION IN OLIGODENDROCYTE PRECURSOR CELL PROLIFERATION By Mateusz Dobrowolski A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland November, 2018 Abstract Oligodendrocyte specification, proliferation and differentiation are tightly controlled by extrinsic signals; however, proteins that modulate cellular response to these factors remain unclear. Six-transmembrane GDEs are emerging as central regulators of cellular differentiation via their unique ability to shed GPI-anchored proteins from the cell surface. We show here that GDE3 controls the pace of oligodendrocyte generation by negatively regulating oligodendrocyte precursor cell (OPC) proliferation. GDE3 inhibits OPC proliferation by stimulating CNTF-mediated signaling through release of CNTFRα, the ligand-binding component of the gp130/LIFRβ CNTF receptor complex that can function as a soluble factor. GDE3 releases CNTFRα by GPI-anchor cleavage from the plasma membrane and from extracellular vesicles (EVs) after co-recruitment of CNTFRα in EVs. These studies uncover new physiological roles for GDE3 in gliogenesis and identify GDE3 as a key regulator of CNTF-dependent inhibition of OPC proliferation through bi-modal release of CNTFRα. Thesis Advisor: Dr. Shanthini Sockanathan, Ph.D, Professor of Neuroscience, The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine Reader: Dr. Alex Kolodkin, Ph.D, Professor of Neuroscience, The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine ii Table of Contents Abstract ...................................................................................................................... ii Table of Contents ..................................................................................................... iii List of Tables ........................................................................................................... vii List of Figures ......................................................................................................... viii Chapter 1 Introduction .................................................................................................. 1 1.1 CNS cell diversity and oligodendrocyte development................................. 2 1.1.1 Development of CNS ..................................................................................... 2 1.1.2 Oligodendrocyte development ....................................................................... 3 1.1.2.1 Oligodendrocyte Proliferation ................................................................. 4 1.1.2.2 Oligodendrocyte Migration ..................................................................... 5 1.1.2.3 Oligodendrocyte Differentiation .............................................................. 5 1.1.3 Gp130 family of cytokines in CNS development ............................................ 7 1.1.3.1 Gp130 signaling in neuroepithelial precursor cells ................................. 8 1.1.3.2 Gp130 signaling in glia ........................................................................... 9 1.2 GDEs – Six-transmembrane domain GDPD containing proteins .............. 10 1.2.1 GDE2........................................................................................................... 10 1.2.2 GDE3........................................................................................................... 11 1.3 Extracellular vesicles ................................................................................... 12 1.3.1 1.3.1 Biogenesis of EV ................................................................................ 13 1.3.2 Release mechanisms of EV ........................................................................ 14 1.3.3 EV content and signaling ............................................................................. 14 iii 1.4 Summary and Specific Aims ....................................................................... 16 Chapter 2 GDE3 regulates OPC proliferation and differentiation in vivo ............... 22 2.1 Introduction .................................................................................................. 23 2.2 Results .......................................................................................................... 23 2.2.1 GDE3 regulates cell cycle kinetics of ventral progenitors in spinal cord ............................................................................................................................... 23 2.2.2 Loss of Gde3 results in increased OPC production and differentiation in vivo ........................................................................................................................ 24 2.3 Summary....................................................................................................... 26 Chapter 3 GDE3 regulates proliferation and differentiation of purified OPCs ....... 34 3.1 Introduction .................................................................................................. 35 3.2 Results .......................................................................................................... 35 3.2.1 GDE3 is necessary to inhibit OPC proliferation ....................................... 35 3.2.2 GDE3 controls the maturation of OPCs .................................................... 36 3.2.3 GDE3 is sufficient to inhibit OPC proliferation ......................................... 36 3.3 Summary....................................................................................................... 37 Chapter 4 Identifying pathways that mediate GDE3-dependent regulation of OPC proliferation ................................................................................................................. 41 4.1 Introduction .................................................................................................. 42 4.2 Results .......................................................................................................... 42 4.2.1 RNA-seq approach ...................................................................................... 42 4.2.2 Functional annotation of differentially enriched genes ........................... 43 iv 4.2.3 CNTFRα, a candidate GPI-AP mediating cytokine signaling ................... 44 4.3 Summary........................................................................................................ 45 Chapter 5 GDE3 regulates CNTF-CNTFRα signaling in proliferating OPCs ........... 52 5.1 Introduction .................................................................................................. 53 5.2 Results .......................................................................................................... 53 5.2.1 CNTF inhibits OPC proliferation in a GDE3 dependent manner ............. 53 5.2.2 Neutralization of CNTFRα-GP130 signaling increases OPC proliferation .......................................................................................................... 54 5.3 Summary....................................................................................................... 55 Chapter 6 GDE3 mediates bimodal release of CNTFRα ........................................... 58 6.1 Introduction .................................................................................................. 59 6.2 Results .......................................................................................................... 59 6.2.1 GDE3 releases CNTFRα via two separate mechanisms .......................... 59 6.2.2 Specific domains of GDE3 regulate CNTFRα release and other GPI-APs ............................................................................................................................... 61 6.2.3 GDE3’s release of CNTFRα correlates with inhibition of OPC proliferation .......................................................................................................... 63 6.2.4 GDE3 is present on EVs and is enzymatically active ............................... 64 6.2.5 Exogenous CNTFRα rescues hyperproliferation defects of Gde3 KO OPCs ..................................................................................................................... 65 6.3 Summary....................................................................................................... 66 Chapter 7 Summary and Future Directions .............................................................. 76 v 7.1 Summary....................................................................................................... 77 7.2 Discussion and Future Directions .............................................................. 79 7.2.1 GDE3 function in OPC proliferation and differentiation .......................... 79 7.2.2 GDE3 regulation of CNTF signaling .......................................................... 80 7.2.3 GDE3 and EV release .................................................................................. 83 Materials and Methods ................................................................................................ 87 References ................................................................................................................... 95 Biography .................................................................................................................. 108 vi List of Tables Table 1.1 gp130 Cytokines and their receptor components. ...........................