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Circadian Control of Adult Neural Progenitor Cell Behavior in Vivo and in Vitro

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Title:
Circadian Control of Adult Neural Progenitor Cell Behavior in Vivo and in Vitro a Molecular and Genetic Screen of Regulatory Candidates for Neuroregenerative Therapies
Creator:
Hoang-Minh, Lan B.
Place of Publication:
[Gainesville, Fla.]
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University of Florida
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Language:
english
Physical Description:
1 online resource (181 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
Ormerod, Brandi K.
Committee Members:
Ogle, William
Carney, Paul R
Reynolds, Brent
Graduation Date:
12/15/2012

Subjects

Subjects / Keywords:
Adults ( jstor )
Cell cycle ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Dentate gyrus ( jstor )
Neurogenesis ( jstor )
Neurons ( jstor )
Neuroscience ( jstor )
Progenitor cells ( jstor )
Rats ( jstor )
Biomedical Engineering -- Dissertations, Academic -- UF
cell -- circadian -- cycle -- estradiol -- genes -- hippocampus -- neurogenesis -- progenitors
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
Biomedical Engineering thesis, Ph.D.

Notes

Abstract:
Identifying the mechanisms that either stimulate or allowneurogenesis in the adult hippocampus could lead to the development of excitingnew strategies for treating neurodegenerative diseases and injuries. Chapter 1discusses the history, significance, and potential of endogenous andtransplantable stem cell strategies for CNS repair. Previous work suggests thatthe level of hippocampal neurogenesis is potentiated in nocturnal adult miceduring the dark (active) versus light (active) phase of the light-dark (LD) cycle.The experiments described in Chapter 2 directly tested whether neuralprogenitor cell (NPC) division, neuronal differentiation, and/or survival ofNPC progeny vary across the LD cycle. These experiments show that, during thedark cycle, more dividing NPCs are detected, and more surviving neurons areproduced in the hippocampal dentate gyrus than during the light cycle. Interestingly,more dividing cells were detected at night in the substantia nigra, but noevidence of neuronal differentiation among new cells was observed. Theexperiments in Chapter 3 show that the rate of NPC division in vivo is increased during the dark phase and the expression of cell cyclegenes in the hippocampus varies between light and dark phases. Chapter 3 experiments also show that moreprogenitor cells exit the multipotential state at night, and the expression levelsof genes stimulating neuronal differentiation and survival are significantly increasedin the hippocampi of mice harvested during the dark versus light phase,differentially between female and male adult mice. Next, in vitro experiments were conducted to test whether the NPC proliferationand/or daughter cell differentiation varied with diurnal-like changes ofpro-mitotic estradiol (which increases in mammals during the active versusinactive phase of the LD cycle; Chapter 4).In vitro analysis demonstrated thatNPC proliferation increases in the presence of a high versus low dose ofestradiol, while estradiol exposure has no effect on young neurondifferentiation or survival after 1 week in culture. Chapter 5 summarizes the circadiancycle-induced changes in components of neurogenesis that were identified inthis thesis and how they might influence normal hippocampal function, neuroregenerativestrategies, and potentially even strategies for combating brain tumors. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Ormerod, Brandi K.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31
Statement of Responsibility:
by Lan B. Hoang-Minh.

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UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
12/31/2014
Classification:
LD1780 2012 ( lcc )

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1 CIRCADIAN CONTROL OF ADULT NEURAL PROGENITOR CELL BEHAVIOR IN VIVO AND IN VITRO : A MOLECULAR AND GENETIC SCREEN OF REGULATORY CANDIDATES FOR NEURO REGENERATIVE THERAPIES By LAN B. HOANG MINH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Lan B. Hoang Minh

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3 To my parents, Mau and Phuong To my brother and sister, Dany and Bich Thu

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4 ACKNOWLEDGMENTS I would like to express my sincere and deepest gratitude to my thesis advisor, Dr. Brandi Ormerod; I am greatly thankful for her support and guidance throughout my graduate experience. I am grateful for the time she spent discussing experiments, papers, an d reviewing my publications, oral and poster presentations, and dissertation. Thanks to her mentorship, I have gained invaluable knowledge of how to be an effective and rigorous scientist and will always be grateful for her academic as well as moral suppor t. I would also like to thank Dr. Paul Carney, Dr. Brent Reynolds, and Dr. William Ogle who devoted time from their busy schedules to serve on my committee and mentor me. I am very grateful for their continuing support and very valuable advice throughout my graduate studies. My research experience in the Ormerod research group was very enjoyable, particularly thanks to the company and friendship of my fellow group members, Dr. Crystal Stephens, Rachel Speisman, Vikram Munikoti, and Aditya Asokan, and I am very grateful for all their help and support in the laboratory. I would also like to thank Christina Bonarrigo, James Su, Steve Noutong, and Angie Posada for assisting with various projects and being wonderful mentees. The Biomedical Engineering Department is a friendly place where professors and students are always helpful, and I would like to give special thanks to Dr. Hans Van Oostrom, Dr. Bruce Wheeler, and Dr. Ben Keselowsky for their support and valuable discussions. Thanks also go to Dr. Jamal Lewis and Dr. Phil Barish for their support and assistance with RNA analysis, Jennifer Lee, Natalia Dolgova, and Joe Uzarski for lending a helping hand whenever I needed, members of the Foster laboratory,

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5 especially Dr. Ashok Kumar, the Cohn laboratory, especial ly Dr. Zheng Fu and Marissa Gredler, and Tifiny McDonald and Kathryn Whitesides for all their logistic help and kindness during my graduate studies in the Biomedical Engineering Department. I am grateful to members of the HHMI Science for Life program, pa rticularly Director Dr. David Julian and Dr. Gabriela Waschewsky, for providing me with the opportunity to be part of a wonderful and progressive undergraduate teaching effort. Thanks also go to Matt Hintze and the TutoringZone team for their support and a llowing me to help hundreds of students succeed in their biochemistry course. I would like to give special thanks to my Gainesville tennis (and soccer) teammates for their friendship and for contributing to providing me with healthy and enjoyable outlets I cannot list everyone here, but I would like to particularly thank the following people, who were always present for me on and off the courts: Robin, Vanessa, Tahereh, Basma, Fran, Alexis, Thomas, Yann, Abdoulaye, and Dave. I would also like to express my gratitude to my friend Delphine Mico for her valuable friendship and for being a great role model of kindness and perseverance Thanks also go to Mr. and Mrs. Whitener for being my second family in Florida (and for great food, art activities, and fun scalloping and fishing trips!). Finally, I would like to express my heartiest gratitude to the most important people in my life. I am very thankful to Chase Whitener for being a pillar of support, always being there for me through my tribulations, frustrations, and joys, and supporting me in every way possible. My family has always been present, supportive, and encouraging during my journey at the University of Florida, and I cannot thank them enough. Without them, nothing I have accomplished to date would have been possible. I

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6 would like to particularly thank my sister, as well as my brother and sister in law, my grand parents, aunts and my close family members living in the United States, France, and Canada. Above all, I would like to express my deepest gratitude to my parents for shaping me into the person I have become. Their endless love, support, and encouragement have kept me s trong.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 16 CHAPTER 1 NEURAL PROGENITOR CELLS, ADULT NEUROGENESIS, AND THE CIRCADIAN CLOCK ................................ ................................ ............................... 18 Neural Progenitor Cells and Adult Neurogenesis ................................ .................... 19 The Neurogenic Niche ................................ ................................ ............................ 24 Regulation of Adult Neurogenesis ................................ ................................ .......... 27 Proliferation and Differentiation (Table 1 1) ................................ ...................... 27 Survival, Maturation, and Integration of Newborn Neurons (Table 1 1) ............ 32 Functional Implications of Adult Neurogenesis ................................ ....................... 34 Circadian Cycle and Cell Division ................................ ................................ ........... 35 Neurogenesis and Cell Cycle ................................ ................................ .................. 38 Circadian Cycle and Neurogenesis ................................ ................................ ......... 39 Summary ................................ ................................ ................................ ................ 42 2 PROGENITOR CELLS DIVIDING DURING THE DARK PHASE OF THE LIGHT DARK CYCLE GENERATE MORE HIPPOCAMPAL NEURONS IN ADULT MICE BUT NO NIGRAL NEURONS ................................ .......................... 44 Introduction ................................ ................................ ................................ ............. 44 Experimental Procedures ................................ ................................ ........................ 46 Subjects ................................ ................................ ................................ ............ 46 Experiment Design ................................ ................................ ........................... 46 Histological procedures ................................ ................................ .................... 47 Immu nohistochemistry ................................ ................................ ...................... 47 Data Analysis ................................ ................................ ................................ ... 50 Statistical Analysis ................................ ................................ ............................ 52 Results ................................ ................................ ................................ .................... 52 NPC proliferation in the dentate gyrus and the substantia nigra is increased with activity and during the dark phase of the LD cycle ................................ 52 Neuronal differentiation of daughter cells in the dentate gyrus is increased among progenitor cells dividing during the dark versus light phase and in a nimals exposed to a running wheel ................................ ............................. 54

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8 Cell survival in the substantia nigra is increased among progenitor cells labeled with BrdU during the dark phase in animals exposed to a running wheel, with no neuronal differentiation ................................ .......................... 59 Discussion ................................ ................................ ................................ .............. 60 3 VARIATIONS IN HIPPOCAMPAL PROGENITOR CELL PROLIFERATION AND DIFFERENTIATION BETWEEN THE LIGHT (SLEEP) AND DARK (WAKE) PHASES MIGHT BE RELATED TO CHANGES IN CELL CYCLE KINETICS AND GENE EXPRESSION IN ADULT MICE ................................ .......................... 65 Introduction ................................ ................................ ................................ ............. 65 Experimental Procedures ................................ ................................ ........................ 68 Subjects ................................ ................................ ................................ ............ 68 Experiment Design ................................ ................................ ........................... 69 Bromodeoxyuridine (BrdU) preparation, injection and histology ...................... 71 Immunofluorescence ................................ ................................ ........................ 72 Cumulative 5 Bromo Deoxyuridine (BrdU) Labeling ................................ ..... 75 Immunohistochemistry ................................ ................................ ...................... 77 Pyknotic Cell Analysis ................................ ................................ ...................... 78 Quanti tative RT PCR ................................ ................................ ....................... 79 Statistical Analysis ................................ ................................ ............................ 80 Results ................................ ................................ ................................ .................... 80 More Cells are Found in G 2 M and S Phases at Night versus Day in the Hippocampus of Female Adult Mice ................................ .............................. 80 Cell Death Does not Influence Observed Cell Proliferation Variations between Light and Dark Phases ................................ ................................ ... 84 Cell Cycle Gene Expression Differs between Night and Day in the Hippocampus of Female and Male Adult Mice ................................ .............. 84 Neural progenitor cells produced at night exit the multipotential, undifferentiated state faster than those produced during the day. ................. 87 Four week old neurons generated at night have more extensive branching than those produced during the day. ................................ ............................. 89 Differentiation gene expression differs between night and day in the hippocampus of female and male adult mice ................................ ................ 90 Discussion ................................ ................................ ................................ .............. 94 4 ESTRADIOL INCR EASES THE PROLIFERATION BUT NOT DIFFERENTIATION OF ADULT FEMALE NEURAL PROGENITOR CELLS IN CULTURE ................................ ................................ ................................ ............. 104 Introduction ................................ ................................ ................................ ........... 104 Experimental Procedures ................................ ................................ ...................... 106 Experiment Design ................................ ................................ ......................... 106 In Vitro I mmunofluorescence ................................ ................................ .......... 109 Confocal Microscopy ................................ ................................ ...................... 111 Cell Cycle Kinetics ................................ ................................ .......................... 112 Statistical Analysis ................................ ................................ .......................... 112

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9 Result s ................................ ................................ ................................ .................. 113 High Dose Estradiol Exposure Increases DNA Replication and Entry into G 0 Phase of NPCs in Culture ................................ ................................ ........... 113 High Dose Estradiol Exposure Decreased Synthesis Phase and Total Cell Cycle Lengths ................................ ................................ ............................. 115 Estradiol Dose and Exposure Duration Affect the Expression of Estrogen Receptors ................................ ........................... 116 Estradiol Exposure does not Influence the Differentiation or the Survival of NPCs in Culture 1 Week after Plating ................................ ......................... 118 Discussion ................................ ................................ ................................ ............ 119 5 GENERAL DISCUSSION ................................ ................................ ..................... 126 The Circadian Cycle Influences Cell Proliferation, Cell Cycle Kinetics, and Cell Cycle Gene Expression ................................ ................................ ........ 127 Estradiol Influences the Cell Proliferation of Neural Progenitors In Vitro ........ 131 The Circadian Cycle Influences the Differentiation and Survival of Daughter Cells and Maturation Associated Gene Expression ................................ .... 132 A Hypothetical Model of the Interactions between Circadian Timing System, Estradiol, and Cell Cycle Machinery ................................ ............................ 13 3 Functional Implications of Adult Neurogenesis ................................ ............... 139 Circadian Rhythms and Tumor Growth ................................ .......................... 140 Implications and Approaches for Gene Delivery and Stem Cell Based Strategies for Neuroregenerative Therapies ................................ ............... 141 Implications for Cancer Therapy ................................ ................................ ..... 145 Summary ................................ ................................ ................................ ........ 145 LIST OF REFERENCES ................................ ................................ ............................. 148 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 181

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10 LIST OF TABLES Table page 1 1 Factors shown to influence NPC proliferation, differentiation, and/or survival modulated by the circadian rhythm ................................ ................................ ..... 32

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11 LIST OF FIGURES Figure page 1 1 Sagittal photomicrograph of mouse brain stained with Cresyl Violet .................. 21 1 2 Neurogenesis in the SVZ ................................ ................................ .................... 23 1 3 Cartoon showing neurogenesis in the adult hippocampal dentate gyrus ............ 24 1 4 Development of newborn neurons in the SGZ ................................ .................... 34 2 1 Timeline of BrdU injections and perfusions ................................ ........................ 48 2 2 Photomicrographs of BrdU positive cells ................................ ............................ 53 2 3 Total number of BrdU positive cells ................................ ................................ .... 53 2 4 Confocal images of new neurons and astrocytes in the dentate gyrus of an adult mouse. ................................ ................................ ................................ ....... 54 2 5 Total number of BrdU positive cells (+/ S.E.M.) 1 week after BrdU injections ... 55 2 6 Total number of BrdU positive cells (+/ S.E.M.) 4 weeks after BrdU injections ................................ ................................ ................................ ............ 57 2 7 Confocal image of dopaminergic neurons in the substantia n igra of an adult mouse. ................................ ................................ ................................ ................ 59 3 1 Experimental timeline and cell cycle markers for cell cycle distributio n and cell cycle kinetics study ................................ ................................ ...................... 70 3 2 Timeline of BrdU injections and per fusions for morphological study ................... 71 3 3 Representative light photomicrographs from granule cell layer of dentate gyrus ................................ ................................ ................................ ................... 79 3 4 The light dark cycle alters the cell cycle distribution of subgranular zone (SGZ) neural progenitor cells phase ................................ ................................ ... 81 3 5 Cumulative BrdU labeling shows that total cell cycle duration and synthesis phase lengths are decreased at night versus day in the hippocampus of female adult m ice ................................ ................................ ............................... 83 3 6 Genes related to cell cycle regulation are differentially expressed between day and night in female adult mice ................................ ................................ ..... 86 3 7 Genes related to cell cycle regulation are differentially expressed between day and night in male adult mice ................................ ................................ ........ 87

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12 3 8 Neural progenitor cells produced at night exit the multipotential, undifferentiated state faster tha n those produced dur ing the day ....................... 89 3 9 Four week old neurons generated at night display more extensive branching than those generated during the da y ................................ ................................ .. 90 3 10 Genes related to neuronal differentiation are differentially expressed between day and night in female adult mice ................................ ................................ ..... 91 3 11 Genes related to neuronal differentiation are differentially expressed between day and night in male adult mice ................................ ................................ ........ 93 3 12 Diagram of cell cycle genes up regulated (green) or down regulated (red) at nighttime versus daytime in the h ippocampus of adult female mice ................... 98 4 1 In vitro experiment timeline ................................ ................................ ............... 109 4 2 Estradiol dose concentration and exposure time alter the cell cycle distribution of neural progenitor cells in vitro ................................ .................... 114 4 3 Estradiol dose concentration and exposure time alter cell cycle kinetics of neural progenitor cells (NPCs) in vitro ................................ .............................. 116 4 4 Estradiol dose concentration and exposure time alter the expression of estrogen receptor in neural progenitor cells (NPCs) in vitro ................................ ................................ ................................ ................... 117 4 5 Adult hippocampal NPCs, whether exposed or not to estradiol in vitro, produce similar proportions of neurons with an immature phenotype and few astrocyt es 1 week after plating ................................ ................................ ......... 119 5 1 A Hypothetical Model of the Interactions between the Circadian Timing System, Estradiol Gene Reg ulation Pathway s, and the Cell Cycle Machinery 138

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13 LIST OF ABBREVIATIONS BDNF Brain Derived Neurotrophic Factor BrdU Bromo Deoxyuridine CA1 Cornu Amnion's region 1 CA3 Cornu Amnion's region 3 CLOCK Circadian Locomotor Output Cycles Kaput CNS Central Nervous System Cy3 Indocarbocyanine 3 Cy5 Indodicarbocyanine 5 DAB Diaminobenzidine DAPI 4',6 Diamidino 2 Phenylindole DCX Doublecortin DNA Deoxyribonucleic Acid E2 Estradiol Est ERE Estrogen Response Element EGF Epidermal Growth Factor EGF r Epidermal Growth Factor FGF Fibroblast Growth Factor FGF 2 Fibroblast Growth Factor 2 bFGF basic Fibroblast Growth Factor FITC Fluorescein g grams

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14 GABA Gamma Aminobutyric Acid GCL Granule Cell Layer GFAP Glial Fibrillary Acidic Protein h hour HCl Hydrochloric acid i.p. Intraperitoneal IGF Insulin Like Growth Factor IgG Immunoglobulin G ir immunoreactive LD Light Dark NaCl Sodium Chloride NDS No rmal Donkey Serum NeuN Neuronal Nuclear Antigen NMDA N Methyl D aspartate NPC Neural Progenitor Cell PBS Phosphate Buffered Saline PHH3 Phosphohistone H3 Prox1 Prospero homeobox 1 PVA DABCO Polyvinyl Alcohol Diazobicyclooctane RNA Ribonucleic Acid mRNA messenger RNA RT Room Temperature s.c. Subcutaneous

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15 SCN Suprachiasmatic Nucleus SGZ Subgranular Zone Shh Sonic Hedgehog SN Substantia Nigra Sox2 (Sex determining region Y) box 2 SRE Steroid Response Element SVZ Subventricular Zone TBS Tris Phosphate Buffered Saline TGF Transforming Growth Factor TH Tyrosine Hydroxylase VEGF Vascular Endothelial Growth Factor

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16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillmen t of the Requirements for the Degree of Doctor of Philosophy CIRCADIAN CONTROL OF ADULT NEURAL PROGENITOR CELL BEHAVIOR IN VIVO AND IN VITRO : A MOLECULAR AND GENETIC SCREEN OF REGULATORY CANDIDATES FOR NEURO REGENERATIVE THERAPIES By Lan B. Hoang Minh December 2012 Chair: Brandi K. Ormerod Major: Biomedical Engineering Identifying the mechanisms that either stimulate or allow neurogenesis in the adult hippocampus could lead to the development of exciting new strategies for treating neurodegenerative d iseases and injuries. Chapter 1 discusses the history, significance and potential of endogenous and transplantable stem cell strategies for CNS repair. Previous work suggests that the level of hippocampal neurogenesis i s potentiated in nocturnal adult mic e during the dark (active) versus light (active) phase of the light dark (LD) cycle. The experiments described in Chapter 2 directly tested whether neural progenitor cell (NPC) division neuronal differentiation and/or survival of NPC progeny vary across the LD cycle. The se experiments show that during the dark cycle more dividing NPCs are detected and more surviving neurons are produced in the hippocampal dentate gyrus than during the light cycle Interestingly, more dividing cells were detected at night in the substantia nigra but no evidence of neuronal differentiation among new cells was observed The experiments in Chapter 3 show that the rate of NPC division in vivo is increased during the dark phase and the expression of cell cycle genes i n the hippocampus varies between light and dark phases Chapter 3

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17 experiments also show that more progenitor cells exit the multipotential state at night and the expression levels of genes stimulating neuronal differentiation and survival are significantl y increased in the hippocampi of mice harvested during the dark versus light phase differentially between female and male adult mice Next, in vi tro experiments were conducted to test whether the NPC proliferation and /or daughter cell differentiation varied with diurnal like ch anges of pro mitotic estradiol (which increases in mammals during the active versus inactive phase of the LD cycle; Chapter 4) I n vitro analysis demonstrate d that NPC proliferation increase s in the presence of a high versus low dose of estradiol while e stradiol exposure has no effect on young neuron differentiation or survival after 1 week in culture Chapter 5 summarizes the circadian cycle induced change s in components of neurogenesis that were identified in this thesis and ho w they might influence normal hippocampal function neuro regenerative strategies and potentially even strategies for combating brain tumors

