Wip1 regulates proliferation of adult neural progenitors through p53 dependent g2 cell cycle control

WIP1 REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53-DEPENDENT G2 PHASE CELL CYCLE CONTROL ZHU YUNHUA NATIONAL UNIVERSITY OF SINGAPORE 2009 WIP1 REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53-DEPENDENT G2 PHASE CELL CYCLE CONTROL ZHU YUNHUA 2009 WIP1 REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53-DEPENDENT G2 PHASE CELL CYCLE CONTROL ZHU YUNHUA (B.Sci.(Merit), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENT First, I would like to thank my supervisors: Associate Professor Xiao ZhiCheng, for taking me as a graduate student in his lab, for his continuous guidance and supervision throughout the past three years. I would like to express my gratitude to Associate Professor Dmitry Bulavin, for excellent mouse models provided and insightful discussions. I feel much indebted and express my big thank to my supervisor Associate Professor Ng Yee Kong, for the key helps and encouragement during the last stage of my candidature and the critical reading and corrections on this thesis, without which I would not be able to submit this thesis. I am especially thankful to the Head of the Department, Professor Bay Boon Huat, for his kind understanding and help in accepting me into the department, and the great assistance and support provided during my candidature. Without him, I would neither be able to join the department nor to complete my Ph.D work and thesis. I am especially thankful to the administrative help and advice rendered by Ms Geetha Sreedhara Warrier and Madam Ang Lye Geck, Carolyne. I would like to thank the members of my Thesis Advisory Board, Associate Professor Yang Xiaohang, Dr. He Beiping and Dr. Gavins Stewart Dawe, for their invaluable discussions and suggestions towards the completion of this project. I would like also thank Associate Professor Tay Sam Wah, Samuel and Dr. Fu Jiang for their help in trouble shooting problems regarding culturing neural stem cells. I am grateful to my colleagues in Professors Xiao’s and Dmitry’s laboratories, for their encouragement and suggestions to this project. Special thank goes to Dr. i Zhang Chengwu for providing assistance in Western blot technique, Xavier and Jocelyn who read and provided critical comments and suggestions to the thesis. I would also thank my friend Dr. Yu Faxing for the encouragement, the information and the pressure that make the completion of the thesis possible. Last but not least, I deeply appreciated my family, especially my father, mother and my wife, who support me, encourage me throughout the course of my study. ii Table of Contents Acknowledgement i Table of Contents iii List of Figures viii Abbreviations x Publication and Awards xiv Summary xv Chapter 1, General Introduction 1.1 Preface 1.2 Adult neurogenesis 1.2.1 Ontology of adult NSCs 1.2.2 Processes in adult neurogenesis 1.2.3 Functional significance of adult neurogenesis 1.2.4 NPCs in aging and transformation 1.2.5 Principle of common used methods in the study of adult neurogenesis 1.2.5.1 BrdU short- and long-term labeling 1.2.5.2 Neurosphere assays 11 1.2.5.3 Differentiation assay 11 1.3 Wip1 phosphatase 12 1.3.1 General features of Wip1 12 1.3.2 Wip1 knockout mice 15 1.3.3 Wip1 in tumorigenesis 15 iii 1.3.4 Wip1 in DNA repair and cell cycle regulation 17 1.3.5 Possible role of Wip1 in neurogenesis 18 1.4 Wip1 related cell cycle regulators in adult neurogenesis 21 1.4.1 A general theme of cell cycle regulation 21 1.4.2 Regulation of NPCs by DNA damage signaling molecules 22 1.4.2.1 ATM in hippocampal neurogenesis 22 1.4.2.2 p53-p21 in NPC proliferation and self-renewal 23 1.4.2.3 Bax in NPC spontaneous apoptosis 26 1.4.3 Regulation of NPCs by p38MAPK stress signaling molecules 29 1.4.3.1 p38MAPK in hippocampal neurogenesis 29 1.4.3.2 Bmi1-p16Ink4ap19Arf in proliferation and self-renewal 30 1.4.4 NPCs in aging and transformation 33 1.4.4.1 Wip1 related molecules in aging 33 1.4.4.2 NPC transformation 36 1.5 Aims, hypothesis and