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18 CHAPTER 1 NEURAL PROGENITOR CELLS ADULT NEUROGENESIS AND THE CIRCADIAN C LOCK A dult neural progenitor cells (NPCs) exhibit limited clonality and potential. They divide ~ 60 times and generate limited neuronal and astrocyte phenotypes under standard culture conditions (Palmer et al. 1997; Stephens et al. 2011) T he intrinsic epigenetic and environmental cues that most likely interact to guide their behavior are still being identified (Ming & Song, 2011a) M ajor efforts in stem cell research have been devoted to understanding the regulation of NPCs, because of the potential of these cells as endogenous or exogenous sources for replacement therapies in adult neurodegenerative diseases and injuries. For instanc e, NPCs, which are present in all areas of the brain, might be utilized to replace degenerating or dead neurons, as well as astrocytes and oligodendrocytes (glial cells), following neurological disorders or injuries. In the developing brain and specific ad ult brain regions (SVZ and SGZ), these cells are found to proliferate, migrate to specific regions, integrate into existing networks, and ultimately differentiate into specific neurons in a timely fashion (Alvarez Buylla & Garcia Verdugo, 2002; Gage, 2002) process also found transiently following injury in other brain regions This course of regeneration is regulated by intrinsic or extrinsic cues found in the microenvironment surrounding the NPCs, such as chemokines, cytokines, and growth factors. The development of new treatments for neurodegenerative diseases (administrati on of neuroprotective factors, exogenous stem cell transplants, etc.) requires identifying the mechanisms that contribute to neurogenesis in adult mammals, and a several internal and external factors have been shown to regulate adult hippocampal neurogenes is. However, the majority of molecular mechanisms and

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19 are largely unknown and have been an important focus of neural stem cell research. Further elucidation of crucial molecular regulators could lead to more effective approaches in utilizing the exciting phenomenon of adult neurogenesis for the treatment of neurodegenerative diseases and injuries, as well as for neuroprotective strategies using cell based delivery of dru gs or neuroprotective growth factors (Ormerod et al. 2008) Neural Progenitor Cells and Adult Neurogenesis Neural stem cells are characterized by their ability to self renew and generate all cell types found in the nervous system (Gage, 2000) Experimentally, dividing NSCs and NPCs can be identified using injections of [ 3 ] H bromo 2 deoxyuridine (BrdU) followed by antibodies against markers for early differentiation Both compounds are incorporated into DNA during the S phase of the cell cycle and can be detected in the dividing cells after a short survival period and their daughter cells after longer survival times (Altman, 1962; Miller & Nowakowski, 1988) Two theories have emerged concerning neural stem cell lineages; one is that a neural stem cell could give rise to one pluripotent progenitor cell that could produce all three typ es of cells of the adult brain; the second theory would be that three unipotent, or perhaps bipotent, progenitors of restricted lineage can generate only one or two types of cells (Alvarez Buylla et al. 2001; Hack et al. 2005; Kohwi et al. 2005; Merkle et al. 2007) Stem cel ls are defined by their abilities to 1) self renew and 2) differentiate into multiple cell types in different germ layers Some evidence suggests that NPC s are multipotent that is, they have some capacity for differentiation (hence, the term

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20 and could mature to one of three major cell types of the adult brain: neurons, oligodendrocytes, or astrocytes. Potentially multipotent NPCs have been isolated from most regions throughout the adult mammalian brain (Palmer et al. 1995a; Weiss et al. 1996; Gage, 2000; Arsenijevic et al. 2001) NPCs harvested from human fetal and adult brains as well as human blastocysts have been propagated as genetically stable cel l lines that can form specific neuronal or glial subtypes (Svendsen et al. 1998; Carpenter et al. 1999; Vescovi et al. 1999; Amit et al. 2000; Thomson et al. 2012) NPCs can be harvested from the entire adult mammalian CNS, but only two regions of CNS have been confirmed to host ongoing adult neurogenesis, a process defined as the production of new neurons. These neurogenic areas include the subgranular zone (SGZ) of the hippocampal dentate gyr us (DG) and the subventricular zone (SVZ) bordering the lateral ventricles (Fig 1 1 ). Although advances have been made in identifying the intrinsic and extrinsic signals that control the maintenance proliferation, and differentiation of NPCs as well as the integration of their daughter cells, neurons and glia into existing circuitry, we still cannot fully control neurogenesis in the adult brain (Palmer et al. 2000; Song et al. 2002a; Alvarez Buylla & Lim, 2004; Morrison & Spradling, 2008)

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21 Figure 1 1. Sagittal photomicrograph of mouse brain stained with Cresyl Violet showing known neurogenic regions in the dentate gyru s subgranular zone (SGZ; yellow) and subventricular zone (SVZ; yellow). Neural progenitor cells in the SGZ migrate into the granule cell layer of the dentate gyrus where they become mature granule neurons. NPCs from the SVZ migrate through the rostral migr atory stream (yellow) and become granule neurons or periglomerular neurons in the olfactory bulb NPCs resident to the SVZ or to the hippocampal SGZ generate significant numbers of neurons each day (Cameron et al. 1993; Alvarez Buylla & Garcia Verdugo, 2002) while NPCs located in other CNS regions primarily generate glia (Gould & Gr oss, 2002; Spalding et al. 2005; Geha et al. 2010) unless injury is induced, which appears to stimulate the production of transient neuronal populations (Gu et al. 2000; Magavi et al. 2000a; Kernie et al. 2001; Arvidsson et al. 2002; Parent et al. 2002) Neurons are the functional components of the nervous system and are responsible for information processing and transmission, while astrocytes and oligodendrocytes are known as glia and play supporting roles essential for the proper functioning of the nervous system.

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22 In vitro, adult NPCs can self renew and differentiate into all types of neural cells, including neurons, astrocytes and oligodendrocytes (Palmer et al. 1995a) The stem cell properties of adult NPCs have been demonstrated in vitro via neurosphere and adherent monoculture layers (Reynolds & Weiss, 1992b; Gage et al. 1995) In vivo 3H thymidine (Altman & Das, 1965; Altman, 1969) and later BrdU labeling (Kempermann et al. 1997; Eriksson et al. 1998) have been used to demonstrate endogenous cell proliferation. Subsequent studies combining BrdU labeling and immunohistochemistry shed light on the identity and function of these new BrdU labeled cells, most of which acquire a neuronal phenotype (Cameron & McKay, 2001b; van Praag et al. 2002; Zhao et al. 2006) Three types of NPCs (type B, A, and C; Fig 1 2 ) have been identified in the ependyma that lines the SVZ (Reynolds & Weiss, 1992b; Chiasson et al. 1999) Type B cells are considered to be the most nave NPC s and are thought to potentially a symmetrically differentiate into Type C cells (neuroblasts) which then potentially differentiate into Type A cells that chain migrate through the rostral migratory stream and differentiate fully into GABAergic granule neurons and dopaminergic periglomerul ar neurons upon reaching the the olfactory bulb s (Bedard & Parent, 2004; Curtis et al. 2007; Lledo et al. 2012) Maturation and migration processes of these NPCs are governed by cell cell, cell extracellu lar matrix interactions, and a host of other signals from their environment (Bonfanti & Theodosis, 1994; Wu et al. 1999; Conover et al. 2000; Duan et al. 2007)

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23 Figure 1 2. Neurogenesis in the SVZ. The adult SVZ consists of transit amplifiers (type C cel ls, blue) that give rise to neuroblasts organized as chains (type A cells, red), ensheathed by the processes of slowly dividing SVZ astrocytes (type B cells, green). Ependymal cells (type E cells, white) line the ventricle and are sometimes displaced by ty pe B cells that interlock between the ependyma to contact the ventricle (adapted from Zhao C et al., Cell 2008). In vivo studies have identified 4 NPC phenotypes in the hippocampal SGZ of the hippocampal dentate gyrus Type 1 radial glia like (GFAP + ) cells are thought to give rise to T ype 2 a (GFAP/Sox2 + ) transiently amplifying progenitor cells which give rise to Type 2b (Sox2/Prox1 + ) neuronally committed progenitors that mature or differentiate into Type 3 (Prox1/DCX + ) neuroblasts. These phenotypes ha ve been described based upon their morphologies and their expression of specific molecular markers associated with developmental stages of cell maturation (Kempermann et al. 2004) (Fig.1 3) Neuroblasts are thought to migrate deeper into the granule cell layer of the dentate gyrus before fully differentiating into granule neurons that receiv e input from the

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24 entorhinal cortex and rapidly extend axons to synapse with pyramidal cells in the CA3 region (Hastings & Gould, 1999; Kempermann & Gage, 2000; Fukuda et al. 2003; Suh et al. 2007) Knowledge of the internal and external stimuli that regulate the dynamic processes of neural progenitor proliferation and neurogenesis in the adult brain is crucial in developing strategies for treating neurodegenerative dise ases. Figure 1 3 Cartoon showing n eurogenesis in the adult hippocampal dentate gyrus. First, type 1 radial glia like (GFAP + ) cells in the subgranular zone (SGZ) of the dentate gyrus are thought to give rise to t ype 2 a (GFAP/Sox2 + ) transiently amplifying progenitor cells (proliferation and fate determination phases) which give rise to type 2b (Sox2/Prox1 + ) neuronally committed progenitors that mature or differentiate into type 3 (Prox1/DCX + ) neuroblasts. Neuroblasts then migrate i nto the granule cell layer of the dentate gyrus (migration phase). Finally, immature neurons mature into new granule neurons, receive input from the entorhinal cortex, and extend projections into CA3 (integration phase) CA3 (Fukuda et al. 2003; Kempermann et al. 2004; Lie et al. 2004; Suh et al. 2007) The Neurogenic Niche Although NPCs can be harvested from many areas of the adult nervous system, adult neurogenesis has only been consistently found in the SVZ and SGZ in vivo (Kempermann et al. 1 997; Eriksson et al. 1998; Alvarez Buylla & Garcia Verdugo, 2002) Whether the NPCs in these regions are unique compared to those in other adult

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25 CNS regions or the microenvironments located in the SVZ and SGZ are unique is unclear. However, there is evidence that signals provided by the microenvironments of the SGZ and SVZ, making up the neurogenic niche, regulate the maintenance, proliferation, differentiation, and integration of new neurons (Zhao et al. 2008) In both regions, adult NPCs are in close proximity to other neuro ns (mature and immature), astrocytes, oligodendrocytes, a variety of interneurons and vasculature. Whether SVZ and SGZ NPCs represent unique NPC populations is unclear but there are similarities between their niches that may instruct similar behaviors. A d ult SGZ NPCs reside in close contact with a dense layer of granule cells ( that includes mature and newborn granule neurons), but also with astrocytes, oligodendrocytes, and a variety of interneurons Recent evidence points to astrocytes as integral to the neurogenic process in the SGZ as well as SVZ including neural progenitor cell proliferation and differentiation (Sanai et al. 2004; Barkho et al. 2006) Specific roles that astrocytes play in the brain include trophic factor delivery, which promotes cell growth and neuronal environment maintenance and neuronal signaling (Bergami et al. 2008; Filosa et al. 2009) I n vitro, where they have been shown to actively regulate adult neurogenesis both by instructing neuronal fate commitment and by promoting proliferation of adult neural stem cells (Song et al. 2002b) through the secretion of different factors, such as interleukins. Using in vitro NPC differentiation assays, results in dicated that two interleukins, IL 1 beta and IL 6, and a combination of factors that included these two interleukins could promote NPC neuronal differentiation, whereas insulin like growth factor binding protein 6 and decorin secreted by astrocytes inhibit ed neuronal differentiation of adult NPCs (Barkho et al. 2006)

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26 Astrocytes also play an important role in adult neurogenesis in vivo. One mechanism is through Wnt signaling, and overexpression of Wnt3 is sufficient to increase neurogenes is from adult hippocampal progenitor cells in vitro and in vivo ; by contrast, blockade of Wnt signaling reduces neurogenesis from progenitor cells in vitro and abolishes neurogenesis almost completely in vivo (Lie et al. 2005) Using mouse models, it has been suggested that astrocytes act as both up and down regulators of neurogenesis, depending on the region in which they are located (Song et al. 2002a) Specific examples of neurogenic regulatory factors produced by astrocytes include Ephrin A2, a neurogenic inhibitor found mainly in areas of the adult mouse brain outside of the neurogenic hippocam pus and subventricular zone (SVZ), and Sonic hedgehog (Shh), a neurogenic promoter found mostly within the hippocampus and SVZ (Han et al. 2008; Jiao et al. 2008) This suggests that, depending on their location, astrocytes can assist in part as up or down regulators of neurogenesis. Therefore, signals provided by the cellular environment surrounding NPCs in the SGZ, particularly those produced by astrocytes, play a major role in regulating the maintenance, proliferation, and differentiation of ne w neurons Differences between NPC niche components in the SGZ and SVZ may instruct different NPC behaviors, such as migration to the olfactory bulb through the rostral migrator stream for NPCs located in the SVZ but not in the SGZ, as well as NPC prolifer ation and differentiation. For instance, p rogenitors in the SVZ are found adjacent to ependymal cells of the lateral ventricles. These ependymal cells express Noggin that may promote neurogenesis by antagonizing bone morphogenetic proteins (Lim et al. 2000) In addition, dopaminergic signaling through dopaminergic afferents may promote

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27 SVZ progenitor proliferation through D2 like dopamine receptors in vivo (Hoglinger et al. 2004) In addition, blood vesse ls that are closely associated with NPCs also release factors that increase adult neurogenesis in the SGZ and SVZ (Palme r et al. 2000) such as vascular endothelial growth factor (VEGF) which was found to promote cell proliferation in the SVZ and SGZ after infusion in vivo (Cao et al. 2004) The exact anatomical and functional components within the neurogenic niche in the SGZ and SVZ still remain to be determined. Since any diffusible molecules produced by local or distant cells can influence NPCs, adult neurogenesis is subject to complex extrinsic regulation. Regulation of Adul t Neurogenesis Proliferation and Differentiation (Table 1 1) Several environmental factors have been shown to modulate hippocampal proliferation and differentiation, such as learning (Gould et al. 1999) environmental enrichment (Kempermann et al. 1997) and voluntary wheel running, especially in the middle of the dark period (van Praag et al. 1999c; Holmes et al. 2004) The number of adult generated neurons was found to double in the rat dentate gyrus following associative learning tasks dependent on t he hippocampus such as trace eyeblink conditioning and Morris water maze spatial navigation but no change in new cell number was found after training on hippocampal independent learning tasks such as delayed classical eyeblink conditioning and cue train ing in the Morris water maze (Gould et al. 1999 ) Short term exposure to an enriched environment lead to a striking increase in new neurons, along with a substantial improvement in behavioral performance (Kempermann et al. 1997) Exercise, especially runn ing, was also found to be a powerful inducer of neurogenesis in the adult and aged rodent hippocampus

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28 (van Praag et al. 19 99b; van Praag et al. 1999c; van Praag et al. 2005) and the effect of exercise induced increased cell proliferation was mediated in part by an increase in BDNF and VEGF levels (Koyanagi et al. 2003; Olson et al. 2006; Hetland et al. 2008) On the other hand, neurogenesis has been shown to decline with aging and stress. While neurogenesis persists across lifespan in mam mals, it decreases with age in rodents, dogs, and non human primates (Aizawa et al. 2011; Speisman et al. 2012c) both in the SGZ and SVZ. In aged mice living in an enriched environment from the age of 10 to 20 months, adult hippocampal neurogenesis became fivefold higher than in contro ls. This increase was accompanied by significant improvements of learning parameters, exploratory behavior, and locomotor activity (Kempermann et al. 2002a) In vitro studies suggest that the self renewal capacity and the number of SVZ progenitors in neurosphere cultures decrease significantly in aged animals (Kuhn et al. 1996; Drapeau et al. 2003; Driscoll et al. 2006; Molo fsky et al. 2006) Neurogenesis can be partially restored in aged animals by voluntary exercise and fully restored by adrenalectomy (Cameron & McKay, 1999; van Praag et al. 2005) suggesting that NPCs in aged animals are capable of responding to extrinsic stimuli. Stress is another known major negative regulator of neurogenesis. Numerous studies have demonstrated that a variety of chronic stressors cause a reduction in NPC proliferation in the SGZ (Gould et al. 1997; Tanapat et al. 1998; McEwen, 1999; Karten et al. 2005) In contrast, the effect of acute stress on SGZ cell proliferation depends on the specific type of stress as well as the sex of the animal. For instance, acute odor stress exposure suppressed both cell proliferation and death in adult male rats but not in any group of females (Falconer & Galea, 2003) Elevation of corticosterone

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29 (glucocorticoid) levels by the activated hypothalamic pituitary adrenal axis is a main mechanism for stress mediated suppression of SGZ cell proliferation. Corticosterone decreases cell proliferation whereas adrenalectomy increases SGZ neurogenesis, and the glucocorticoid level is increased by a variety of stress situations, while adrenalectomy preven ts the stress induced suppression of SGZ cell proliferation ( McEwen, 1994; 1996; Montaron et al. 2003; Brummelte & Galea, 2010) As mentioned, adrenalectomy can even restore neurogenesis in aged rodents. Therefore, a variety of environmental factors have been found to regulate neurogenesis, most likely through the modulation of specific molecular pathways. Since NPCs reside in a microenvironment of complex ne ural networks in the SGZ, neurotransmitters and other neural peptides and lipids directly influence neural progenitor behavior at the molecular level in that area. Within the dentate gyrus, NMDA receptor dependent activity was inversely correlated with the level of hippocampal proliferation (Nacher et al. 2003) GABA was shown to promote the differentiation of type 2 h ippocampal progenitors (Tozuka et al. 2005) and calcium channel antagonists and agonists respectively decreased and increased neuronal differentiation in the hippocampus (Deisseroth et al. 2004) Other neurotransmitters such as serotonin, noradrenal ine, and acetycholine have all been shown to increase NPC proliferation (Kulkarni et al. 2002; Banasr et al. 2004; Mohapel et al. 2005) In addition, growth factors such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) have been shown to be powerful factors for the maintenance of NPCs in vitro (Reynolds & Weiss, 1992b; Palmer et al. 1995a; Doetsch et al. 2002) They also promote NPC proliferation in the SVZ in vivo and FGF2 increases the

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30 number of new neurons in the olfactory bulb (Kuhn et al. 1997) Deletion of the FGF receptor fgfr1 in the hippocampus also decreases neurogenesis, indicating that FGF2 mediated signaling is involved in SGZ proliferation (Jin et al. 2003; Zhao et al. 2007) Signaling through the Sonic hedgehog pathway has also been implicated in the r egulation of neurogenesis, participating in the establishment and maintenance of adult neurogenic niches and regulating the proliferation of neuronal or glial precursors in several brain areas (Machold et al. 2003; Ahn & Joyner, 2005; Banerjee et al. 2005; Palma et al. 200 5) BDNF has also been shown to be one of the key pos itive regulators of neurogenesis, increasing the proliferation of NPCs in the SVZ and NPC survival in the SGZ (Sairanen et al. 2005; Scharfman et al. 2005; Henry et al. 2007; Young et al. 2007) Moreover intr acellular mechanisms implicated in the regulation of neurogenesis involve intracellular signaling pathways downstream of the growth factors, neurotrophins, and morphogens, along with a whole host of transcription factors, such as TLX, Bmi 1, Pax6, and Olig 2, that play critical roles in postnatal neurogenesis (Shi et al. 2004; Hack et al. 2005; Molofsky et al. 2005; Sun et al. 2007) Adult neurogenesis is also controlled by epigenetic regulation (Zhao et al. 2003b) as well as genes involved in cell cycle regulation (Kippin et al. 2005; Gil Perotin et al. 2006; Molofsky et al. 2006) Hormones, such as thyroid hormone, prolactin and estradiol, have been found to promote new neuron survival (Hidalgo et al. 1995) and enhance neurogenesis in the forebrain in the case of prolactin (Shingo et al. 2003) and dentate gyrus (Perez Martin et al. 2003) In particular, estradiol has been shown to increase neurogenesis in the dentate gyrus by increasing the survival of young granule neurons (Ormerod et al.

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31 2003; 2004b) Therefore, a variety of molecular signals including neurotransmitters, neural peptides and lipids such as growth factors, transcription factors, and hormones directly influence neural progenitor behavior in the SGZ and SVZ neurogenic regions of the adult brain. Finally, neurogenesis levels seem to vary based on genetic background. For instance, two strains of recombinant inb red mice based on C57BL/6, which are good learners and show high baseline levels of neurogenesis, and DBA/2, which are known to be poor learners and exhibit low levels of adult neurogenesis, showed a 26 fold difference in the number of newly generated neur ons per hippocampus. Over all strains, there was a significant correlation between the number of new neurons generated in the dentate gyrus and parameters describing the acquisition of the water maze task (slope of the learning curves) (Kempermann & Gage, 2002; Epp et al. 2011b) Therefore, adult neurogenesis is controlled by a host of intrinsic and extrinsic factors, from genes to proteins an d lipids. The majority of these factors and signaling pathways still remain largely unknown and need to be elucidated in order to optimize stem cell based treatments for neurodegenerative diseases or injuries.

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32 Survival, Maturation, and Integration of Newborn Neurons (Table 1 1) Table 1 1. Factors shown to influence NPC proliferation, differentiation, and/or survival modulated by the circadian rhythm (+, variations across the circadian cycle; no variations). Proteins Prol ifer atio n Su rvi va l Diff eren tiati on Circadia n studies /variation s References Growth factors FGF2 + No (Zheng et al. 2004) EGF + + Yes/ + (Scheving et al. 1989; Craig et al. 1996) Notch1 + No (Hitoshi et al. 2002; Chojnacki et al. 2003) CNTF + No (Emsley & Hagg, 2003; Enwere et al. 2004) TGFalpha + Yes/ + (Enwere et al. 2004; Van der Zee et al. 2005; Tournier et al. 2007) SHH + No (Machold et al. 2003; Palma et al. 2005) APP + No (Caille et al. 2004) Ephrin A2 No (Lim et al. 2008) VEGF + Yes/ + (Louissaint et al. 2002; Koyanagi et al. 2003; Hetland et al. 2008) BDNF + + Yes (Ahmed et al. 1995; Bova et al. 1998; Schaaf et al. 2000; Lee et al. 2002; Begliuomini et al. 2008) IGF 1 + + Yes/ (Jorgensen et al. 1990; Aberg et al. 2000; Trejo et al. 2001; Noble et al. 2007) NGF + + Yes/+ (Gnahn et al. 1983; L evi Montalcini, 1998; Bersani et al. 2004) Neurotransmitters 5 HT (serotonin) + + Yes/+ (Quay, 1963; Hery et al. 1972; Akerstedt & Levi, 1978; Banasr et al. 2004; Holmes et al. 2004) Noradrenaline + Yes/+ (Kulkarni.Rd et al. 1965; Manshard.J & Wurtman, 1968; Akerstedt & Levi, 1978) ACh (acetylcholine) + Yes/+ (Hanin et al. 1972; Marrosu et al. 1995; Mohapel et al. 2005) Glutamate (NMDA) + / + No (Gould et al. 1997; Arvidsson et al. 2001) Hormones Estrogen +/ + Yes/+ (Roy & Wilson, 1981b; Ormerod et al. 2003; Perez Martin et al. 2003; Ormerod et al. 2004b; Cai et al. 2008a) Prolactin + Yes/+ (Sassin et al. 1972; Ehara et al. 1975; Plant, 1981; Garcia Bonacho et al. 2000; Shingo et al. 2003) Thyroid hormone + Yes/+ (Fisher, 1996; Lemkine et al. 2005; Chan et al. 2008)

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33 Newborn neur ons in the dentate gyrus go through several developmental stages with distinctive physiological and morphological properties; Fig 1 4). Initial depolarization by GABA plays a critical role in the maturation of newborn granule cells, which display typical features of mature granule cells at 4 weeks of age (Ge et al. 2006) and express factors that change drastically as they mature. Physiological and pathological conditions also affect neuronal maturation, such as accelerated maturation following voluntary exercise, but the molecular pathways media ting these effects still remain unknown (Zhao et al. 2008) Many ne wborn neurons die within 4 weeks after birth, and the survival of 1 to 3 week old newborn neurons is heavily influenced by environment al factors, such as spatial learning and exposure to an enriched environment (Kempermann et al. 1997; Tashiro et al. 2006; Epp et al. 2007; Kee et al. 2007; Epp et al. 2011a) Other factors, such as signaling through the NMDA receptor and BDNF, also play a major role in the maintenance of normal neuron survival (Sairanen et al. 2005; Platel et al. 2010) In addition, hormones such as estradiol have been shown to incre increased spatial memory (Ormerod et al. 2003) Therefore, a variety of environmental stimuli such as neurotransmitters, growth factors, and hormones play a role in the survival and maturation of newborn neurons in the dentate gyrus, but t he majority of these factors still remain unknown.

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34 Figure 1 4. Development of newborn neurons in the SGZ, as characterized by expression of specific markers, morphogenesis, synapse formation, electrophysiological properties, and functional integration. Abbreviations are as follows: MSB, multiple synapse boutons; SSB, single synapse boutons; LTP, long term potentiation; IEG, immediate early gene (from Zhao et al., 2008). Functional Implications of Adult Neurogenesis Correlation, ablation, and computational modeling studies have uncovered some function of SGZ neurogenesis in learning and memory. A correlation between hippocampal neurogenesis and learning in spatial memory task (using the Morris water maze) was observed in mice of different strains (Kempermann et al. 1997; Kempermann & Gage, 2002; Deng et al. 2010) and mutant mice with decreased SGZ neurogenesis have decreased performance on hippocampus depe ndent learning tasks (Zhao et al. 2003b; Shimazu et al. 2006; Zhao et al. 2007) Manipula tions that chronically attenuate neurogenesis are associated with impaired learning and memory (Madsen et al. 2003; Raber et al. 2004; Rola et al. 2004; Winocur et al. 2006) while those that increase neurogenesis are associated with better performances on hippocampus dependent tasks (Ormerod et al. 2004a; van Praag et al. 2005) Neurogenesis was also correlated with learning abilities in anti mitotic drug treated rats

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35 that failed to form conditioned responses in trace eyeblink or trace fear conditioning (Shors et al. 2001) and in irradiated or ganciclovir treated rats or mice (Saxe et al. 2006; Winocur et al. 2006) Studies correlating neurogenesis with spatial learning and memory remain contradictory, with some showing impaired spatial learning and memory in the Barnes maze b ut not the Morris maze, and others the opposite (Raber et al. 2004; Rola et al. 2004) Place and object recognition memories were also impacted by ablated neurogenesis in irradiated rats (Winocur et al. 2006) and in anti mitotic drug treated animals (Bruel Jungerman et al. 2005) in enriched environments. Neurogenesis has also been implicated in mood regulation in many studies, particularly by Gould and colleagues (Gould et al. 1992) who showed that SGZ progenitor cell division is suppressed by corticosteroids, which are elevated in depressed a nd stressed patients. SGZ neurogenesis might also be necessary for various antidepressant functions, as demonstrated by Santarelli and colleagues (Santarelli et al. 2003) Greater understanding of the physiological and molecular mechanisms regulating in vivo neurogenesis would therefore help in restoring normal neurological functions in cases of functional loss accompanying neurodegenerative diseases or brain injuries. Circadian Cycle and Cell Division Most natural processes exhibit temporal periodicity, and all aspects of mammalian life are affected by a wide variety of time clocks: the annual cycles of activ ity and metabolism, the monthly physiological clocks of women and men, and the 24 h diurnal rhythms, to name a few (O'Malley, 2009) The most studied and understood mammalian clock is the 24 h diurnal rhythm in mammals, controlled by hormones such suprachiasmatic nucleus (SCN) of the hypothalamus (Reppert & Weaver, 2001; Ga chon

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36 et al. 2004) and peripheral clocks located in most cells. At the molecular level, circadian activators or other proteins that are able to feedback as well as inhibit their own expression and are organized in a complex transcriptional translational regulatory network (Dunlap, 1999; Hunt & Sassone Corsi, 2007) The SCN is anatomically located to receive visual input from the light dark cycle and non photic information from other neuronal tracts. Photosensitive retinal ganglion cells send photic signals to the SCN through the retinohypothalamic tract (Dibner et al. 2010) In addition to receiving photic signals, the SCN integrates internal signals from feeding, locomotor activity, and photobiotic hormones such as melatonin. In turn, neuronal connections from the SCN to the pineal gla nd regulate rhythms of melatonin secretion and the adrenal cortex those of cortisol/corticosterone (Dibner et al. 2010) T he master circadian pacemaker is therefore networked with feedback interactions which assist SCN neurons in regulating th eir function (Greene, 2012) The master circadian clock located in the SCN is composed of a transcriptional and post translational feedback loop. In mammals, the positive end of the transcriptional loop are the transcription factors, CLOCK ( Circadian Locomotor Output Cycles Kaput protein) and BMAL1 (Aryl Hydrocarbon Re ceptor Nuclear Translocator like 1 protein) or NPAS2 ( Neuronal PAS domain containing protein 2 ) that initiate transcription of the Cryptochrome ( Cry ) Period ( Per ) genes and other clock gene products. The Cry and Per proteins in turn act as negative regulators by interacting with CLOCK and/or BMAL1 in the nucleus (Chen et al. 200 9) The core transcriptional loop is also regulated by two nuclear receptors: the retinoic acid receptor (or

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37 NR1D1; nuclear receptor subfamily 1, group D, mem ber 1) which can directly regulate the expression of BMAL1, NPAS2, and CLOCK (Gui llaumond et al. 2005; Crumbley et al. 2010; Crumbley & Burris, 2011) In addition to the master circadian clock in the SCN, circadian clocks in other parts of the brain and peripheral organs are also composed of a transcriptional and post translational feedback loop with many of the same molecular components, as confirmed by circadian oscillation in gene expression in cultured cells as well as tissue explants from liver, lung, kidney, spleen, pancreas, heart, stomach, skeletal muscle, lung, cornea and thyroid gland (Yoo et al. 2004) Therefore, the SCN but also several regions of the brain and peripheral organs contain circadian clocks that are regulated by multiple molecular components that still remain relatively unexplored. There is substantial e vidence that circadian rhythms affect the timing of cell divisions in mammals in vivo For instance, day night variations in both the mitotic index, DNA synthesis, and cell cycle time occur in many tissues, including the oral mucosa, tongue keratinocytes, intestinal epithelium, skin, and bone marrow, with a DNA synthesis peak usually occurring during the active (night time) period in rodents (Scheving et al ., 1978; Laerum et al ., 1988; Scheving et al ., 1989; Brown, 1991; Buchi et al ., 1991; Bjarnason et al ., 1999; Garcia et al. 2001; Bjarnason & Jordan, 2002; Smaaland et al ., 2002; Wille et al. 2004) Some of these variations persist even in constant darkness (Scheving et al. 1974) The circadian clock has also recently been shown to control the temporal behavior of epidermal stem cells (Janich et al. 2011) and disrupting this clock genetically affected the homeostasis of dormant hair follicle stem cells and increased

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38 the predisposition to tumorigenesis. The expression of cell cycle proteins also displays circadian variations in several organs. For example, in the regenerating liver of mice, the circadian clock controls the expression of cell cycle related genes which modulate the expression of active Cyclin B1 Cd c 2 kinase, a key regulator of mitosis (Matsuo et al. 2003) Circadian rhythms also regulate cell differentiation in sev eral organs. For instance, adipogenesis and osteogenesis are under the control of circadian genes and associated transcription factors (Kawai et al. 2010; Kawai & Rosen, 2010) Therefore, circadian rhythms play a key role in most biological processes, including the expression of genes and proteins involved in metabolism, cell cycle progression, and cell fate in many organs in mammals in vivo However, the underlying mechanisms of the timing of cell divisions by the circadian clock are not fully understood and have not significantly been studied in adult neurogenic regions. Neurogenesis and Cell Cycle Fetal and adult neurogenesis is correlated to the rate of proliferation of NPCs in the neurogenic SGZ and SVZ regions, which is directly linked to the proliferating cs (Takahashi et al. 1993b; Calegari et al. 2005; Arai et al. 2011) Using a high dose of the S phase marker bromodeoxyuridine (BrdU) along with a second S phase marker, [(3)H]thymidine, Cameron and McKay (2001) found that, in young adult rats, the hippocampal dentate gyrus has 9,400 dividing cells proliferating with a cell cycle time of 25 hours, which would generate about 9,000 new cells each day, or more than 250,000 per month (6% of the total size of the granule cell population). Studies in adult mice show that estimates of the cell cycle and its components in the rat and mouse SGZ are very different For example, in rats versus mice, the total cell cycle length is 25 versus

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39 12 14 h and S phase length is 9 versus 7h Furthermore, in rats versus mice, the percentage of the ce ll cycle devoted to S phase is 38 versus 60% and to Gap2 and mitosis (G 2 /M) is 18 versus 35% (Cameron & McKay, 2001a; Hayes & Nowakowski, 2002; Burns & Kuan, 2005; Mandyam et al. 200 7) Species differences and even intraspecies differences in NPC proliferation likely relate to variable levels of gene expression among cell division/cycle regulators. However, this aspect of NPC cell cycle kinetics has been largely unexplored In Chapter 3, we examine whether factors controlling cell cycle kinetics are expressed differ entially in mice during the light (sleep) versus the dark (sleep) phase of the circadian cycle of nocturnal mi ce. Circadian Cycle and Neurogenesis Circadian rhythm and cell cycle progression interact at the level of genes, proteins and biochemical signals (Bjarnason et al. 1999; Gru ndschober et al. 2001; Matsuo et al. 2003) and most factors that have been found to regulate neurogenesis in the adult nervous system are themselves controlled by diurnal rhythms (Table 1 1). Various externa l factors have been shown to enhance hippocampal neurogenesis, such as voluntary wheel running, especially in the middle of the dark period (van Praag et al. 1999c; Holmes et al. 2004) suggesting that cell proliferation and neurogenesis are modulated by both circadian phase and amount of daily activity. The effect of exercise induced increased cell proliferation might be due to an increase in VEGF levels, which appear to also fluctuate across the light dark (LD) cycle (Koyanagi et al. 2003; Hetland et al. 2008) In addition, other growth factors, such as epi dermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), are powerful factors for the maintenance of NPCs in vitro (Reynolds & Weiss, 1992b; Palmer et al. 1995a; Doetsch et al. 2002) EGF a nd FGF2 also promote NPC proliferation in the SVZ in vivo and FGF2 increases the

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40 number of new neurons in the olfactory bulb (Kuhn et al. 1997) E GF receptor levels have been sh own to vary throughout the circadian cycle in the liver (Scheving et al. 1989) although no study has examined diurnal variations in FGF receptor levels. BDNF is also one of the key positive regulators of neurogenesis, increasing neurogenesis, proliferation of NPCs in the SVZ, and survival in the SGZ (Sairanen et al. 2005; Scharfman et al. 2005; Henry et al. 2007; Young et al. 2007) BDNF mRNA expression and plasma BDNF levels have been shown to vary with the LD cycle (Bova et al. 1998; Schaaf et al. 2000; Begliuomini et al. 2008) Moreover, neurotransmitters such as serotonin, noradrenaline, and acetycholine that all increase NPC proliferation (Kulkarni et al., 1965; Banasr et al., 2004; M ohapel et al., 2005) also show levels varying across the LD cycle (Quay, 1963; Manshard.J & Wurtman, 1968; Marrosu et al., 1995). For instance, serotonin, a positive regulator of neurogenesis in vertebrate species (Gould, 1999) shows levels that fluctuate on a circadian rhythm in many organisms (Castanon Cervantes et al. 1999) In addition, hormones, such as thyroid hormone, prolactin and estradiol, have been found to p romote new neuron survival (Hidalgo et al. 1995) and enhance neurogenesis in the forebrain (Shingo et al. 2003) and the dentat e gyrus by increasing the survival of young granule neurons (Ormerod et al. 2003; 2004b) Fluctuations in the levels of these hormones across the circadia n cycle have been well documented (Sassin et al. 1972; Ehara et al. 1975; Szafarczyk et al. 1980; Roy & Wilson, 1981b; Cai et al. 2008a) Melatonin, a neurohormone promotes differentiation and survival of new neurons and is inhibited by light and secreted at night in a circadian fashion (Ramirez Rodriguez et al. 2009; Rennie et al. 2009) The expression of receptor

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41 proteins for hormones in the CNS also fluctuates across the circadian cycle. For instance, estrogen receptor levels decrease in darkness (Cai et al. 2008a) and prolactin levels are highest during the daytime (Sassi n et al. 1972; Garcia Bonacho et al. 2000) Furthermore, adult neurogenesis is controlled by genes involved in cell cycle regulation (Kippin et al. 2005; Gil Perotin et al. 2006; Molofsky et al 2006) which themselves show coordination with the circadian cycle (Lowrey & Takahashi, 2004) More recently, clock genes have been shown to regulate neurogenic transcription factors and the neuronal dif ferentiation of adult neural NPCs in vitro (Kimiwada et al. 2009 b) In vivo clock controlled Period2 gene expression has been linked to the intrinsic control of neural progenitor cells proliferation, cell death, and neurogenesis in the dentate gyrus of adult mice (Borgs et al. 2009c) which suggests a strong influence of components of the circadian cycle on neurogenesis at the gene level. Therefore, many factors, including growth factors, neurotransmitter s hormones, and genes, which have been found to regulate neurogenesis in the adult nervous system, are themselves controlled by diurnal rhythms, strongly suggesting that neurogenesis might also be regulated by components of the circadian cycle. In addition sleep studies have shown that total sleep deprivation, rapid eye movement sleep deprivation, and sleep fragmentation all have negative effects on neural progenitor cell proliferation, differentiation, and/or survival (Guzman Marin et al. 2012; Hairston et al. 2012) with up to 50% reduction of hipp ocampal cell proliferation with rapid eye movement sleep deprivation, which suggests the strong influence of circadian rhythms on normal neurogenesis. Moreover, several studies have

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42 shown a direct correlation between learning and memory and adult neurogene sis (Gould et al. 1999; van Praag et al. 1999b; Shors et al. 2001) Time of day variation in learning and memory has been described in many species, including humans (Dijk et al. 1992) and synaptic excitability and long term potentiation, major cellular mechanisms underlying le arning and memory, have also shown diurnal rhythms in vivo and in vitro (Chaudhury et al. 2005) suggesting the potential influence of the cir cadian cycle on learning and memory processes, and thus, neurogenesis. In summary, circadian rhythms play a key role in most biological processes, including the expression of genes, growth factors, neurotransmitters, and hormones, and most factors that ha ve been found to regulate neural progenitor cell division, differentiation, and survival in the adult central nervous system are themselves controlled by diurnal rhythms, strongly suggesting that neurogenesis might be regulated by the light dark cycle. How ever, many of the circadian factors and signaling pathways affecting neurogenesis are still unknown. Once the circadian variations and mechanisms of the transcriptional clock and gene expression in neurogenic regions are fully understood, neural stem cell research can be directed toward altering the gene clock or cell cycle kinetics in ways that could implement favorable regulation of normal neural progenitor activity. Summary Neural progenitor cells that are found in the subventricular zone and dentate gyr us subgranular zone of the adult brain could be useful in cell replacement therapies for neurological disorders. The development of treatments and/or cures that would increase production of new neurons requires the identification of endogenous or natural m olecular regulators of adult neurogenesis. Indeed, t

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43 aging, other neurodegenerative diseases and brain trauma could involve administering factors (orally, intravenously or through gene therapy and stem cell based treatments ) that would stimulate endogenous neurogenesis for the replacement of degenerating neurons. Robust circadian cycle induced changes in neurogenesis are a powerful means for exploring the molecular mechanisms that regulate cell cycl e behavior and neural progenitor cell proliferation, without submitting the brain to injury or other external manipulation. Relationships between neural progenitor cell behavior, cell cycle regulators, and the circadian clock would also have important impl ications in chronotherapy and the development of the optimal and timely administration of neuro therapeutic drugs in the case of neurodegenerative diseases, or anti c ancer drugs for brain tumors. In the present study, we aim at uncovering extrinsic and end ogenous factors linked to the circadian cycle including genetic regulators of cellular activity in the scope of better understanding the mechanisms regulating adult neurogenesis. We will be focusing on the signaling pathways (genes, hormones) regulated b y the light dark cycle as these pathways hold the potential for the development of novel strategies for neural stem cell based therapies as well as cancer prevention and treatment.

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44 CHAPTER 2 P ROGENITOR CELLS DIVIDING DURING THE DARK PHASE OF THE LIGHT DARK CYCLE GENERATE MORE HIPPOCAMPAL NEURONS IN ADULT MICE BUT NO NIGRAL NEURONS Introduction Neural progenitor cells (NPCs) resident to the mammalian subventricular zone and hippocampal subgranular zone spontaneously ge nerate significant numbers of neurons each day throughout life (Cameron et al. 1993; Alvarez Buylla & Garcia Verdugo, 2002) NPCs harvested from other CNS regio ns that include the substantia nigra (Reynolds & Weiss, 1992a; Palmer et al. 1995b; Pencea et al. 2001; Lie et al. 2002; Nunes et al. 2003; Rive rs et al. 2008; Lee & Blackshaw, 2012) produce abundant neurons in vitro but are described as gliogenic in vivo (Geha et al. 2010) unless stimulated to generate transient neurons by injury (Hoehn et al. 2005; Van Kampen & Robertson, 2005; Shan et al. 2006; Bertilsson e t al. 2008; Peng et al. 2008; Su et al. 2009) or more permanent neurons by targeted cell death (Magavi et al. 2000b) Controversial reports of spontaneous low level neuro gen esis in extra hippocampal and olfactory bulb central nervous system (CNS) reg ions that include the substantia nigra (Zhao e t al. 2003a; Lee & Blackshaw, 2012) are exciting because the development of methods that better detect and potentiate neurogenesis in these regions could lead to novel endogenous repair strategies. Voluntary exercise and exposure to environmental and social enrichment increase adult hippoc ampal neurogenesis (Kempermann et al. 1997; van Praag et al. 1999a) P revious work sugge sts that the effect of voluntary wheel running on NPC proliferation may be potentiated at night in nocturnal mice (Kempermann et al. 1997; Goergen et al. 2002; Holmes et al. 2004) The circadian clock is known to orchestrate

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45 cellular functions most mammalian tissues (Scheving et al. 1978; Bjarnason & Jordan, 2002; Smaaland et al. 2002; Matsuo et al. 2003; Wille et al. 2004) and Circadian Locomotor Output Cycles Kaput ( Clock ) genes have been shown to regulate the ex pression of neurogenic transcription factors in vivo and the n euronal differentiation of adult NPCs both in vitro and in vivo (Borgs et al. 2009b; Kimiwada et al. 2009a) Levels of f actors that regulate hippocampal neurogenesis in vivo such as serotonin, brain derived neurotrophic factor (BDNF) epidermal growth factor receptor (EGF r ), and transforming growth factor (TGF) fluctuate in a circadian and ultradian fashion (Scheving et al. 1989; Bova et al. 1998; Castanon Cervantes et al. 1999; Van der Zee et al. 2005; Tournier et al. 2007; Lindley et al. 2008; Ming & Song, 2011b) Therefore, variations in rates of hippocampal neurogenesis across the light dark cycle would be unsurprising, and, by taking advantage of factors that potentiate hippocampal neurogen esis, low level spontaneous rates of neurogenesis in extra hippocampal CNS regions may be boosted to the point of better detectability. Here, we tested whether NPC proliferation in the hippocampus and substantia nigra varies across the light versus dar k phase of the light dark cycle in adult nocturnal C57Bl/6 mice and whether the neuronal differentiation and survival of cells produced in each phase varies. We detected more BrdU + cells in the hippocampi and substantia nigra of mice injected with BrdU dur ing the dark versus light phase of the light dark cycle. In the hippocampus, more new cells produced during the dark cycle exhibited neuronal phenotypes and survived than new cells produced during the light phase. No new neurons were detected in the substa ntia nigra.