strategies 37 1.5.1 Aims and perspectives 37 1.5.2 Hypothesis 37 1.5.3 Questions to address 37 1.5.4 Strategies 38 Chapter 2, Materials and Methods 39 2.1 In vivo methods 40 2.1.1 Animals 40 2.1.2 Antibodies 43 2.1.3 BrdU injection 43 iv 2.1.4 Immunohistochemistry 44 2.1.5 Counting of immuno-positive cells 45 2.1.6 TUNEL staining 46 2.1.7 Brain dissection 47 2.2 In vitro methods 47 2.2.1 Neurosphere experiments 47 2.2.2 Immunocytochemistry and quantification 49 2.2.3 CFSE pulse labeling 50 2.2.4 PI labeling and flow cytometry 50 2.3 Molecular and biochemical methods 51 2.3.1Real-time PCR 51 2.3.2 Western blot 55 2.4 Data analysis 55 Chapter 3, Results 56 3.1 Wip1 deficiency decreases new neuron formation and NPC activity in vivo 57 3.1.1 Wip1 deficiency decreased new neuron formation in olfactory bulb 57 3.1.2 Wip1 deficiency reduced the number of SVZ NPCs in vivo 63 3.1.3 Wip1 deficiency reduced SGZ neurogenesis in hippocampus 68 3.2 Functional analysis of Wip1 ko NPCs in vitro 71 3.2.1 Wip1 deficiency decreased amplification of NPCs 71 3.2.2 Wip1 deficiency impaired self-renewal of NPCs 77 3.2.3 Wip1 deficiency diminished neuronal differentiation in vitro 79 3.3 The G2/M transition is impaired in Wip1 deficient NPCs 81 v 3.3.1 Wip1 ko NPCs exhibited prolonged cell cycle 81 3.3.2 G2 to M phase transition was compromised upon knocking out Wip1 83 3.4 Molecular abnormalities in Wip1 ko NPCs 85 3.4.1 Phosphorylation of p53 and expression of cell cycle inhibitors are both elevated in neurospheres and SVZ 85 3.4.2 The elevation of cell cycle inhibitors was p53-dependent 89 3.5 p53 mediates the repressed cell cycle abnormalities in Wip1 ko NPCs 91 3.5.1 Knocking out p53 released the restriction on M phase entry 91 3.5.2 Knocking out p53 drove NPC cell cycle shorter than normal 91 3.6 Wip1 modulates NPC amplification in a p53-dependent manner in vitro and in vivo 93 3.6.1 In vitro phenotypes were rescued by knocking out p53 93 3.6.2 In vivo phenotypes were rescued by knocking out p53 in forebrain 98 3.6.3 In vivo phenotypes were rescued by knocking out p53 in hippocampus 102 3.7 Physiological regulation of Wip1-p53 pathway during aging 107 3.7.1 Wip1 expression decreased while phospho-p53 increased during aging 107 3.7.2 Transgenic expression of Wip1 increased neurogenesis in aged mice 112 Chapter 4, Discussion and Conclusion 115 4.1 G2/M transition is important in mammalian NPCs 116 vi 4.2 Wip1-p53 pathway is critical for adult hippocampal neurogenesis 122 4.3 ATM is probably not involved in NPC proliferation 123 4.4 p38MAPK may not mediate the function of Wip1 in NPCs 127 4.5 Implications of the Wip1/p53 pathway on NPC aging and transformation 128 4.5.1 The change of Wip1-p53 balance during aging may contribute to age related functional decline of NPCs 128 4.5.2 A proposed model of NPC aging 131 4.5.3 A possible role of Wip1 in brain tumor formation 133 4.6 Conclusion 134 4.7 Remaining questions 134 4.7.1 What are upstream regulators of p53? 134 4.7.2 How does Wip1/p53 regulate NPC self-renewal? 135 4.7.3 What regulates the activity of Wip1? 136 References 138 vii Gil-Perotin S, Marin-Husstege M, Li J, Soriano-Navarro M, Zindy F, Roussel MF, Garcia-Verdugo JM and Casaccia-Bonnefil P (2006) Loss of p53 induces changes in the behavior of subventricular zone cells: implication for the genesis of glial tumors. 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Nat Neurosci 9(2): 268-275. 161 [...]... presentation Wip1 regulate proliferation of adult neural progenitors through p53 dependent G2 cell cycle control Singapore General Hospital 17th Annual Scientific Meeting 2008 Date: 25 April 08, Friday Abstract published in SGH Proceedings 17(1):45-48 Poster Presentation Wip1 regulates neural stem cell/ progenitor proliferation through p53 dependent G2 cell cycle control Institute of Molecular and Cell Biology... amplification of Wip1- null NPCs were both reversed in Wip1/ p53 double-null genotype to that comparable to p53- null genotype Functional rescues by Wip1/ p53 double null genotype were reproduced