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46 Experimental Procedures Subjects Mice used as subjects in this study were treated in accordance with St anford University and federal regulations regarding the ethical use of animals for experimentation. Every effort to minimize animal number s and their suffering was undertaken. Adult female C57Bl/6 mice (n=6 0; 7 weeks old upon arrival from Taconic Farms ) were group housed (n=5 per cage) in shoebox cages ( 10.5 X 19 X 8 accommodate running wheels ) in a colony room with a 12:12 h light dar k cycle (lights off at 21 00 h) The mice were provided with Prolab Mouse 3000 chow (PMI Nutrition International, St. Louis, MO) and tap water ad libitum for the duration of the experiment To increase hippocampal neurogenesis (van Praag et al. 1999a; van Praag et al. 1999c) perhaps differentially during the light versus dark phase of the light dark cycle, and potentially stimulate neurogenesis in the substantia nigra to detectable levels, freely moving running wheels were introduced to some cages of mice and immobilized wheels were introduced to others Experiment Design One week after freely moving or immobilized running wheels were introduced to their home cages the mice were given either a single or began a series of intraperitoneal (i.p.) bromodeoxyuridine (BrdU; Sigma Aldrich, St. Louis, MO ) injections. BrdU was disso lved in freshly prepared isotonic saline (10mg/ml) just before use and injected at the standard 50mg/kg dose used to label dividing progenitors in the hippocampi of adult m ice (Kempermann & Gage, 2000) me ice were injected with BrdU once or daily for 6 days 2h after lights on (0900h) under standard

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47 under red light. NPC proliferation was quantified in daytime and nighttime injected non runners and runners perfused 2h after a single BrdU Injection (n=5 per group). The % of BrdU + cells expressing neuronal and glial phenotypes was quantified in daytime and nighttime injected runners and non runners that were perfused the day af ter the last of 6 daily BrdU injections (n=5 per group). The survival of new neurons was confirmed in daytime and nighttime injected non runners perfused 3 weeks after the last of 6 daily BrdU injections (n=5 per group). The experiment timeline is shown in Fig 2 1. Histological procedures Mice were anaesthetized deeply with a ketamine (100mg/kg)/xylazine (15mg/kg) cocktail injected i.p. before being perfused transcardially with ice cold saline and then 4% paraformaldehyde. Brains were extracted, stored ov er night in perfusate and then equilibrated in 30% sucrose intervals through their rostral caudal extent using a freezing stage sledge microtome (AO Corporation Model 860; Buffalo, NY, USA) Six sets of 1 in 6 series of sections through the hippocampus and substantia nigra were collected and stored at 20C in cryoprotectant ( 30% ethylene glycol, 25% glycerin, and 45% 0.1 M sodium phosphate buffer (vol/vol/vo l) ) until immunostained. Immunohist ochemistry Immunohistochemical staining was conducted as described previously (Speisman et al. 2012b) Free floating s ections were rinsed repeatedly with tris buffered saline (TBS; pH 6.0) before processing and between immunohistochemical step s Total BrdU + and BrdU/TH + cell numbers were estimated stereologically on every 12 th uniformly distributed section through the hippocampus and substantia nigra immunostained to reveal TH and BrdU enzymatically.

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48 Figure 2 1. Timeline of BrdU injections and perfusions. Adult female C57Bl/6 mice exposed to running wheels for 1 week were injected with the cell synthesis marker bromodeoxyuridine (B rdU; 50 mg/kg/day; Sigma) once per day for 6 were not exposed to running wheels. On day 7 or day 28 after injection, animals were deeply anesthetized with ketamine/xylazin e and perfused transcardially with 4% paraformaldehyde. Brains were extracted following perfusions, stored overnight in perfusate at 4 C, equilibrated in 30% sucrose and then sectioned through the entire brain at 40 m. The sectio ns were stored at 20C u ntil i mmunostained. Sections were then processed immunohistochemically so that total new cell number could be estimated stereologically and new cell phenotype assessed using confocal microscopy. One of the 6 sets that these sections were obtained from was randomly chosen to ensure the first section of the immunostained set could randomly be the 1 s t through 12 th section for each mouse. The sections were were blocked in 100mM Levamisole in 3% normal donkey serum (NDS; Jackson Immunoresearch, West Grove, PA, USA) with 0.3% Triton X in TBS for 30 min, followed by mouse anti tyrosine hydroxylase (TH) primary antibody (1:500; Sigma Aldrich, St. Louis, MO) overnight at 4C to reveal

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49 dopamine producing neurons. The next day, sections were rinsed and incubated for 4 hours in biotinylated anti mouse IgG (1:500; Jackson Immuno r esearch), followed by alkaline phosphatase streptavidin complex (Vectastain ABC AP; Vector Laboratories, Burlingame, CA) for 2h, then Vector Red substrate (Vector Laboratories) to reveal TH befo re being f ixed in 4% paraformaldehyde The sections were then rinsed with 0.9% NaCl, followed by 2M HCl for 20 min at 37C to denature DNA and then blocked in 3% NDS for 30 min to block non specific antigen binding and then incubated overnight in rat anti BrdU (1:500; Accurate Chemical, Westbury, NY). The following day, sections were incubated in 1:500 biotinylated anti rat IgG antibody (Jackson ImmunoResearch) for 4h, followed by avidin biotin peroxidase complex (ABC Elite; Vector Laboratories) for 2h. Fin ally, the sections were DAB to reveal BrdU, mounted on glass microscope slides, dried overnight, dehydrated in an alcohol series and then coverslipped under H istoclear (National Diagnostics, Atlanta, GA) The neuronal and glial phenotypes of BrdU + cells were confirmed on 3 5 randomly selected hippocampal sections, and the presence of a TH + neuronal phenotype among BrdU + cells was examined on 3 5 nigral sections on which fluorescent secondary antibodies revealed BrdU and pro teins of interest. S ections were rinsed with 0.9% NaCl, followed by 2M HCl for 20 min at 37C to denature DNA and then blocked in 3% NDS (Jackson Immunoresearch ) for 30 min before being incubated overnight at 4C in rat anti BrdU (1:500; Accurate Chemical) The following day the sections were incubated for 4h at RT in Cy3 conjugated anti rat IgG (1:500; Jackson Immunoresearch) and then fixed in 4% paraformaldehyde. The sections were then incubated overnight in 1) goat anti doublecortin (DCX; 1:500; Santa C ruz

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50 Biotechnology, Santa Cruz, CA) and mouse anti neuronal nuclei (NeuN; 1:500; Chemicon International) to reveal immature and mature neurons, respectively, 2) mouse anti TH (1:500; Sigma Aldrich) to reveal dopaminergic neurons or 3) rabbit anti NG2 (1:100 0; Millipore, Billerica, MA) and guinea pig anti glial fibrillary acidic protein (GFAP; 1:500; Advanced Immunochemical, Inc., Long Beach, CA) to reveal astrocytes/radial glial like progenitor cells and oligodendrocyte precursors, respectively. The followin g day, the sections were incubated for 4h at RT in the appropriate fluorescein FITC or Cy5 conjugated secondary antibody (1:500; Jackson ImmunoResearch) DAPI stained (1:10,000 ; Calbiochem, San Diego, CA, USA ) for 10 min mounted on microscope glass slides and coverslipped under PVA DABCO ( 2.5% diazobicyclooctane in TBS with 10% polyvinyl alcohol and 20% glycerol ). Data Analysis Stereological quantification of BrdU + cel l numbers Analyses were conducted on coded slides t o blind the experimenter to the treatment conditions. BrdU + and BrdU/TH + cells were counted on sections immunostained enzymatically under 40 magnification using a Zeiss Axio Observer Z1 inverted microscope (Thornwood, NY, USA) and estimates of total cell numbers were generated using stereological principles (Gundersen et al. 1988; West et al. 1991; Kempermann et al. 2002a; Speisman et al. 2012a) In the hippocampus, we counted round or oval BrdU + cells in both the SGZ and GCL through the rostral caudal extent of the dentate gyrus of each mouse (~ 8 sections spaced 240 m apart per mouse). BrdU + cells were counted exhaustively because they are typically s ituated irregularly through the SGZ and GCL In the substantia nigra (SN), BrdU + and TH/BrdU + cells were counted on every 12 th section (4 5 sections per mouse

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51 spaced 240 m apart). BrdU + cell coun ts obtained from the hippocampal SGZ and GCL and from the SN was multiplied by 12 (the section interval in each set) to generate a stereological estimate of the total number of dividing NPCs (in tissue collected 2h after a single BrdU injection) and the to tal number of new cells surviving 1 or 4 weeks in tissue collected at the later time points. AxioVision software (version 4.6.3) was used to measure the areas (in mm 2 ) on which BrdU + c ells were counted under a 20x objective. Analysis of Phenotypes On se ctions obtained 24h or 3 weeks after the final of 6 BrdU injections, at least 100 BrdU + cells were scanned through their x y and z planes to confirm their neuronal or glial phenotypes using a Zeiss LSM 710 fully spectral laser scanning microscope equipp ed with 405 (used to excite DAPI) 488 (used to excite FITC) 510 543 (used to excite Cy3) and 633 (used to excite Cy5) nm laser lines under a 40x objective (with 2.3x digital zoom). In hippocampal sections (2 3 sections examined per staining set) a f + cells revealing clear expression of D CX or NeuN confirmed a neuronal phenotype or a glial phenotype when astrocyte marker GFAP or the oligodendrocyte marker NG2 was clearly expressed. In nigral sections (5 6 sections ex amined per mouse) a BrdU/DAPI + cell was classified as expression through the nucleus. The % of BrdU + cells in the hippocampus expressing each phenotype and the total number of new neurons, astrocytes and oligodendrocyte precursors (estimated BrdU + cell number x the % of each phenotype) was calculated. The % of BrdU+ cells in the substantia nigra expressing TH was calculated.

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52 Statistical Analys i s Statistical analyses were perfo rmed using Statistica software (StatSoft; Tulsa, OK U.S.A. ) T he effects of the independent variables (running wheel exposure and/or light dark cycle phase) on the dependent variables ( BrdU + cell number, % of new neurons or glia, total new neurons or glia) were analyzed using an analysis of variance (ANOVA) and explored with Newman Keuls post hoc analyses All data shown are group means S.E.M. and levels were set at P < 0 .05 for all stati stical tests Results NPC proliferation in the dentate gyrus and the substantia nigra i s increased with activity and during the dark phase of the LD cycle Figure 2 2 shows dividing NPCs (A) and new cells surviving 1 (B) and 4 (C) weeks in the dentate gyri of adult female mice. An ANOVA revealed an effect of group on the number of dividing NPCs detected 2h after lights on or lights off (F 3,16 = 5.22, P < 0. 0 1 ; Fig. 2 3 A ). Relative to the number of dividing NPCs detected in non run ning mice during the day, we tended to observe more in non runners at night ( P = 0.08), and observed significantly more dividing NPCs in runners either during the day ( P = 0.01) or at night ( P = 0.01). A light dark cycle effect on cell proliferation was on ly observed in non runners. We also detected a statistically significant effect of group on the number of dividing NPCs in the substantia nigra ( F 3,16 = 11.58 P < 0 05; Fig 2 3 B). Specifically, relative to the number of dividing NPCs detected in non runners during the day, significantly more dividing NPCs were detected in non runners at night ( P < 0.001) or in runners during either the day ( P < 0.05) or night ( P < 0.01).

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53 Figure 2 2 Photomicrographs of BrdU positive cells in an animal which r eceived wheel access A) 2 hours, B) 1 week, and C) 4 weeks after the last BrdU injection performed Figure 2 3 Total number of BrdU positive cells (+/ S.E.M.) 2 hours after BrdU injection in A) the hippocampus and B) subtantia nigra in animals receiving wheel P 0 .05 ** P 0 .01 *** P 0 .005. C) Diagram showing hippocampal and nigral regions analyzed (in blue and pink, hippocampal dentate gyrus and subgranular zone, respectively; in green, subtantia nigra).

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54 Figure 2 4. C onfocal images of new neurons and astrocytes in the dentate gyrus of an adult mouse. Representative examples of approximately 1 week old (BrdU + ) cells (in red) that co express the mature neuronal marker NeuN (in green; B) and/or the immature neuronal marke r DCX (in blue; A), or the oligodendrocyte marker NG2 (in blue; D), or the astrocyte marker GFAP (in green; C). Scale Neuronal differentiation of daughter cells in the dentate g yrus is increased among progenitor cells dividing during the dark versus light phase and in animals exposed to a running wheel Phenotypic differentiation of surviving cells was evaluated using triple label immunofluorescence for BrdU, astrocytic marker GFAP and oligodendrocyte precursor marker NG2, or BrdU, neuronal marker NeuN and immature neuron al marker doublecortin (DCX) (Fig. 2 4). Total number of cells for each phenotype was calculated

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55 by combining the total number of BrdU labeled cells and the fraction of BrdU labeled cells of each phenotype 1 and 4 weeks afte r BrdU injections. One week after BrdU injections r unning animals had a significantly increased number of dividing NPC s compared with non runners whether they were injected with BrdU during the dark or light phase ( P < 0 05; Fig. 2 5 A ). We also detecte d a statistically significant effect of group on the proportion of differentiated neuronal cells (F 3,15 = 3.70, P < 0.05 ). The proportion of new mature neurons was significantly increased in runners versus non runners injected with BrdU during the light phase ( P < 0.05; Fig. 2 5 C ). We also observed a statistically significant effect of group on net new neuron number ( F 3 ,1 2 = 3.48, P < 0.05 ; Fig. 2 5 D ) one week after BrdU injections Net new neuron number was significantly increased in running versus non running animals, whether animals were injected during the light or dark phase ( P < 0 01 ). Net new neuron numbers were also significantly increased in animals injected with BrdU during the dark versus light phase in running and non running animals ( P values less than 0 01 ). No significant differences in the proportions of new oligodendrocytes or astrocytes were detected between groups one week after BrdU injections ( F 3 16 = 0.39 P > 0 05 ). Figure 2 5 Total number of BrdU positive cells (+/ S.E.M.) 1 week after BrdU injections in A) the hippocampus and B) subtantia nigra in animals C ) At least 100 new (BrdU + ) cells per mouse were examined to determine the proportion that new cells expressing markers of immature (DCX + ), transitioning (DCX+NeuN + ), or mature (NeuN + ) neurons, revealed using fluorescent immunohistochemistry and confirmed under confocal microscopy (40x objective and 2x digital zoom). D) Total new neuron numbers (BrdU+ DCX or NeuN + ) P 0 .05 ** P 0 .01.

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56

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57 Four weeks after BrdU injections, diurnal phase was found to have a significant effect on the diff erentiation of daughter cells. The proportion of new mature neurons was significantly increased in animals injected at night versus day ( P < 0 001; Fig. 2 6 B ) and n et new neuron number was significantly increased in animals injected during the dark versus light phase ( P < 0.01; Fig. 2 6 D ). The proportion of surviving cells was similar between animals injected during the light or dark phase ( P > 0.05). No sig nifi cant differences in the proportions of new oligodendrocytes or astrocytes were detected between groups 4 weeks after BrdU injections ( P > 0 05 ). Figure 2 6. Total number of BrdU positive cells (+/ S.E.M.) 4 weeks after BrdU injections adminis C) At least 100 new (BrdU + ) cells per mouse were examined to determine the proportion that new cells expressing markers of immature (DCX + ), transitioning (DCX+NeuN + ), or mature (NeuN + ) neurons, revealed using fluorescent immunohistochemistry and confirmed under confocal microscopy (40x objective and 2x digital zoom). D) Total new neuron numbers (BrdU+ DCX or NeuN + ) were calculated in P 0 .05 ** P 0 .01 *** P 0 .005.

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59 Cell survival in the substantia nigra is increased among progenitor cells labeled with BrdU during the dark phase in animals exposed to a running wheel, with no neuronal differentiation One week and 4 weeks after BrdU injections we detected a significant effect of the circadian phase when the mice were injected with BrdU on cell survival in the substantia nigra. Running animals injected with BrdU at night had a significantly greater number of dividing NPCs than runners injected during the day both 1 week ( P < 0.05; Fig. 2 5 B ) and 4 weeks ( P < 0 .001; Fig. 2 6 B ) after BrdU injections. No new dopaminergic neuronal cells or glia were observed in the substantia nigra in any of the groups (Fig. 2 7) Figure 2 7 Confocal image of dopaminergic neurons in the substantia nigra of an adult mouse. Inset: representative example of an approximately 1 week old (BrdU + ) cell (in red) that does not co express the dopaminergic neuronal marker TH

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60 Discussion The results of the present experiment confirm earlier reports that running activity significantly increases cell proliferation and total number of new neurons born in the dentate gyrus of adult mice (van Praag et al. 1999c; Holmes et al. 2004) Animals allowed wheel access for one week before BrdU injections had significantly increased lev els of neurogenesis in the hippocampus. Furthermore, animals that were allowed wheel access and that were injected with BrdU during the dark phase displayed a statistically significant increased number of new neurons in the dentate gyrus 1 week and 4 weeks after BrdU administration, compared with non running and running animals injected with BrdU during the day. It is interesting to note that in the substantia nigra, there was a significant increase in cell proliferation in animals injected with BrdU at night compared with both runners and non runners injected during the day 2 hours after BrdU injection, as well as increased survival after 1 and 4 weeks. However, none of these new cells adopted a neuronal phenotype, as has been previously ob served (Lie et al. 2002) whether animals were injected with BrdU during the day or night The fact that the circadian phase had a more r obust effect on cell proliferation in the substantia nigra than in the hippocampus suggests a disparity of neurochemical factors influencing cell division between the hippocampus and the midbrain area at night. We also observed a significant increase in ne w neuron survival in runners injected with BrdU at night compared to runners injected during the day. These findings suggest that the light dark cycle can influence neuron production, maturation and survival, independently of physical activity; neurogenic activity seems to not only be dependent on duration or amount of activity (Holmes et al ., 2004) but also to be under the control of the light dark cycle. The effect of exercise induced increased cell

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61 proliferation might be due to an increase in VEGF level s, which appear to also fluctuate across the light dark cycle (Koyanagi et al. 2003; Hetland et al. 2008) Circadian control of cell division and diurnal variation in cell proliferation have been well established in rodent and human studies of several tissues, including bone marrow (Smaaland et al. 2002) gastrointestinal tract mucosa (Bjarnason & Jordan, 2002) epidermis (Brown, 1991) liver (Matsuo et al. 2003) and cornea (Scheving & Pauly, 1967) The fact that neurogenic activity in the hippocampus and cell division in the s ubstantia nigra are enhanced at night may provide insight into the factors regulating neurogenesis in the adult brain. In fact, most factors that have been found to regulate neurogenesis in the adult nervous system are themselves controlled by diurnal rhyt hms. Neurotransmitters such as serotonin, noradrenaline and acetycholine all increase neural progenitor cell proliferation (Kulkarni.Rd et al. 1965; Banasr et al. 2004; Mohapel et al. 2005) and show levels varying across the LD cycle (Quay, 1963; Manshard.J & Wurtman, 1968; Marrosu et al. 1995) Serotonin, a regulator of neurogenesis in vertebrate species (Gould et al. 1999; Brezun & Daszuta, 2000) shows levels that fluctuate on a diurnal cycle in many organisms (Castanon Cervantes et al. 1999; Grimes et al. 2000) Plasma corticosterone levels rise late in the rest phase of the circadian cycle and peak early in the active phase, in both nocturnal and diurnal animals (Ottenweller et al. 1979; Weber et al. 2000) and mice living in an enriched environme nt have significantly decreased corticosterone levels and increased adult hippocampal neurogenesis (Kempermann et al. 2002b ) Therefore, moderate levels of corticosterone that vary in a

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62 circadian fashion might be associated with both increased cell survival and number of new neurons. In addition, growth factors such as epidermal growth factor (EGF) and fibroblast growth fac tor 2 (FGF2) are powerful factors for the maintenance of neural progenitor cells in vitro (Palmer et al. 1995a; Doetsch et al. 2002) They also promote proliferation in the SVZ in vivo and FGF2 increases the number of new neurons in the olfactory bulb (Reynolds et al. 1992; Kuhn et al. 1997) EGF has been shown to stimulate DNA synthesis in a circadian dependent manner in the aorta, lung, liver, cornea, tongue, intestines and other organs of adult mice (Scheving et al. 1979; Yeh et al. 1981; Scheving et al. 1987) although no study has examined diurnal variations in FGF receptor levels. BDNF is also one of the key positive regulators of neurogenesis, increasing neurogenesis, proliferation of neural progenitor cells in the SVZ and survival in the SGZ (Sairanen et al. 2005; Scharfman et al. 2005; Henry et al. 2007; Young et al. 20 07) BDNF mRNA expression and plasma BDNF levels have been shown to vary with the light dark cycle (Bova et al. 1998; Schaaf et a l. 2000; Begliuomini et al. 2008) Hormones, such as thyroid hormone, prolactin and estradiol, have also been found to promote new neuron survival (Hidalgo et al. 1995) and enhance neurogenesis in the forebrain and in the dentate gyrus by increasing the survival of young granule neurons (Shingo et al. 2003; Ormerod et al. 2004b) These hormones were also found to exhibit variations across the circadian cycle (Sassin et al. 1972; Ehara et al. 1975; Szafarczyk et al. 1980; Roy & Wilson, 1981b) For instance, estrogen receptor levels decrease in darkness (Cai et al. 2008a) and prolactin levels are highest during daytime (Sassin et al. 1972; Garcia Bonacho et al. 2000) In addition, t he significant expression of clock related genes in the hippocampus

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63 (Guilding & Piggins, 2007) suggests that the daily regulation of progenitor cell divisions and neurogenesis may be dictated by those circadian clocks localizing in the hippocampus. Recently, Per2 gene, a clock gene expressed in a circadian fashion in the suprachiasmatic nucleus in the mammalian brain, was found to correlate negatively with the DNA synthesis activity of neural stem cells in vitro (Moriya et al. 2007) Other Clock genes ( Clock or Bmal1 ) have also been shown to regulate neurogenic transcription factors and the neuronal differentiation of adult neural progenitor cells (Kimiwada et al. 2009a) In vivo clock c ontrolled Period2 gene expression has been linked to the intrinsic control of neural stem/progenitor cells proliferation, cell death and neurogenesis in the dentate gyrus of adult mice (Borgs et al. 2009b) C ircadian rhythm plays a key role in al l biological processes, including the expression of genes, which in turn might impact the levels of factors regulating neurogenesis The present data demonstrate that cell proliferation, neurogenesis and neuron survival in the hippocampus are dependent on circadian phase, independently of physical activity. Although o ur data give some new insight into the regulation of neurogenesis the physiological and behavioral factors explaining the effect of the circadian cycle on neurogenic ac t ivity remain to be eluc idated. Once the variations and the mechanisms of transcriptional clock and gene expression are fully understood, neural stem cell research can be directed toward altering the gene clock or cell cycle kinetics in ways that could implement favorable regulat ion of normal neural progenitor activity. Indeed, the development of treatments and/or cures that would increase production of new neurons requires the identification of endogenous or natural molecular regulators of adult neurogenesis. Robust light dark cy cle induced changes in

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64 neurogenesis could be a powerful means for exploring the mechanisms that regulate cell cycle behavior, without submitting the brain to injury or other external manipulation.

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65 CHAPTER 3 VARIATIONS IN HIPPOCAMPAL PROGENITOR CELL PROLIFERATION AND DIFFERENTIATION BETWEEN THE LIGHT (SLEEP) AND DARK (WAKE) PHASE S MIGHT BE RELATED TO CHANGES IN CELL CYCLE KINETICS AND GENE EXPRESSION IN ADULT MICE Introduction The entire adult brain holds regenerative potential in the form of neural stem/progenitor cells (Palmer et al. 1995a; Weiss et al. 1996; Arsenijevic et al. 2001; Palmer et al. 2001) Progenitor cells resident to the subventricular z one or the hippocampal subgranular zone generate significant numbers of neurons each day (Cameron et al. 1993; Kempermann & Gage, 2000; Alvarez Buylla & Garcia Verdugo, 2002) while progenitor ce lls located in other CNS regions primarily generate glia (Gould & Gross, 2002; Gould, 2007; Geha et al. 2010) unless injury is induced, which appears to stimulate the production of transient neuron populations. Knowledge of the internal and external stimuli that regulate the dynamic processes of progenitor proliferati on and neurogenesis in the adult brain is crucial in developing strategies for treating neurodegenerative diseases and injuries Previous work suggests that neural progenitor cell prolif eration and differentiation are robustly increased after physical acti vity and at night (Kempermann et al. 1997; Ho lmes et al. 2004) Circadian rhythm and cell cycle progression interact at the level of genes, proteins and biochemical signals (Bjarnason et al. 1999; Grundschober et al. 2001; Matsuo et al. 2003) and t here is substantial evidence that, in mammals, circadian rhythms affect the timing of cell divisions in vivo Day night variations in mitotic index, DNA synthesis an d cell c ycle time occur in multiple organs, i.e. oral mucosa tongue intestinal epithelium liver, skin, bone marrow etc. (Tutton, 1973; Scheving et al. 1978; Scheving et al. 1987; Scheving et al. 1989; Brown, 1991; Buchi et al. 1991;

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66 Smaaland, 1996; Bjarnason et al. 1999; Garcia et al. 2001; Smaaland et al. 2002; Matsuo et al. 2003; Wille et al. 2004) some of which persist even in constant darkness (Scheving et al. 1974) Expression of cell cycle proteins has been shown to displa y circadian variations as well. For instance, in the regenerating liver of mice, the circadian clock controls the expression of cell cycle related genes that in turn modulate the expression of active Cyclin B1 Cdc2 kinase, a key regulator of mitosis (Matsuo et al. 2003) Therefore the circadian rhythm plays a key role in all biological processes, including the expression of genes, which suggests it might regulate the levels of factors controlling neural progenitor cell proliferation. P roliferation of neural progenitor cells is tightly controlled by cell cycle kinetics (Nowakowski et al. 1989b; Takahashi et al. 1993b; Cameron & McKay, 2001b; Hayes & Nowakowski, 2002; Caviness et al. 2003; Burns & Kuan, 2005) and, as described above, circadian rhythms affect the timing of cell divisions in vivo in multiple organs However, the influence of the ligh t dark cycle on cell cycle kinetics of neural progenitor cells in the adult brain has not yet been investigated. In experiment 1, w e first examine d whether variations in cell cycle distribution and kinetics in vivo are responsible for the increased neural progenitor cell proliferation at night by using immunofluorescence and a cumulative 5 bromo 2 deoxyuridine (BrdU) labeling protocol developed by Nowakowski et al. (1989 ). In addition, to assess if differences in cell death rate between the light and dark phases might explain the variation in neural progenitor cell proliferation, we quantified the number of new pyknotic cells in the brains of mice injected with BrdU at ni ght or during the day. Second, in order to determine whether differences in cell cycle kinetics between light and dark phases were due to variations in

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67 gene expression, and because cell cycle kinetics are governed by the levels of various proteins (e.g., c yclins and cyclin dependent kinases), light dark cycle induced changes in the expression of 84 genes associated with cell cycle regulation were identified using Real Time PCR array technology RNA from m ale intact and ovariectomized female mice was used to determine the effect of sex hormones on gene expression during the light or dark phase. Differentiation and survival of neural progenitor cells are controlled by various intrinsic and environmental factors. However, the influence of the light dark cycle on these factors has not yet been investigated in the adult brain. In experiment 2, w e first examined whether maturation rates differed early after neurons were generated during the day or night by quantifying the proportions of Sox2 and Prox1 positive ne w cells a few hours after cell division. Sox2 is a transcription factor that is essential to maintain self renewal of undifferentiated neural stem cells (Graham et al. 2003; Ellis et al. 2004; Suh et al. 2007; Favaro et al. 2009) and Prox1 is a transcription factor expressed early during neuronal maturation and required for granule cell maturation and intermediate progenitor maintenance during adult brain neurogenesis (Elkouris et al. 2010; Lavado et al. 2010; Karalay et al. 2011) We also determined whether the morpholog y of doublecortin (DCX) positive neurons produced during the day differ s from those produced at night in 1 and 4 week old neurons, as it reflect the rate of neuronal differentiation (Plumpe et al. 2006) DCX is a microtubule associated protein associated with post mitotic neuronal differentiation that has been linked to cell migration and ot her aspects of maturation (Deuel et al. ; Portes et al. ; Gleeson et al. 1998; Francis et al. 1999) Second, t o determine whether differences in NPC

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68 differentiation and survival between light and dark phases were due to variations in gene expression associated with neurogenesis RNA from the hippocampi of male and adult female mice were harvested 6 h after lights on (0 6 00) or 6 h after lights off (1 8 00 ), and light dark cycle induced changes in the expression of 84 genes associated with NPC maturation and survival were identified using Real t ime PCR array technology. Once the mechanisms and variations in the transcriptional clock and gene expression are better understood, neural stem cell research can be directed toward altering the gene clock or cell cycle kinetics in ways that could implement favorable regulation of normal neural progenitor activity. Experimental Procedures Subjects All mice used as sub jects in this study were treated in accordance with the policies set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health and in accordance with the guidelines established by the U.S. Public H ealth Service Policy on the Humane Care and Use of Laboratory Animals. Every effort was made to minimize number of animal subjects used and their suffering during the study. A dult C57Bl/6 mice were group housed (n=5 per hr light dark cycle (lights off at 1800 h). M ice were given free access to Prolab Mouse 3000 chow (PMI Nutrition International, St. Louis, MO) and tap water ad libitum for the duration of the experiment. All experiments began after animals were allowed a week of habituation.

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69 Experiment Design For experiment 1, t o determine the effects of the light dark cycle on cell cycle distribution and ce ll cycle kinetics, a dult female C57Bl/6 mice (n= 58 ; 7 weeks old upon arrival from Taconic Farms) were given s ingle and cumulative BrdU injections either during the day or night depending on the phase of light dark cycle studied (see Fig 3 1 ) T o test the effects of the light dark cycle on cell cycle gene expression, adult female (n = 1 0 ) and male (n = 10) C57Bl/6 mice (n= 30 total; 8 weeks old upon arrival from Taconic Farms) were used. For Experiment 2, to determine possible differences in the rate of m aturation between neurons produced at night or during the day, two groups of female C57Bl/6 mice ( n= bromo deoxyuridine (BrdU) every 2 h, d Prox1 immunostaining. Another group of female C57Bl/6 mice ( n= 58 total ) were exposed to mobile running wheels to increase neurogenesis (n= 29 ) or immobilized control wheels (n= 29 ) (Zhao et al. 2003a; van Praag et al. 2005; Uda et al. 2006) for 1 week before BrdU injections and doublecortin and NeuN immunostaining 1 or 4 weeks later (see Figure 3 2). T o test the effects of the light dark cycle on cell cycle gene expression, adult female (n = 1 0) and mal e (n = 10) C57Bl/6 mice (n= 2 0 total; 8 weeks old upon arrival from Taconic Farms) were used.

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70 Figure 3 1. Experimental timeline and cell cycle markers for cell cycle distribution and cell cycle kinetics study. (A) Timeline of BrdU injections to determi ne cell cycle kinetics in daytime and nighttime groups. (B) Cell cycle diagram showing the stages of the cell cycle labeled by Ki 67, BrdU, and PHH3 BrdU labels cells in S phase, PHH3 labels cells in G 2 and M phase, and Ki 67 labels all actively dividing cells. (C) Timeline for cell cycle distribution study: mice were injected with BrdU (50 mg/kg) after a 7 day habituation period 2 cycle kinetics study: cumulative BrdU in jections were made every 2 hours for group sacrificed 0.5 hours after each injection. (D) A cumulative labeling procedure with BrdU used for the determination of the length of the cell cycle T C and the DNA synthetic phase T S A best fit slope is linear with an extrapolated y axis intercept (= T C / T S GF). The labeling index (LI) reaches the maximum growth fraction (GF) at an interpolated value corresponding to T C T S (Nowakowski et al. 1989a; Takahashi et al. 1993a) BrdU, bromodeoxyuridine; PHH3 phosphohistone H3.

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71 Figure 3 2 Timeline of BrdU injections and perfusions for morphological study Adult female C57Bl/6 mice exposed to running wheels for 1 week were injected with the cell synthesis marker bromodeoxyuridine (BrdU; 50 mg/kg/day; Sigma) once per ff groups were not exposed to running wheels. On d ay 7 or d ay 28 after injection, animals were deeply anesthetized with ketamine/xylazine and perfused tran scardially with 4% paraformaldehyde. Brains were extracted following perfusions, stored overnight in perfusate at 4 C, equilibrated in 30% sucrose and then sectioned through the entire brain at 40 m. The sections were stored at 20C until inmmunostaine d Sections were then processed immunohistochemically so that total new cell number could be estimated stereologically and new cell phenotype assessed using confocal microscopy. Bromodeoxyuridine (BrdU) preparation, injection and histology BrdU was dissolv ed just prior to injection to a concentration of 10 mg/ml (w/v) in freshly prepared 0.9% saline and was injected intraperitoneally in a volume of 50l/10g body weight. This 50mg/kg BrdU dose is standard for quantifying dividing progenitors and for determin ing the fate and survival of their progeny in mice (Kempermann &

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72 Gage, 2000; Cameron & McKay, 2001a; Clelland et al. 2009) Daily injecti ons were administered under standard fluorescent light after lights on (0700h), and nightly injections were administered under red light after lights off (1900h). At the end of experiments, animals were anesthetized deeply using a single intraperitoneal k etamine (100mg/kg)/ xylazine (15 mg/kg) (Webster Veterinary, Alachua, FL) injection and transcardially perfused with ice cold isotonic saline, followed by 4% paraformaldehyde (Electron Microscopy Sciences; Hatfield, PA) Brains were extracted (approximately 4 the rostral caudal extent of their hippocampi using a freezing stage sledge microtome ( American Optical Corporation Model 860). The sections were then stored at 20C in cryoprotectant ( containing 30% ethylene glycol, 25% glycerin, and 45% 0.1 M sodium phosphate buffer (v/v/v) ) until immunostained. Immunofluorescence In E xperiment 1, t o estimate the effects of the light dark cycle on cell cycle phase distribution of NPCs, d ouble immunofluorescence staining for BrdU and Ki 67 (a marker of proliferating cells) or BrdU and phosphohistone H3 (PPH3; a marker of G 2 M phase) was performed on frozen sections from animals injected with BrdU (50 mg/kg) and sacrificed 2 hours later. BrdU is a marker of dividing cells in synthesis (S) phase of the cell cycle; PHH3 is a marker of G 2 /M phases of proliferating cells, and Ki 67 is a marker of actively dividing cells (Kuan et al ., 2004). Immunofluorescent labeling was done on slide mounted tissue to examine the phenotype of BrdU labeled cells as described previously (Gould et al ., 1999; van Praag et al ., 2002; Ormerod et al ., 2003). Sections were rinsed repeatedly between steps with TBS. Free floating sections were blocked in

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73 3% normal donkey serum (NDS; Jackson Immunoresearch, West Grove, PA, USA) and incubated overnight at 4C in rabbit polyclonal anti Ki67 (1:500; Vision Biosystems ) or rabbit polyclonal anti PHH3 (1:500, 06 570 Millipore ) primary antibodies The following day, sections were incubated in the appropriate FITC conjugated secondary antibody (anti rabbit Ig G; 1:500; Jackson ImmunoResearch, West Grove, PA ) for 4 h at room temperature Sections were then incubated in 2M HCl for 30 min at 37C to denature DNA, followed by rat anti BrdU (1:500; AbD Serotec, Raleigh, NC) primary antibody. The following day, sections were i ncubated in the appropriate Cy3 conjugated secondary antibody (anti rat Ig G; 1:500; Jackson ImmunoResearch, West Grove, PA ) for 4 h at room temperature S ections were then incubated in 4',6 diamidino 2 phenylindole (D API; Calbiochem, San Diego, CA; 1:10,000) for 10 minutes to reveal cell nuclei, then mounted on glass slides and cover slipped under the anti fading agent diazobicyclooctane (DABCO; 2.5% DABCO, 10% polyvinyl alcohol and 20% glycerol in TBS; Sigma Aldrich). The phenotypes of f luorescently labeled cells were confirmed using a Ze iss Met a LSM 710 fully spectral laser scanning confocal microscope (with 405, 488, 510, 543 and 633 laser li nes) using a 63x oil objective (and 2 x digital zoom). To assess the total number of Ki 67, PH H 3 positive cells and BrdU positive cells, every t welve secti on of the entire hippocampus were analyzed. The number of immunopositive cells in the hippocampus was then multiplied by 12 to provide an estimate for the total number of the positive cells in the dentate gyrus. In Experiment 2, i n an effort to understand how NPC maturation is affected by the light dark cycle, we examined the proportions of early differentiation markers (Sox2,

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74 Prox1) 10.5 h after BrdU administration, as well as intermediate and late proliferation markers (DCX, NeuN), and whether the morpho log y of neurons produced at night differ from those produced during the day 1 week and 4 weeks after BrdU administration. Sections were rinsed repeatedly between steps with TBS. Free floating sections per mouse were stained immunohistochemically to detect new (BrdU+) and undifferentiated (Sox2 + ), early immature (Prox1 + ), immature (DCX + ), transitioning (DCX/NeuN + ), and mature (NeuN+) neurons. Sections were blocked in 3% normal donkey serum (NDS; Jackson Immunoresearch, West Grove, PA, USA) for 30 min at roo m temperature, then incubated overnight at 4C in a cocktail of primary antibodies that included rabbit anti Sox2 ( 1:500; Cambridge, MA, USA ) and mouse anti Prox1 ( 1:500; Chemicon, Billerica, MA, USA ) or mouse anti NeuN (1:500; Chemicon, Billerica, MA, USA) and goat anti DCX (1:500; Santa Cruz, Santa Cruz, CA, USA). The following day, sections were incubated for 4 h in a cocktail of Cy5 conjugated secondary anti mouse IgG and FITC conjugated secondary anti rabbit IgG ( 1:500; Jackson ImmunoResearch), or Cy5 conjugated secondary anti goat IgG and FITC conjugated secondary anti mouse IgG (1:500; Jackson ImmunoResearch) respectively S ections were then incubated in 2 N HCl for 2 0 min at 37C to denature DNA, fo llowed by anti rat BrdU (1:500; Accurate, Westbury, NY, USA) primary antibody overnight at 4C. The next day, sections were incubated in 4',6 diamidino 2 phenylindole (DAPI; Calbiochem, San Diego, CA; 1:10,000) for 10 minutes to reveal cell nuclei, then mo unted on glass slides and cover slipped under the anti fading agent diazobicyclooctane (DABCO; 2.5% DABCO, 10% polyvinyl alcohol and 20% glycerol in TBS; Sigma Aldrich).

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75 Fluorescently labeled cells ( at least 100 per mouse) were imaged using a Zeiss meta L SM 710 fully spectral laser scanning confocal microscope with 405, 488, 543 and 6 33 nm laser lines with a 63x oil objective (and 2 x digital zoom) and Zeiss ZEN soma perimeter s and areas, as well as branch quantity, length and node number (Rao & Shetty, 2004) The number of primary branches extending from the soma as well as secondary branches extending from nodes were recorded, because not only are these indicators of the stage of neuronal development, but differences in one of these types of branching could be reflective of different differ entiation processes between day and the soma as well as seconda ry branches extending from nodes the number of secondary branches P roportions of Br dU+ (new) cells with undifferentiated (Sox2 + ) or early immature neuronal (Prox1 + ), or that had acquired immature (DCX + ), transitioning (DCX/NeuN + ), or mature (NeuN + ) neuronal phenotypes were also measured using confocal digital images. Proportions of branc hed DCX+ new neurons were also measured 1 week and 4 weeks after BrdU administration. Cumulative 5 Bromo Deoxyuridine (BrdU) Labeling To estimate the e ffects of the light dark cycle on cell cycle kinetics we used a cumulative BrdU labeling protocol established by Nowakowski et al. (1989) and Takahashi et al. (1993), we estimated the length of cell cycle (T C ), the length of S phase (T S ) and proliferating population of S G Z cells (GF). This protocol requires that the serial BrdU injections be spaced so that the length of non labeled intervals between injections is less than the length of S phase such that all nuclei passing through S phase were labeled (Nowakowski et al., 1989; Takahashi et al ., 1993). Briefly, adult mice were

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76 intraperitoneally injected with BrdU (50 mg/kg, Sigma) at 2 h intervals over a total period of 12 h, either during the dark or light phase respectively At 30 min after each of the BrdU injections, selected mice were killed. Three to 4 animals per time point were killed with a total of 6 time points. For analysis of BrdU labeled cells, every 40th BrdU immunostained coronal section at the level of AP +10.6 mm and AP + 9.2 mm (Paxinos and Watson, 1986) was digitized using a 40x objective o n a Zeiss Observer Z1 inverted microscope and Zeiss Axiovision software (version 4.6.3) 5 Bromo 2 deoxyuridine labele d and total Cresyl Violet (CV) labeled cells along the dentate gyrus were counted on a computer monitor using the Stereo Investigator sof tware ( Microbrightfield Corp., USA) 5 Bromo 2 deoxyuridine labeled and CV labeled cells in each coronal section are presented as the number of the cells /section. Density for the f ive sections per mouse was averaged to obtain a mean density value for eac h brain according to published methods (Kuhn et al ., 1996; Zhang et al ., 2001b). From similar studies of cell proliferation, the cells comprising the proliferative population are assumed to be asynchronously distributed in the cell cycle. It is also assumed that the intrahilar proliferative population is in a steady state growth phase (Nowakowski et al., 1989) For each brain, an average labelin g index (LI), that is, the ratio of BrdU labeled cell to total cell numbers was determined by averaging the LIs of f ive nonadjac ent sections at each time point and was plotted as a function of time after the initial injection (Nowakowski et al. 1989;Takahashi et al. 1993) The GF, that is, the ratio of proliferating cells to the total cells in the population, and the parameters of T C and T S were calculated by using a least squares (LS) line fit to all considered data points (Nowakowski et al ., 1989;Takahashi et al ., 1993) T C and T S were calculated from