in vivo with the number of NPCs and neuroblasts Present data demonstrate that Wip1 regulates the generation of new neural cells in adult olfactory bulb specifically through p53- dependent M phase entry of the cell cycle of. .. Wip1 At cellular level, Wip1 knockout (ko) NPCs exhibited a prolonged cell cycle, an accumulation at G2 to M phase transition and elevated expression of cell cycle inhibitors p21 and Reprimo Two observations suggested that Wip1 regulates NPCs through p53, which are the phosphorylation level of p53 was up-regulated in Wip1 ko NPCs in vivo and in vitro, and transcriptions of p21 and Reprimo was p53- dependent. .. Figure 3.16: Elevations of p21 and Reprimo are p53- dependent in Wip1 ko NPCs 90 Figure 3.17: NPC cell cycle regulation by Wip1 is dependent on p53 92 viii Figure 3.18: The compromised amplification of NPCs in Wip1 ko mice is p5 3dependent 94 Figure 3.19: Regulation by Wip1 on NPC self-renewal is p53- dependent 96 Figure 3.20: Regulation by Wip1 on neurogenesis in vitro is p53- dependent 97 Figure 3.21:... Ultra violet VZ Ventricular zone Wip1 Wild-type p53- induced phosphatase 1 Wip1Tg Wip1 transgenic wt Wild type xiii Publication and Awards: Zhu Yunhua, Zhang Cheng-Wu, Lu Li, Demidov Oleg N., Sun Li, Yang Lan, Bulavin Dmitry V., Xiao Zhi-Cheng (2009) Wip1 Regulates the Generation of New Neural Cells in the Adult Olfactory Bulb through p53- Dependent Cell Cycle Control Stem Cells 27(6):1433-1422 Award Young... deficiency of ATM or p38MAPK does not affect SVZ NPC numbers in vivo 99 Figure 3.22: Regulation of NPC number in vivo by Wip1 is p53- dependent 100 Figure 3.23: Reduction of the OB size in Wip1 ko mice is p53- dependent 101 Figure 3.24: p53 deficiency elevates the number of NPCs in SGZ of adult hippocampus 104 Figure 3.25: p53 knockout rescues the number of NPCs in adult SGZ 105 Figure 3.26: Wip1 regulation... this study, Wip1 is identified as a critical regulator of adult neurogenesis acting through modulating p53- dependent G2 progression in NSCs and progenitors Three components of background information will be reviewed in this chapter: a general theme of adult neurogenesis; a summary of known functions of wild-type p53- induced phosphatase 1 (Wip1) ; and current knowledge of NPC regulation by Wip1 related... of NPCs in adult SVZ 65 Figure 3.4: Characterization of effects of Wip1 ko on different cell types in SVZ 66-7 Figure 3.5: Knocking out Wip1 decreases neurogenesis in adult hippocampus 69 Figure 3.6: Knocking out Wip1 decreases long-term survival of new cells in adult hippocampus 70 Figure 3.7: Wip1 deficiency reduces proliferation of NPCs in vitro 73 Figure 3.8: Neurosphere diameters of Wip1 wt and... Reduction of amplification of Wip1 ko NPCs is not dependent on specific growth factors 75 Figure 3.10: BrdU labeling in vitro 76 Figure 3.11: Impaired self-renewal of Wip1 ko NPCs 78 Figure 3.12: Impaired neurogenesis of Wip1 ko NPCs 80 Figure 3.13: Cell cycle abnormalities of Wip1 ko NPCs 82 Figure 3.14: M phase entry is impaired in Wip1 deficient NPCs 84 Figure 3.15: Molecular abnormalities of Wip1 ko... implication of Wip1/ p53 pathway on aging of NPCs was analyzed by real-time PCR and immunostaining During aging, the number of NPCs was down- xv regulated, which correlated with a marked down-regulation of Wip1 and a drastic upregulation of p53 phosphorylation Based on these data, Wip1/ p53 regulation was proposed to mediate the aging process of NPCs This study led to the identification of the important . REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53- DEPENDENT G 2 PHASE CELL CYCLE CONTROL ZHU YUNHUA 2009 WIP1 REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53- DEPENDENT. WIP1 REGULATES PROLIFERATION OF ADULT NEURAL PROGENITORS THROUGH P53- DEPENDENT G 2 PHASE CELL CYCLE CONTROL ZHU YUNHUA NATIONAL UNIVERSITY OF SINGAPORE 2009 WIP1 REGULATES. the generation of new neural cells in adult olfactory bulb specifically through p53- dependent M phase entry of the cell cycle of NPCs. The implication of Wip1/ p53 pathway on aging of NPCs was