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77 the graphs based on two relationships: (1) the time required to label the GF, that is, the inflection point of the curve, is equal to T C T S ; and (2) the y intercept of the curve is equal to ( T C / T S ) GF (Nowakow ski et al., 1989;Takahashi et al., 1993; Fig. 3 1 ) Immunohistochemistry For cumulative BrdU labeling analysis, 1 in 6 series through the hippocampus were processed free floating BrdU diaminobenzidine (DAB). Sections were washed repeatedly betwee n steps in tris buffered saline (TBS; pH 7.4). Endogenous peroxidase was quenched by incubation in 0.3% H 2 O 2 for 10 min and following several 0.9% NaCl rinses, DNA was denatured in 2N HCl for 20min at 37C. S ections were blocked in a 3% no rmal donkey serum (NDS) solution (3% NDS and 0.1% triton x in TBS; v/v/v) and incubated overnight in rat anti BrdU (1:500; AbD Serotec, Raleigh, NC) at 4C then for 4 h in biotinylated secondary, anti rat IgG (Jackson ImmunoResearch, West Grove, PA; 1:500) at RT. Next, the sections were incubated in avidin biotin horseradish peroxidase (Vector Laboratories, Burlingame, CA) then reacted in a solution of 0.02% 3, diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich, St. Louis MO) followed by 0.5 % H 2 O 2 and finally counterstained with Cresyl Violet in order to visualize neurons and their nuclei Tissue slices were then mounted on glass slides and submitted to stepwise alcohol dehydration before being mounted under P ermount (Fisher Scientific; Pitt sburgh, PA). For analysi s of BrdU labeled cells, every sixth BrdU immunostained coronal section at the level of AP +10.6 mm and AP +9.2 mm (Paxinos and Watson, 1986) was digitized using a 63x oil objective on a Zeiss Observer Z1 inverted microscope. BrdU l abeled and unlabeled Cresyl Violet positive cells along the dentate gyrus were counted on a computer monitor

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78 using the Stereo Investigator software (Microbrightfield Corp., USA) to estimate average labeling index (LI), that is the ratio of Brdu labeled cel ls to total cells Pyknotic Cell Analysis To determine whether the day/night variation in the number of proliferating cells in experiment 1 is influenced by differential light dark cycle induced degeneration of hippocampal NPCs, cell death was measured usi ng pyknotic cell analysis. All slides were coded to blind the experimenter from the treatment conditions. On peroxidase treated tissue used for c umulative BrdU l abeling pyknotic/fragmented cells which were BrdU positive were counted on every 10th section through the granule cell layer and subgranular zone (SGZ; defined as approximately the 50 cell layer and the hilus; Palmer et al., 2000) using a 100 objective with oil on a Zeiss (Thornwood, NY) Axovision light microscope Pyknotic/fragmented cell counts were used as a relative measure of cell death as they might contribute to the decreased rate of NPC proliferation detected in the hippocampi of adult mice during the light phase Cells were co nsidered pyknotic or fragmented if they exhibited a pale or absent cytoplasm, dark spherical chromatin and no nuclear membrane, and BrdU labeled if intensely stained brown (Cameron et al., 1993; Ormerod and Galea, 2001; Ormerod et al., 2003a, b; Fig.3 3 )

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79 Figure 3 3 Representative light photomicrographs from granule cell layer of dentate gyrus showing cells stained with Cresyl Violet and BrdU. (A) Mouse BrdU labeled cell stained brown (arrow; 63X oil objective). (B) Mouse pyknotic (dying) cell sh owing dark, condensed, spherical chromatin (arrow; 63X oil objective). Scale bar is 5 m and applies to A B GCL, granule cell layer Quantitative RT PCR One week after arrival or 24 h following the last injection of EB for the ovariectomized female mice, all animals were anesthetized with CO 2 and decapitated, either 6 h after lights on (n = 5 per group) or 6 h after lights off (n = 5 per group). The brain was quickly removed and placed in ice cold phosphate buffer saline solution. Both hippocampi were remo Hippocampi were pooled according to the time of collection. The RNA extracted from hippocampi w as pooled according to the time at which they were collected. The tissue was minced, treated with RNase fre e DNase I (Ambion), and then RNA was purified using the RNeasy Mini kit (Qiagen). RNA quantity and purity were determined using a NanoDrop ND 1000, and RNA integrity was assessed by determining the RNA integrity number (RIN) a s well as the 28S/18S ratio u sing a Bioanalyzer 2100 (Agilent Technologies). A quantity of 500 ng of high quality RNA (260/280 ratios slightly higher than 2.0 and 260/230 ratios higher than 1.5, RIN > 8.0) for each pooled sample was converted into cDNA using the RT 2 First Strand cDNA Kit (SABiosciences). All qPCR reactions use the RT 2 SYBR Green qPCR Master Mix (SABiosciences). Cell cycle and neurogenesis

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80 gene expression was determined using Cell Cycle PCR Array (PAMM 020, SABiosciences ) and Neurogenesis and Neural Stem Cell PCR Array (PAMM 040, SABiosciences) respectively, and the My iQ5 system (Bio Rad) according to the in this article; the complete list of genes assayed on the array can be found at the e ( http://www.sabioscience.com/rt_pcr_product/HTML/PAMM 020A.html ; http://www .sabioscience.com /rt_pcr_product/HTML/PAMM 040A. html ). Statistical Anal ysis t tests (one dependent variable between groups) or analysis of variances (more than one dependent variable between groups) and explored using Newman Kewls post hoc tests. A lpha levels were set at 0.05. For instance, ANOVAs were run on the data collected from mice perfused after the last of 7 daily BrdU injections (4 groups) and t tests were run on the data collected from mice perfused 3 weeks after the l ast of 7 daily BrdU injections (2 groups). Results More Cells are Found in G 2 M and S Phase s at Night versus Day in the Hippocampus of Female Adult Mice The effect of the light dark cycle on the distribution of hippocampal NPCs across the cell cycle wa s examined. In this experiment, mice in the daytime and nighttime group s received a single BrdU injection (50 mg/ or respectively and sacrificed 2 hours later. In both groups, Ki 67 + PHH3 + and BrdU + NPCs were easily i dentified wit hin cell clusters located along the SGZ of the dentate gyrus. Ki 67, which la bels cells in all active phases of the cell cycle, was unaltered by

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81 the light dark cycle ( P = 0. 18; Fig 3 4 A ) In contrast, the number of PHH3 + NPCs was significant ly increased at nighttime by 53% ( P = 0. 002; Fig 3 4 B), as was the number of BrdU + NPCs which was increased by 50 % ( P = 0. 01; Fig 3 4 C). This demonstrates that, in adult mice the proportion s of hippocampal NPCs in the S and G 2 M phases are increased during the dark phase of the light dark cycle with no ob served effect on the proportion of NPCs in G 1 phase (no difference in Ki 67 + cells between daytime and nighttime) Therefore, T C was shortened at ni ght by 8%. A large portion of this decrease was due to a 24% reduction in S phase duration, in which T S was shortened to 3.8 h A bbreviated T C and T S suggest that progression through the cell cycle is accelerate d at nighttime, leading to an overall increase in NPC pr oliferation during the dark phase The present T S and T C data determined by cumulative BrdU labeling are comparable to previously reported studies for adult mice (Nowakowski et al. 1989a; Cameron & McKay, 2001a; Hayes & Nowakowski, 2002) Figure 3 4. The light dark cycle alt ers the cell cycle distribution of subgranular zone (SGZ) neural progenitor cells. (A) Diagram showing the SGZ (pink) and granule cell layer (blue) of the hippocampal dentate gyrus in which cells were analyzed. (B D) Confocal laser scanning microscope imag es (40 objective with 2 digital zoom) of (B) a Ki 67 labeled cell in dentate gyrus (green, Ki 67; white, DAPI), (C) a PHH3 labeled cell in dentate gyrus (green, PHH3; white, DAPI), and (D) BrdU labeled cells in dentate gyrus (red, BrdU; white, DAPI). Sca le bar is 5 m and applies to B D. (E) Ki 67 stereology data shows that the number of actively dividing cells in the SGZ is not affected by the light dark cycle. (F) PHH3 stereology data shows that the number of dividing cells in G2 and M phases of the cel l cycle is increased during the dark phase. (G) BrdU stereology data demonstrates that the number of dividing cells in S phase of the cell cycle is significantly increased during the dark versus light phase. D: BrdU, bromodeoxyuridine; GCL, granule cell la yer; PHH3,

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82

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83 Figure 3 5 Cumulative BrdU labeling shows that total cell cycle duration and synthesis phase lengths are decreased at night versus day in the hippocampus of female adult mice. (A C) Light photomicrographs (magnification : 10X and 63X for the insets) of dentate gyrus showing BrdU labeled (dark brown) and Cresyl Violet labeled cells (purple) (A) 2.5 h, (B) 6.5 h, and (C) 10.5 h after the first BrdU injection. D) Proportion of BrdU labeled NPC nuclei (BrdU labe ling index) after cumulative BrdU labelling for 0.5, 2.5, 4.5, 6.5, 8,5, and 10.5 h in the dentate gyrus of mice injected during the dark phase beginning 2 (black circles ). Data are the mean LIs for 3 4 animals for each time point; error bars indicate the standard error of the mean at each time point. The solid (daytime group) and dashed (nighttime group) lines are the least squares fits obtained to the conditions for a on e population model as described in the text. Solid and dashed arrows indicate the time point at which the labeling index reaches a plateau (T C T S ) for nighttime and daytime groups, respectively. Scale bar is 10 m and applies to A C GCL, granule cell la yer

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84 Cell Death Does not Influence Observed Cell Proliferation Variations between Light and Dark Phases To rule out the possibility that light dark cycle induced degeneration of hippocampal NPCs is responsible for light dark cycle measured effects on cell cycle distribution and kinetics, new pyknotic cells within the granule cell layer and along the SGZ were analyzed. No new BrdU labeled cells were found to be pyknotic among the cells dividing at night or during the day within 12 h ours after BrdU labeling in the cumulative BrdU experiment. The low level of pyknosis confirms that cell death is not likely responsible for the variation in BrdU and PHH3 positive cell numbers and for changes in cell cycle kinetics between light and dark phase s Cell Cycle Gene Expression Differs between Night and Day in the Hippocampus of Fem ale and M ale Adult Mice We used the quantitative RT2 PCR Profiler Array to monitor the response of 84 genes involved in cell cycle regulation and compared genes expre ssed in the hippocampi of adult female, male and ovariectomized female mice perfused in the middle of the night ( active phase) or day ( sleep phase). In female mice ( Fig. 3 6 ), examination of cell cycle genes revealed a significant increase in the levels o f cell cycle genes for proteins that control entry into S phase of DNA replication (cyclin C / D1 / E1, cell division cycle 25 homolog A, cyclin dependent kinase 2, CDC28 protein kinase 1b, Cohesin subunit SA 1 ; two fold increase) at nighttime versus daytime, as well as an increase in the levels of genes for proteins controlling entry into G 2 /M transition and mitosis ( cyclin B1 / F, Cyclin dependent kinases regulatory subunit 1 CDC28 protein kinase 1b, cell division cycle 25 homolog A, Cohesin subunit SA 1 Adenylate kinase 1; at least two fold increase). A significant increase in several positive regulators of the cell cycle (transcription factors E2 f 1 E2 f 2 E2 f 3 nucleoplasmin 2, Mdm2; at least two fold

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85 increase) and a decrease in negative regulators, inc luding those involved in cell cycle checkpoint and arrest, of the cell cycle ( regulatory subunit of protein phosphatase 2, serine/threonine protein kinase Chk1, breast cancer type 2 susceptibility protein BRCA2, cysteine aspartic acid protease 3; at least two fold decrease) were also detected at nighttime versu s daytime. In terestingly S phase and cell cycle positi ve regulator genes for proteins that have not yet been associated with regulation of NPC cell cycle were increased by up to more than 4 fold at n ighttime versus daytime (cyclin B1, F). In males (Fig. 3 7) examination of cell cycle genes also revealed a significant increase (at least two fold) in the levels of cell cycle genes encoding proteins that control entry into S phase of DNA replication (C yclin E1) or M phase ( never in mitosis gene a related kinase 2 Nek2) as well as a significant increase in the gene coding for transcription factor E2 f 2, a positive cell cycle regulator of cell proliferation at nighttime versus daytime. Cell cycle genes i nvolved in the negative control of cell growth and division and cell cycle checkpoint and arrest were also down regulated at night time versus day time ( Gpr132, Slfn1, Prm1, Cdkn2a, Chek1, Brca1, Trp63 ; at least two fold decrease ).

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86 Figure 3 6 Genes related to cell cycle regulation are differentially expressed between day and night in female adult mice. Graphs show quantitative comparisons of transcript levels in adult female mice perfused at night or during the day using Pr ofiler PCR Array ( SABiosciences ). RNA Changes in transcript level are expressed as gene fold changes in nighttime relative to daytime perfused mice for G 1 /S and S phase ce ll cycle genes (A), G 2 /M and M phase gene (B), positive regulators (C), and negative regulators (D) of the cell cycle.

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87 Figure 3 7 Genes related to cell cycle regulation are differentially expressed between day and night in male adult mice. Graphs show quantitative comparisons of transcript levels in adult male mice perfused at night or during the day using Profiler PCR Array (SABiosciences). RNA Changes in transcript level are expressed as gene fold changes in nighttime relative to daytime perfused mice for G 1 /S and S phase cell cycle genes (A), G 2 /M and M phase gene (B), positive regulators (C), and negative regulators (D) of the cell cycle. Neural progenitor c ells produced at night exit the multipotential, undifferentiated state faster than those produced during the day. Previous studies suggest that neural progenitor cell differentiation into neurons and their survival are robustly increas ed after physical activity and at night

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88 (Kempermann et al., 1997, Goergen et al., 2002, Holmes et al., 2004). In order to compare the rate of differentiation of neurons produced at night versus day Sox2 and Prox1 immunofluorescence was performed on hippoca mpal sections of mice injected Very few new cells were found to be BrdU + /Prox1 + whether in the night or daytime injection group. Prox1 is a transcription factor expr essed early during neuronal maturation and required for granule cell maturation and intermediate progenitor maintenance during adult brain neurogenesis (Elkouris et al., 2010, Lavado et al., 2010, Karalay et al., 2011). On the other hand, the fraction of B rdU + /Sox2 + cells was significantly higher among cells that divided during the day versus night; therefore, the fraction of committed new cells (BrdU + /Sox2 ) was significantly higher among cell that divided at night (54% of new cells at night versus 38% dur ing the day; P < 0.01; Fig. 3 8 ). Sox2 is a transcription factor that is essential to maintain self renewal of undifferentiated neural stem cells (Graham et al., 2003, Ellis et al., 2004, Suh et al., 2007, Favaro et al., 2009). While Prox1, an early marker of neuronal maturation, is not yet expressed 10.5 h after NPC division, more NPCs generated at night exited their undifferentiated state within 10.5 h of cell division (over 50%) compared to those that divided during the day.

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89 Figure 3 8 Neural progeni tor cells produced at night exit the multipotential, undifferentiated state faster than those produced during the day. (A) Confocal photomicrograph of mouse dentate gyrus immunostained with BrdU (red), Sox2 (green), Prox1 (blue), and DAPI (gray) (20X objec tive, 3X digital zoom). White arrowhead points to a new undifferentiated cell (BrdU + /Sox2 + /Prox1 ). (B) Graph showing the fraction of new committed, multipotential cells (BrdU + /Sox2 ) 8.5 h after the first BrdU injection. GCL: granule cell layer. P < 0.0 1. Scale bar: 10 m Four week old neurons generated at night have more extensive branching than those produced during the day. To assess the degree of maturation of newborn neurons in the process of differentiation (Francis et al., 1999, Plumpe et al., 2006 ), we measured various morphological characteristics of new differentiating neuronal (BrdU + /DCX + ) cells, such as soma perimeter, area, branch and node number, total branch length, as well as the proportion of branched and unbranched immature (DCX + ) neurons 1 and 4 weeks after BrdU administration (Fig. 3 9 ). There were no significant differences in morphological characteristics between 1 week old neurons generated during the day or at night. On

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90 the other hand, average branch length was significantly increased in transitioning (DCX + /NeuN + /BrdU + ) neurons generated during the night versus day ( P <0.05), while the proportions of branched neurons between the night and daytime groups were similar. Figure 3 9 Four week old neurons generated at night display more extensive branching than those generated during the day. (A) Confocal photomicrograph of mouse dentate gyrus immunostained with BrdU (red) and DCX (blue) (10X objective, 1X digital zoom). White arrows point to new immature neurons (BrdU+/DCX+). (B D) High magnification photomicrographs (20X objective, 2X digital zoom) of new (BrdU + ; red) immature (DCX + ; blue) neurons. (E) Photomicrograph showing morphological characteristics measured on new immature ne urons (including branch length, soma area and perimeter, and node number). (F) Table showing morphological measurements of 1 and 4 week old neurons produced at night or during the day. Average branch length was significantly greater in 4 week old neurons p roduced at night versus day. P = 0.05. Scale bars: (B D) 20 m (E) 10 m Differentiation gene expression differs between night and day in the hippocampus of female and male adult mice Gene expression been shown to control the timing of neural progenitor cell differentiation in the developing and adult mammalian brain (Sun et al., 2001, Aubert et al., 2002, Ramalho Santos et al., 2002, Graham et al., 2003, Bruggeman et al., 2005,

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91 Kageyama et al., 2005, Sun et al., 2011). In order to determine whether diff erences in NPC differentiation and survival between light and dark phases were due to variations in gene expression, RNA from the hippocampi of male and adult female mice was harvested 6 h after lights on (0600) or 6 h after lights off (1800), and light da rk cycle induced changes in the expression of 84 genes associated with NPC maturation and survival were identified using Real t ime PCR array technology. In female adult mice, the expression of genes associated with the positive regulation of cell motility and migration ( Flna ) as well as cell differentiation ( Ascl1, Ncoa6 ) was up regulated at night versus day (at least two fold; Fig. 3 10 ). Growth factor genes such as GDNF as well as genes involved in synaptogenesis ( Pou4f1 ), cell adhesion ( Sema4d ), cell si gnaling ( Adora1, DVl3 ), and the positive regulation of transcription ( Ncoa6, Ascl1, Mef2c, Mll1, Pou4f1 ) were also up regulated at night versus day (at least two fold). Anti apoptotic genes ( GDNF, Sema4d ) were up regulated during the dark phase (at least t wo fold) as well. This data is in accordance with the increased neurogenesis and survival reported for neurons generated at night versus day. Figure 3 10. Genes related to neuronal differentiation are differentially expressed between day and night in fem ale adult mice. This figure shows quantitative comparisons of transcript levels in adult female mice perfused at night or Profiler PCR Array (SABiosciences ). RNA extraction occurred at ZT6 ( in transcript level are expressed as gene fold changes in nighttime relative to daytime perfused mice for a variety of genes, including genes related to differentiation, synaptogenesis, apopt osis, and signaling.

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92

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93 Figure 3 11 Genes related to neuronal differentiation are differentially expressed between day and night in male adult mice. This table shows q uantitative comparisons of transcript levels in adult male mice perfused at night o r during Profiler PCR gene fold changes in nighttime relative to daytime perfused mice for a variety of genes, including genes related to differentiation, synaptogenesis, apoptosis, and signaling. In male adult mice, the expression of genes associated with the positive regulation of neuronal di fferentiation ( Cdk5r1, Cdk5rap2, Inhba ) was up regulated at night versus day (at least two fold; Fig. 3 11 ). Growth factor and cytokine genes such as

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94 GDNF, EGF, Inhba as well as genes involved in synaptogenesis and synaptic transmission ( Pou4f1, Drd2 ), cell adhesion ( Dll1 ), cell signaling ( Adora1, Adora2, Drd2, Dll1 ), and the positive regulation of transcription ( Pou4f1, Sox3 ) were also up regulated at night versus day (at least two fold). Apoptotic genes, such as Rtn4 were down regulated during the dar k phase (at least two fold). Therefore, different genes associated with neuronal maturation seem to be up regulated depending on the light dark cycle in female and male adult mouse hippocampi, suggesting a role for steroidal hormone in the control of NPC d ifferentiation. Discussion The results of experiment 1 show a significant difference in cell cycle distribution and kinetics between NPCs dividing at night or during the day. T he proportion of cells in S or G 2 M phase show a clear day/night variation, wi th a significant increase during the night while the total number of NPCs in active phases of the cell cycle did not differ between day and night. N o new cells were found to be pyknotic within 12 hours of BrdU injection s at night or during the day, sugges ting that the observed increased new cell number is not due to decreased new cell death at night but most likely due to a difference in cell cycle kinetics or cell cycle entry between light and dark phases. In order to determine whether the increased numb er of cells in S and M phases observed at night w as due to longer S or M phases or accelerated entry into these phases, we measured the length s of total cell cycle and S phase using a cumulative BrdU labeling method (Nowakowski et al. 1989a) We found that T C and T S are shorter at night versus daytime, which suggests that an accelerated progression into S or G 2 M phases is most likely responsible for the increased proportion of cells in S and G 2 M ph ases observed

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95 at night, and not increased phase lengths. Therefore, we hypothesize that G 1 /S and G 2 /M transition of progenitors is promoted during the night, which result s in an increase of S and M phase cell numbers at night. T o determine whether this acc elerated cell cycle progression was due to genes controlled by the light dark cycle, 84 genes involved in cell cycle regulation were monitored using the quan titative RT 2 PCR Profiler Array. Universal regulatory mechanisms have been shown to control cell division, and protein kinase and phosphorylation are central to the orderly timing of cell division events. The cell cycle consists of 4 main stages: S phase (DNA synthesis doubles the DNA amo unt in the cell; RNA and proteins are also synthesized), G 2 phase (no DNA synthesis; RNA and protein synthesis continue) M phase (mitosis or nuclear division and cytokinesis or cell division yield two daughter cells) and G 1 phase (RNA and proteins are sy nthesized; no DNA synthesis). After mitosis and into G 1 a cell can continue through another division or cease to divide, entering a quiescent phase (G 0 ) that may last hours to the lifetime of the cell. Differentiated cells that have acquired their special ized function and form remain in the G 0 phase. Progression through the cell cycle is regulated by the cyclin dependent protein kinases ( C dk s ). These act at specific points in the cell cycle, phosphorylating key proteins and modulating their activities. The catalytic subunit of C dk s is only active when associated with the regulatory cyclin subunit. For instance, cyclin E C dk 2 activity peaks near the G 1 S phase boundary, as the active enzyme triggers synthesis of enzymes required for DNA synthesis in S phase. Cyclin A C dk 2 activity rises during the S and G 2 phases then drops sharply in the M phase, as cyclin B C dk 1 peaks. C dk s and cyclin synthesis is regulated by

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96 extracellular signals such as growth factors and cytokines (inducers of cell division). These sign als induce the synthesis of specific nuclear transcription factors (like Jun and Fos) essential to the production of DNA synthesis enzymes, as well as a variety of gene product s including cyclins, C dk s and E2 f E2 f in turn, controls production o f several enzymes essential for the synthesis of deoxynucleotides and DNA and entry of the cell into S phase. In the present study, genes involved in G 2 /M and G 1 /S phase transitions were up regulated at night versus daytime, particularly in female adult mice, whic h might explain the increased number of cells in G 2 M and S phase observed at night. In female adult mice, we detected a significant (at least two fold) increase in the levels of cell cycle genes that control entry into S phase of DNA replication as well as an increase in the levels of several genes contro l l ing entry into G 2 /M transition and mitosis at nighttime compared to daytime A significant increase in several positive regulators of the cell cycle and a decrease in negative regulators, including those involved in cell cycle checkpoint and arrest of the cell cycle, were also detected at nighttime versus daytime. In particular, S phase and cell cycle positi ve regulator genes that have not yet been ass ociated with regulation of the neural progenitor cell cycle were increased by up to more than 4 fold at night time versus day time ( cyclin B1, F ). These data confirm the results from the cell cycle distribution and kinetics studies, that is an increased rate of G 1 /S and G 2 /M transitions and increased cell proliferation during nighttime versus daytime. We also found an increase in genes controlling G 1 /S and G 2 /M phase transitions as well as the positive regulation of the cell cycle in male mice, but to a lesse r extent than in female mice. Therefore, it appears that genes controlling the G 1 /S

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97 and G 2 /M phase transitions of the cell cycle were up regulated at night, which increased the number of cells in S and M phase and resulted in accelerated cell proliferation during the dark (wake) versus light (sleep) phase. Interestingly, such a circadian regulation of cell division is observed in certain types of tissues including tongue epithelium, intestinal epithelium, and skin (Tutton, 1973; Schevi ng et al. 1978; Scheving et al. 1987; Scheving et al. 1989; Brown, 1991; Buchi et al. 1991; Smaaland, 1996; Bjarnason et al. 1999; Garcia et al. 2001; Smaaland et al. 2002; Matsuo et al. 2003; Wille et al. 2004) implying that t he temporal regulation of progenitor divisions seems to be important for animals living under light dark cycles. For instance, in the gastrointestinal tract, a difference of up to 12 hours is found between day and night in total cell cycle length (Tutton, 1973) A variety of cellular events are controlled in a circadian manner because of endogenous circadian clocks that reside in various tissues. C ircadian clock related genes such as Cryptochromes and Periods have been reported to have inhibitory roles for mitotic divisions in the liver and other organs As clock related genes are significantly expressed in the hippocam pus, it is expected that the daily regulation of progenitor divisions and neurogenesis may be dictated by circadian clocks localize d in the hippocampus. Based on our results we propose a model for the diurnal regulation of NPC s in the hippocampus in whic h they enter the cell cycle irrespective of the time of day. The progression of these cells into S and M phase s is suppressed during the day and stimulated at night (the wake phase), thereby giving rise to more neuronal progeny. Indeed, several of the genes showing increased levels at night versus day are involved in pathways that directly affect the progression through the G 2 /M and G 1 /S phases of the cell cycle ( Fig. 3 12 ).

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98 Figure 3 12. Diagram of cell cycle genes up regulated (green) or down regulated (red) at nighttime versus daytime in the hippocampus of adult female mice. Cyclin/cyclin dependent kinase complexes are directly involved in the progression through the various phases of the cell cycle and are up regulated during the dark versus light phase.

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99 In experiment 2 we found that neural progenitor cells generated at night had a higher rate of maturation than those produced during the day. NPCs produc ed at night had exited the multipotential, undifferentiated state (Sox2+) earlier compared to cells produced during the day. Neurons generated at night also had more extensive branching than those produced during the day 4 weeks after division. Considering the observed accelerated entry into the differentiation stage as well as maturation of NPCs generated at night versus day, we quantified the expression of gene related to maturation processes in the hippocampi of mice harvested during the night or day. We found that the expression of genes related to differentiation of NPCs significantly differed between night and day, and differentially between the hippocampi of female and male adult mice. An in vitro study of NPCs in culture revealed that estradiol expos ure does not influence the differentiation or the survival of NPCs 1 week after plating. In mammals, Sox2 is a transcription factor essential in maintaining the pluripotency of embryonic stem cells (Masui et al ., 2007, Adachi et al ., 2010) but also the sel f renewal of undifferentiated neural stem cells derived from the embryo or the adult (Episkopou, Graham et al., 2003, Ellis et al., 2004, Suh et al ., 2007, Favaro et al., 2009), partly through the regulation of Shh (Favaro et al., 2009). Prox1 (Tomarev et al ., 1998) is a transcription factor expressed early during neuronal maturation and required for granule cell maturation and intermediate progenitor maintenance during adult brain neurogenesis (Elkouris et al ., 2010, Lavado et al., 2010, Karalay et al ., 2011). Prox1 plays an essential role in cell fate decisions during mouse embryonic development in a number of tissues, including the central nervous system (Wigle and Oliver, 1999, Sosa Pineda et al ., 2000, Yamamoto et al ., 2001, Bermingham McDonogh et al ., 2006,

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100 Mishra et al., 2008). Overexpression of Prox1 is capable of driving neuronal precursors III tubulin; however, terminal differentiation is not fully achieved as it requires the function of Ngns or Mash1 (Mishra et al., 2008). Prox1 is highly expressed during the G2 phase where it might act as a determinant for NPCs to enter asymmetric cell divisions occurring during the M phase of the cell cycle (Dyer, 2003, Dyer et al. 2003). To assess the rate of entry into maturation of cells produced at night versus day, Sox2 and Prox1 immunofluorescence was performed on sections of mice injected with BrdU every 2 h, few new cells were BrdU+/Prox1+, whether from the night or daytime injection group, suggesting that no new cells had yet entered the neuronal differentiation state 8.5 h after division. More than 50% of the cells th at were produced at night were BrdU+/Sox2 by that time point, versus 34% for the cells generated during the day. This data suggests that NPCs generated at night enter the differentiation state faster than those produced during the day and might be under the regulation of circadian signals accelerating this process. M orphological analysis revealed that neurons generated at night also had more extensive branching than those produced during the day 4 weeks after division, suggesting accelerated maturation of neurons produced at night (Francis et al., 1999, Kempermann et al., 2004, Plumpe et al., 2006). This data correlates with previous work showing that neural prog enitor cell differentiation into neurons and their survival are robustly increased after physical activity and at night (Kempermann et al., 1997,

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101 Goergen et al., 2002, Holmes et al., 2004). These findings are not surprising, as the circadian clock has been shown to regulate stem cell differentiation, which is essential for homeostasis, in several other organs. For instance, it was recently discovered that, in the hair follicle bulge where epidermal stem cells are located, two subpopulations of stem cells ex ist and differentially express the clock machinery core components, which results in the differential expression of genes known to control stem cell differentiation, such as TGF Arnal and Sassone Corsi, 2011). Th us, one epidermal stem cell subpopulation might be more prone to differentiation at a particular time of day (Janich et al., 2011). Bone formation from osteoblast cells is inhibited by clock genes, and mice deficient in clock genes show increased bone mass (Fu et al., 2005), while embryonic fibroblasts lacking certain clock genes differentiate into adipocytes (Shimba et al., 2005, Grimaldi et al., 2010). In the hippocampus, clock genes, which produce transcription factors that have a central role in the cir cadian pacemaker, have also been shown to regulate neurogenic transcription factors and the neuronal differentiation of adult neural NPCs in vitro (Kimiwada et al., 2009). In vivo, clock controlled Period2 gene expression has been linked to the intrinsic c ontrol of NPC neurogenesis in the dentate gyrus of adult mice (Borgs et al., 2009). Therefore, the circadian rhythm plays a key role in all biological processes, including the expression of genes, and, in turn, the levels of factors controlling NPC differe ntiation. Our gene expression studies in male and female mouse hippocampi confirmed the previous findings, as an up regulation of several genes controlling cell migration, fate determination, synaptogenesis, and signaling, all playing a part in the differ entiation process, was observed during the dark versus light phase, differentially between the

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102 hippocampi of female and male adult mice. This suggests an acceleration of the maturation mechanisms of NPCs generated at night versus daytime, as well as a poss ible hormonal regulation for the differential gene expression between female and male mice. In summary, we found that the G 1 /S or G 2 /M phase transition s of hippocampal progenitors are promoted during the night, which results in an increase of S and M pha se cell numbers at night versus daytime This correlates with a significant increase in the expression leve l of cell cycle genes that control entry into the S phase of cell division as well as those controlling G 2 /M transition and mitosis detected in fema le adult mice hippocampi a t night compared to daytime T his increase was much higher in female compared to male adult mice, suggesting that these genes might be under the control of steroid hormones Therefore, increased cell division at night seems to be accelerated through the up regulation of specific cell cycle genes that are under the control of the circadian clock and possibly steroid hormones We also found that exit out of the multipotential state, entry into the differentiation stage, as well as the maturation process of new granule neurons might be accelerated among NPCs dividing at night versus day. These phenomena correlates with the increased expression of several genes related to NPC dif ferentiation detected at night versus day time these genes being differentially expressed between female and male mice. Further studies might examine the possible presence of Bmal1/Clock binding sites in the promoters of differentiation genes found to be u p regulated at night, as well as the role of hormones such as estradiol t hat could explain the differential expression of these genes between

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103 male and female mice as it has been shown to regulate NPC proliferation and survival in vivo D ay/night induced changes in neurogenesis allowed us to explore mechanisms that regulate progenitor cell behavior without submitting the brain to injury or other external manipulations It is important that we decipher the molecular mechanisms relating circadian rhythms an d NPC differentiation, as these could lead to new targets for manipulating the differentiation and function of adult NPCs in regenerative treatments for neurodegenerative diseases or injuries. Manipulation of NPC cell cycle and timely administration of neu rotrophic therapies might be used to optimize regenerative strategies for the treatment of neurodegenerative diseases.

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104 CHAPTER 4 ESTRADIOL INCREASES THE PROLIFERATION BUT NOT DIFFERENTIATION OF ADULT FEMALE NEURAL PROGENITOR CELLS IN CULTURE Introduc tion The entire adult brain holds regenerative potential in the form of neural stem/progenitor cells (Arsenijevic et al. 2001; Palmer et al. 1995; Palmer et al. 2001; Weiss et al. 1996) Progenitor cells resident to the subventricular zone or the hippocampal subgranular zone generate significant numbers of neurons each day (Cameron et al. 1993; Alvarez Buylla and Garcia Verdug o, 2002) while progenitor cells located in other CNS regions primarily generate glia (Geha et al. 2009; Gould, 2007; Gould and Gross, 2002) unless injury is induced, which appears to stimulate the production of transient neuron populations. Knowledge of the internal and external stimuli that regulate the dynamic processes of progenitor proliferation and differentiation in the adult brain is c rucial in developing strategies for treating neurodegenerative diseases and injuries. We confirmed p revious work that suggests that neural progenitor cell differentiation into neurons and their survival are robustly increased after physical activity and at night (Kempermann et al. 1997; Goergen et al. 2002; Holmes et al. 2004) The circadian rhythm plays a key role in all biological processes, including the expr ession of genes, which suggests it might regulate the levels of factors controlling neural progenitor cell differentiation Most factors that have been found to regulate neural progenitor cell (NPC) differentiation and survival in the adult nervous system are themselves controlled by diurnal rhythms. For example, serotonin, a known regulator of NPC survival in vertebrate species (Brezun & Daszuta, 1999; Gould, 1999) shows levels that fluctuate on a circadian cycle in many organisms (Quay, 1963; Hery et al.

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105 1972; Akerstedt & Levi, 1978; Banasr et al. 2004) Estradiol, a hormone that has also been shown to modulate NPC proliferation and survival (Perez Martin et al. 2003; Ormerod et al. 2004a) also shows considerable levels of diurnal fluctuation (Roy & Wilson, 1981a; Cai et al. 2008a) The expression of several growth factors known to stimulate neuronal cell maturation and/or survival increases with activity and at night, such as vascular endothelial growth factor, brain derived neurotrophic factor, epidermal growth factor receptor, and nerve growth factor (Bova et al., 1998; Scheving et al., 1989; Tournier et al., 2007;Van der Zee et al., 2005;Lindley et al., 2008) Differentiation and survival of neural progenitor cells are controlled by various intrinsic and environmental factors. However, the influence of the factors influenced by the light dark cycle has not yet been investigated in adult NPCs E stradiol 2 ) is known to regulate neurogenesis in rodent brains in vivo (McEwen, 1996; Tanapat et al. 1999; Ormerod & Galea, 2001) and estradiol and its receptors display a diurnal rhythm in the adult mammalian brain (Roy & Wilson, 1981a; Ba o et al. 2003; de la Iglesia & Schwartz, 2006; Cai et al. 2008a) In order to assess whether diurnal variations in NPC proliferation and cell cycle ge ne expression in vivo might be du e in part to changes in estradiol levels, adult female mouse NPCs were exposed to low or high concentrations of estradiol (10 8 M or 10 5 M, respectively ) for 4 h or 24 h along with BrdU (5 M) 4 h prior to fixation. Cells were then analyzed immunohistochemically for cell cycle and estrogen receptor markers 4 h and 24 h after estradiol administration. Last, t o assess whether variations in neuronal differentiation survival, and gene expression betwe en light and dark phases are related to diurnal changes in estradiol

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106 levels, adult neural progenitor cells were exposed to low (10 8 ) and high (10 5 ) concentrations of estradiol for 1 week beginning at or 12 h after plating Estradiol (E 2 ) has been s hown to promote neurogenesis in rodent brains in vivo (McEwen, 1996; Tanapat et al. 1999; Ormerod & Galea, 2001) and estradiol and its receptors display themselves a diurnal rhythm in t he adult brain (Roy & Wilson, 1981a; Bao et al. 2003; de la Iglesia & Schwartz, 2006; Cai et al. 2008a) Once the variations and the mechanisms in the transcriptional clock and gene expression are fully understood, neural stem cell research can be directed toward alter ing the gene clock in ways that could implement favorable regulation of normal neural progenitor activity. Experimental Procedures Experiment Design T o test the effects of estradiol on cell cycle kinetics proliferation, and differentiation in adult NPCs in vitro NPCs were isolated from adult female mouse hippocampi. Mice were deeply anesthetized using a single intraperitoneal ketamine (100mg/kg)/ xylazine (15 mg/kg). Animals were decapitated, the brains were removed, freed of meninges, and the hippocampi were diss ected from the brains as described previously (Palmer et al., 1999). The brains were bisected longitudinally, and each hippocampal lobe was separated from the overlaying cortical white matter using the natural separation line along the alveus hippocampus. Hippocampi were dropped into a dry sterile 6cm tissue culture dish and finely mince using two sterile razor blades. As described previously (Gage et al., 1995, Palmer et al., 1995, Palmer et al., 1999), tissues were finely minced and digested in a solutio n of papain (2.5 U/ml; Worthington, Freehold, NJ), DNase (250 U/ml, Worthington) and neutral protease (1 U/ml Dispase; Boehringer Mannheim, Indianapolis, IN) dissolved in HBSS. Cells and tissue fragments

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107 were washed three times with DMEM containing 10% fet al bovine serum (FBS) (Hyclone, Logan, UT). Whole digested tissue was then suspended in DMEM 10% FBS, filtered through a sterile 107 mm nylon mesh and thoroughly mixed with an equal volume of Percoll solution. The Percoll solution was made by mixing nine p arts of Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) with one part 103 PBS (Irvine Scientific, Santa Ana, CA). The cell suspension was then fractionated by centrifugation for 30 min, 18C, at 20,000 x g. Cell fractions were harvested and washed fr ee of Percoll by three or more rinses in DMEM 10% FBS (Palmer et al ., 1999). Cells were re suspended in growth medium, Neurobasal A (Invitrogen), with 2 mM L 27 without vitamin A (I nvitrogen), 20 ng/ml fibroblast growth factor 2 (PeproTech), and 20 ng/ml epidermal growth factor (PeproTech), and plated onto T75 flasks coated with fibronectin (EMD Biosciences, SanDiego, CA). NPCs were grown for >10 passages on T75 flasks coated with fi bronectin (EMD Biosciences, SanDiego, CA) composed of 2 0 ng/ml fibroblast growth factor (FGF; Peprotech, Inc, Rocky Hill, NJ), 20 ng/ml epider mal growth factor (EGF; Sigma) in Neurobasal A medium ( Invitrogen ) and 2% B27 without vitami n A (Invitrogen); half of the growth medium was replenished every 2 d and cells were passaged at 2 10 6 cells per flask every 7 days In experiment 1, t o test whether estradiol concentration and exposure time have an effect on the proliferation, cell cy cle kinetics, and estrogen receptor expression of adult mouse NPCs in culture cells were passaged using non enzymatic cell dissociation buffer (Fisher Scientific) into 8 well fibronectin coated LabTek chambers at 25,000 cells per well ( ~ 4000/mm 2 ) for imm unofluorescence analysis Cells were treated

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108 with 0 (control), 10 5 M (high dose) or 10 8 M (low dose) estradiol (E2) for 4 h or 24 h 4 h before fixation for proliferation assays. In experiment 2, to test whether estradiol concentration and exposure time a ffect NPC differentiation of adult female mouse NPCs in culture cells were passaged into LabTek wells at 25,000 cells per well ( ~ 4000/mm 2 ) using non enzymatic cell dissociation buffer (Fisher Scientific) The passaged cells were re trans retinoic acid (Acros/Thermo Fisher), 10 ng/ml brain derived neurotrophic factor (BDNF; Peprotech, Inc), 10 ng/ml neurotrophin 3 (NT 3; Peprotech, Inc.), and 1% (v/v) fetal bovine serum in Neurobasal A medium ( Invitrogen ) and 2% B27 without vitamin A (Invitrogen), to drive progeny more toward a neuronal versus glial fate (Takahashi et al. 1999) Cells were treated with 0 (control), 10 5 M (high dose), or 10 8 M (low dose) estradiol (E2) starting at the beginning (T0) or 12 h after the beginning of the experiment (T12). BrdU s added to LabTek wells for 12 h at the start of the experiment. After 1 week in culture, cells were fixed for immunohistochemical processing ( Fig. 4 1)

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109 Figure 4 1 In vitro experiment timeline. Adult female NPCs were harvested and cultured in growth media for >10 passages before being plated onto fibronectin coated 12 h at the start of the experiment. Cells were treated with 0 (control) 10 5 M (high dose), or 10 8 M (low dose estradiol (E2) starting at the beginning (T0) or 12 h after the beginning of the experiment (T12). After 1 week in culture, cells were fixed for immunohistochemical processing. In Vitro Immunofluorescence LabT ek plated cells were fixed with 4% paraformaldehyde for 10 min and rinsed repeatedly between immunostaining steps with TBS (pH 7.4). In experiment 2, c ells were blocked in 3% normal donkey serum (NDS; Jackson Immunoresearch, West Grove, PA, USA ) and incuba ted overnight at 4C in a cocktail of primary antibodies that included rabbit polyclonal anti PHH3 (1:500; 06 570 ; Millipore; to visualize cells in G 2 M phase ) and mouse monoclonal anti Ki67 (1:500; 06 570 ; Novocastra ; to visualize cell all active phases of the cell cycle, G 1 S, G 2 and mitosis ) or rabbit anti 1:500; Santa Cruz Biotechnology ) and mouse anti estrogen receptor

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110 1:500; Santa Cruz Biotechnology ) The fo llowing day, cell s were incubated in the appropriate FITC or Cy5 conjugated secondary antibody (anti rabbit Ig G or anti mouse Ig G, respectively ; 1:500; Jackson ImmunoResearch) for 4 h. Cells were then incubated in 2 N HCl for 2 0 min at 37C to denature DNA, followed by rat anti BrdU (1:50 0; AbD Serotec, Raleigh, NC) primary antibody overnight at 4C The following day, cell s were i ncubated in the appropriate Cy3 conjugated secondary antibody (anti rat Ig G; 1:500; Jackson ImmunoResearch, West Grove, PA ) for 4 h at room temperature Cell s were then incubated in 4',6 diamidino 2 phenylindole (D API; Calbiochem, San Diego, CA; 1:10,000) for 10 minutes to reveal cell nuclei, then the LabTek mounted on glass slides and cover slipped under the anti fading agent diazobicyclooctane (DABCO; 2.5% D ABCO, 10% polyvinyl alcohol and 20% glycerol in TBS; Sigma Aldrich). Fluorescently labeled cells were analyzed using a Ze iss meta LSM 710 fully spectral laser scanning confocal microscope (with 405, 488, 510, 543 and 633 laser li nes) using a 63x oil object ive (and 2 x digital zoom). In experiment 2, c ells were blocked in 3% normal donkey serum (NDS; Jackson Immunoresearch, West Grove, PA, USA) and incubated in a cocktail of primary antibodies that included rabbit anti Tubulin (1:500; Covance; to visualize immature neurons) and mouse anti neuronal nuclei (NeuN; 1:500; Chemicon International; to visualize mature neurons) or guinea pig anti glial fibrillary acidic protein (GFAP; 1:750; Advanced Immunochemical; to visualize astrocytes) and rabbit anti NG2 (1:1 000; Chemicon International; to visualize oligodendrocyte precursors). The following day, cells were incubated in the appropriate FITC or Cy5 conjugated secondary antibody (anti rabbit Ig and anti mouse Ig, or guinea pig Ig and rabbit Ig respectively ; 1 :500;

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111 Jackson ImmunoResearch) for 4 h at room temperature Cells were then incubated in 2 N HCl for 2 0 min at 37C to denature DNA, followed by rat anti BrdU primary antibody (1:500; AbD Serotec, Raleigh, NC) overnight at 4C The following day, cell s were i ncubated in the appropriate Cy3 conjugated secondary antibody (anti rat Ig G; 1:500; Jackson ImmunoResearch, West Grove, PA ) for 4 h at room temperature Finally, cell s were incubated in 4',6 diamidino 2 phenylindole (D API; Calbiochem, San Diego, CA; 1:10 ,000) for 10 minutes to reveal cell nuclei, then the LabTeks were mounted on glass slides and cover slipped under the anti fading agent diazobicyclooctane (DABCO; 2.5% DABCO, 10% polyvinyl alcohol and 20% glycerol in TBS; Sigma Aldrich). Fluorescently labe led cells were analyzed using a Zeiss laser scanning confocal microscope. Confocal Microscopy Confocal i mages (512 512 pixels) of Lab T ek plated cultures were taken using a Zeiss LSM 710 fully spectral Laser Scanning Confocal Microscope (with 405, 440, 48 8, 532, 635 laser lines) with a 63 objective ( 2 digit al zoom) through the z plane ( 3 stacks) of DAPI positive cells Visible laser line intensities were maintained below 1 0 %. Cell densities and specific marker s were determined from images taken in 8 10 non overlapping fields of view taken from the center of 4 wells (> 500 cells per well) per condition. For experiment 1, t he density of DAPI+ and BrdU/DAPI + cells ( total and S phase cells per mm 2 respectively) was measured, as well as the proportion of DAPI+ cells that were label ed with BrdU alone (S phase), PHH3 alone (G 2 /M phase), double labe led with BrdU and PHH3 (S phase cells that had moved into G 2 /M phase within 4 h) BrdU but not Ki67 (new cells that moved to G 0 within 4 h) The fractions of B rdU labeled (new) cells co and/or

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112 in BrdU positive cells was performed using confocal microscopy and the For experimen t 2, t he densities of DAPI+ and BrdU/DAPI+ cells (total and new cells per mm 2 respectively) w ere measured, as well as the proportion of BrdU+ cells tubulin and/or NeuN) or glial (GFAP or NG2) markers. Cell Cycle Kinetics S phase an d total cell cycle length were calculated according to previously reported equations based on total cell number in each phase 4 h after BrdU injection (Seifert et al. 2010a ) BrdU labels cells approximately 30 min after injection (Cameron et al ., 2001) and is metabolized in approximately 2 h (Nowakowski et al 1989; Cameron et al ., 2001) Given that S phase in adult mouse and rat dentate gyr i in vivo is at least 8 h (Nowakowski et al 1989; Cameron et al ., 2001) a 4 h interval between injection and collection was chosen to allow BrdU label ed NPCs to transition from S phase to G2/M phase. Although this may result in slight under representation of cells that transiti oned from G1 to S phase after BrdU metabolism, and cells in late S phase would be BrdU positive in early G2, we consider the percentages reported for each phas e as accurate using this label ing scheme on the basis of the relative nature of the proportion c alculations Statistical Analysis Group differences were tested using the Statistica software (StatSoft, Inc., 1997). All group differences in our dependent variables were revealed using Student's t tests (one dependent variable between groups) or analysis of variances (more than one dependent variable between groups) and explored using Newman Kewls post hoc te Levels were set at 0.05.

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113 Results High Dose E stradiol Exposure Increases DNA Replication and Entry into G 0 Phase of N PC s in Cultur e The effect s of low and high doses of estradiol as well as exposure time on the distribution of hippocampal NPCs across the cell cycle w ere examined in vitro Cells were treated with 0 (control), 10 5 M (high dose), or 10 8 M (low dose) estradiol (E2) for 4 h or 24 h 4 h before fixation to mark dividing cells in synthesis (S) phase of the cell cycle. ANOVA s revealed a significant effect of dose and exposure time on the number of new BrdU + cells (F 5,13 = 17.42; P < 0.0001; Fig 4 2 A ). More new cells were found in cultures treated with a high dose (10 5 M) of E2 for 24 h compared with all other conditions ( P values < 0.001 ) There were no significant differences among other groups. An ANOVA also revealed a significant effect of dose and exposure time on the number of new Ki 67 ve cells, i.e., in G 0 (non dividing) phase (F 5,13 = 39.30; P < 0.0001; see Fig. 4 2 B ). More cells in the quiescent G 0 phase were found in cultures treated with a high dose (10 5 M) of E2 for 24 h compared with all other conditions ( P values < 0.001 ) No significant differences in the fraction of G 0 phase cells were detected among other groups. Finally, an ANOVA revealed a significant effect of dose and exposure time on the number of new PHH3 + (G 2 M phase) cells (F 5,13 = 5.54; P < 0.01; see Figure 4 2 C ). Significantly fewer cells in G 2 M phase were found in cultures treated with a high dose (10 5 M) of E2 after 4 h or 24 h compared with all other conditions ( P values < 0.05 ) The density of DAPI cells was calculated for each condition and no significant differences were found between estradiol dose and exposure time conditions ( P > 0.05).

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114 Fig ure 4 2 Estradiol dose concentration and exposure time alter the cell cycle distribution of neural progenitor cells in vitro. (A) Confocal data demonstrates that the fraction of dividing BrdU + cells in S phase is significantly increased in cultures expose d to a high (10 5 M) dose of estradiol for 24 h. (B) Ki 67 confocal data shows that the fraction of new cells in the quiescent G0 phase (Ki 67 ve) is significantly increased in cultures exposed to a high (10 5 M) dose of estradiol for 24 h. (F) PHH3 confoc al data shows that the fraction of dividing cells in G2 and M phases of the cell cycle is significantly decreased in cultures exposed to a high (10 5 M) dose of estradiol for 4 h or 24 h. ( D) Representative confocal laser scanning microscope images (40 ob jective with 2 digital zoom) of neural progenitor cells in vitro that express DAPI (cell nucleus; in white), Ki 67 (actively dividing cells; in blue), PHH3 (cells in G 2 M phase; in green), and BrdU (cells in S phase; in red). Scale bar is 20 m. BrdU, bromodeoxyuridine; PHH3, phosphohistone H3. P P 0.001

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115 High Dose E stradiol Exposure D ecreased S ynthesis Phase and T otal C ell C ycle Lengths The effect s of low and high doses of estradiol as well as exposure time on hippocampal synth esis (S) phase and total cell cycle lengths were examined in vitro Cells were treated with 0 (control), 10 5 M (high dose), or 10 8 M (low dose) estradiol (E2) for 4 h or 24 h 4 h before fixation to mark dividing cells in synthesis (S) phase of the cell cycle. S phase (T S ) and total cell cycle (T C ) lengths were calculated for each condition as previously described (Seifert, et al., 2010) (Seifert et al. 2010b) ANOVA s revealed a significant overall effect of estradiol dose and exposure time on T S and T C ( F 5,13 = 5.537645, P < 0.05 and F 5, 13 = 6.038330, P < 0.05, respectively) T S was found to be significantly shorter among NPCs which were exposed to 10 5 M estradiol for 4 h or 24 h compared to all other conditions ( P values < 0.05 ; Fig 4 3 A ) There were no significant differences in T S between other groups. T C was also significantly decreased for NPCs exposed to 10 5 M estradiol for 24 h compared with all other conditions ( P values < 0.05 ; Fig 4 3 B ) T C was significantly increased compared to other conditions when NPCs were exposed to a low (10 8 M) estradiol. T C was significantly decreased after 24 h versus 4 h in culture when exposed to a high (10 5 M) estradiol compared to other conditions ( P values < 0.005 )

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116 Figure 4 3 Estradiol dose concentration and exposure time alter cell cycle kinetics of neural progenitor cells (NPCs) in vitro (A) Confocal data demonstrates that cell cycle synthesis pha se duration (T S ) is significantly decreased for NPCs exposed to a high (10 5 M) dose of estradiol for 4 h and 24 h in culture (B) Total cell cycle length (T C ) is significantly decreased for NPCs exposed to a high (10 5 M) dose of estradiol for 24 h in culture and significantly increased for NPCs exposed to a low (10 8 M) dose for 24 h P E stradiol Dose and Exposure Duration Affect the Expression of Estrogen Receptors and Cell Division estradiol a s well as exposure time on the expression of e strogen r (ER ) (ER ) were examined in vitro Cells were treated with 0 (control), 10 5 M (high dose), or 10 8 estradiol (E2) for 4 h or 24 4 h before fixation to mark dividing cells in synthesis (S) phase of the cell cycle. The fraction of + cells in synthesis phase (BrdU + ) was similar for all conditions. An ANOVA revealed a significant effect of es tradiol dose on the proportion of ER + in S phase ( F 5, 12 = 2.430110 ; P < 0.05; Fig. 4 4 B) The fraction of ER + cells in synthesis phase (BrdU + ) was significantly increased when exposed to a high (10 5 M) dose of estradiol for 24 h in culture compared with all other conditions ( P < 0.05 ) fraction s s over total cells in culture (F5,12 = 6.056174; P < 0.05 and F5, 12 = 8.464373; P < 0.05, respectivel y)

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11 7 decreased when cells were exposed to estradiol (high or low concentration) for 24 h compared with no estradiol exposure and when exposed to high or low concentrations of estradiol during 24 h versus 4 h (P < 0.05 ; Fig. 4 4 C was decreased when cells were exposed to high estradiol concentration for 24 h versus no estradiol and when exposed to a high (10 5 M) dose of estradiol during 24 h versus 4 h in culture (P < 0.05 ; Fig. 4 4 D ) Figure 4 4 Estradiol dose concentration and exposure time alter the expression of estrogen receptor in neural progenitor cells (NPCs) in vitro (A) Confocal data demonstrates th at the fraction of + cells in synthesis phase (BrdU + ) is similar in all conditions (B) The fraction of + cells in synthesis phase (BrdU + ) is significantly increased when exposed to a high (10 5 M) dose of estradiol for 24 h in culture. ( C) The fract ion of + cells over total cells is decreased when c ells are exposed to estradiol (high or low concentration) for 24 h. ( D) The fraction of + cells is decreased when cells are exposed to a low (10 8 M) dose of estradiol for 4 h and a high (10 5 M) dose of estradiol for 24 h P P

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118 Estradiol E xposure does not I nfluence the D ifferentiation or the S urvival of NPCs in C ulture 1 W eek after P lating Cultures were fixed and stained 1 week after plating and the proportions of NPC tubulin+), differentiated neuron (NeuN+), astrocyte (GFAP+), and oligodendrocyte (NG2+) phenotypes were measured ( Fig. 4 5 ). Oligodendrocytes were not detected in any condition. An ANOVA on nge significantly across conditions. While the percentage of NPC derived immature neurons was high (68.9 90.6%; n.s.), the proportion of astrocytes was consistently and relatively low (8.3 19.4%; n.s.) in all conditions. The densities of DAPI+ and BrdU+ cells were also measured 1 week after plating. An ANOVA on densities revealed that the density (per mm2) of DAPI+ or BrdU+

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119 Figure 4 5 Adult hippocampal NPCs, whether exposed o r not to estradiol in vitro, produce similar proportions of neurons with an immature phenotype and few astrocytes 1 week after plating. The proportions of adult hippocampal NPC tubulin) and mature (NeuN) neuronal marker s or astrocyte (GFAP) and oligodendrocyte precursor (NG2) glial markers 1 week after plating, as well as exposure to no, low, or high estradiol concentration starting at T0 or T12, were quantified on confocal laser scanning microscope digital images (20 objective with 2 digital zoom; n > 500 cells in 3 wells per staining condition). No NG2 + cells were detected. (A) Adult NPCs generate primarily neurons with an immature phenotype and few astrocytes. No significant differences in the proportions of immatu re neurons or astrocytes were detected between conditions. (B C) show confocal images of NPCs that co express BrdU (in red) and the immature tubulin (in blue) 1 week after plating (B) or BrdU (in red) and the astroglial marker GFAP (in gr een) 1 week after plating (C). Scale bar: (B C) 100 Discussion We previously found a n increase in neural progenitor cell proliferation at night versus day, a diurnal variation in cell cycle kinetics in adult female mice, as well as a significant increa se in the day/night variation of cell cycle gene expression levels in

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120 female compared to male adult mice, suggesting that these genes might be under the control of steroid hormones which are themselves regulated by the light dark cycle. S teroid hormones ( estrogen, progesterone, and cortisol for instance) have been shown to regulate gene transcription through binding to specific receptors in the nucleus, which are then capable of interacting with specific regulatory sequences in DNA, the hormone responsive elements, thus altering gene expression. This regulation of gene transcription by steroid hormones might explain the differen tial cell cycle gene expression levels between female and male mice. Moreover, estrogen, the primary female sex hormone, was shown to modulate the expression of clock genes, such as Per1 and Per2 in reproductive as well as non reproductive tissues (Nakamura et al. 2005; Nakamura et al. 2008; Nakamura et al. 2010) and clock genes themselves regulate the expression of estrogen and estrogen receptors (ER) in various tissues (Gery et al. 2007; Cai et al. 2008a; Damdimopoulou et al. 2011; Shimizu et al. 2011) suggesti ng that estrogen might play a role in the circadian control of cell cycle gene expression in those tissues as well as the hippocampus. We therefore investigated the potential role of estradiol (E2 estradiol), one of the three major naturally occurri ng estrogens in females, on NPC proliferation and cell cycle kinetics in vitro as concentrations of estradiol to adult females NPCs in culture for 4 h or 24 h durations, followed by BrdU to mark dividing cells. M ore cells in S phase of the cell cycle were found in cultures treated with a high dose (10 5 M) of E2 for 24 h com pared with all other conditions, while no significant differences were detected among other groups suggesting that a high dose of estradiol

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121 and minimum exposure duration ( at least 4 h ) are necessary for increased cell division possibly through decreased overall cell cycle phase duration or accelerated G 1 to S phase entry. The latter hypothesis is in a greement with our previous in vivo gene expression data showing a significant increase in the levels of genes stimulating the G 1 /S phase transition as well as S phase in female compared to male adult mice at night versus during the day. More cells in the q uiescent G 0 phase were also found in cultures treated with a high dose (10 5 M) of E2 for 24 h compared with all other conditions while n o significant differences in the fraction of G 0 phase cells were detected among other groups. This finding suggests th at a high dose of E2 exposure for a minimum duration ( at least 4 h) might decrease total cell cycle duration and/or induce dividing cells to exit the cell cycle, reach a quiescent non dividing state, and begin terminal cell differentiation. Finally, s ignif icantly fewer cells in G 2 M phase were found in cultures treated with a high dose (10 5 M) of E2 after 4 h or 24 h com pared with all other conditions, suggesting that a high dose of E2 exposure for a short (4 h) or long (24 h) duration might accelerate the G 2 and M phases and thus entry into the G 1 phase. This finding is in agreement with our previous in vivo gene expression data showing a significant increase in the levels of genes stimulating the G 2 /M phase transition as well as M phase in female compared to male adult mice at night versus during the day. Therefore, our data suggest that high (10 5 M) but not low (10 8 M) levels of estradiol might increase cell division through increased S phase entry, accelerated G 1 to S phase transition, and accelerated G 2 and M phases. High (10 5 M) but not low (10 8 M) levels of estradiol might also increase cell differentiation by inducing neural progenitor cells to enter the G 0 phase and exit the cell cycle.

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122 S ynthesis phase ( T S ) and total cell cycle (T C ) lengths were calculated for each condition and of estradiol dose and exposure time on T S and T C T S and T C w ere significantly decreased for NPCs exposed to a high ( 10 5 M ) estradiol dose for 24 h compared with all other conditions and T C was significantly increased when NPCs were exposed to a low (10 8 M) dose of estradiol. These findings of decreased synthesis phase and total tell cycle lengths might explain the previous results showing that more cells in S phase and more cells in the quiescent G 0 phase were found in cultures treated with a high dose (10 5 M) of estradiol for 24 h com pared with all other conditions. Th ese finding s suggest that a high dose of estradiol expo sure for a minimum duration (at least 4 h) might decrease total cell cycle duration through accelerated G 1 to S phase transition, S phase, and /or accelerated G 2 and M phases. estradiol as well as exposure time on th e expression of e strogen r (ER ) (ER ) were also examined. The fraction of + cells in synthesis phase (BrdU + ) was similar for all conditions. The fraction of ER + cells in synthesis phase was significantly increased when exposed to a high (10 5 M) dose of estradiol for 24 h in culture com pared with all other conditions This suggest s that ER might be the main estradiol receptor responsible for the transcription of genes governing the entry of NPCs into S phase, acting as a transcripti on factor interacting with specif ic regulatory sequences in DNA ( hormone responsive elements ) and altering cell cycle gene expression at high estradiol concentration and minimum exposure time (at least 4 h) It has been shown previously that the increased estradiol is

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123 inhibited by an ER antagonist, showing an involvement of ERs in the cell division process (Brannvall et al. 2002) and that, although ERs are expressed in different brain regions during adult life (Pfaff & Keiner, 1973; Li et al. 1997; Shughrue et al. 1997) ER is mostly found in the hypothalamus, while ER is present in the cerebellar cortex, hippocampus (one of the two adult regio ns where proliferative NPCs are located), and other brain areas (Shughrue et al. 1990) Thi s correlates with our findings showing the involv e ment of ER (but not ) in NPC proliferation in the presence of high estradiol concentration. It was also shown that ER but not ER is controlled by circadian clock proteins. ER mRNA levels fluctuate in different peripheral tissues following a robust circadian pattern with a peak at the beginning of the dark phase (Cai et al. 2008b) suggesting that the previously observed increased NPC prolif eration at night versus day (Goergen et al. 2002; Holmes et al. 2004) might be due to an increase in ER signaling at night in response to nocturnal increase in estradiol levels. Estradiol dose and duration dependently changed the to tal + cells This fraction decreased when cells were exposed to estradiol (high or low concentration) for 24 h compared with no estradiol exposure and when exposed to high or low concentration s of estradiol during 24 h versus 4 h. The fraction of total + cells was decreased when cells were exposed to high estradiol concentration for 24 h versus no estradiol and when exposed to high (10 5 M ) dose of estradiol during 24 h versus 4 h in culture These data suggest a possible negativ e feedback in and ER expression of NPCs exposed to estradiol, particularly at high concentration and minimal exposure duration (at least 4 h). Therefore, even though ER seems to stimulate DNA synthesis in NPCs exposed to a high (10 5 M ) dose of estra diol for a minimum duration,

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124 this effect might decrease over time due to negative feedback regulation of ER by estradiol. Previous work showed that NPC neuronal differentiation was increased at night versus day, and that the increase in differentiation r elated gene expression was greater in female versus male mice at night. We expanded our in vivo work to assess whether estradiol and that has been shown to promote neurogenesis in rodent brains in viv o (McEwen, 1996 : Tanapat et al., 1999 : Ormerod and Galea, 2001 : Perez Martin et al ., 2003 : Ormerod et al ., 2004) and display a circadian rhythm in the adult brain (Somerville, 1971 : Roy and Wilson, 1981 : Bao et al ., 2003 : de la Iglesia and Schwartz, 2006 : Cai et al ., 2008), might be the cause of the variation in maturation related gene expression between male and female mice. Our in vitro study of female NPCs in culture revealed that low or high dose estradiol exposure at the beginning or 12 h after the sta rt of the experiment does not affect the differentiation nor the survival of NPCs 1 week after plating, and the majority of cells in culture 1 week after division are immature neurons, with a minority of new astrocytes, in accordance with previous studies (Brannvall et al ., 2002). Although, in vivo Ormerod et al. showed that neurogenesis was increased through increased survival of young granule neurons (Ormerod et al ., 2004), our in vitro study failed to show these results which might be due to the short amount of time of NPCs in culture (1 week in our in vitro study versus 2 weeks in vivo ) and more broadly to the presence of other factors in vivo not present in our in vitro study. Further studies might include longer term in vitro cultures to assess the e ffect of estradiol and other hormones, such as testosterone, on the differentiation and survival of NPCs.

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125 In summary, our study demonstrates that h igh (10 5 M) but not low (10 8 M) levels of estradiol a steroid hormone up regulated at night, in crease ce ll division in vitro possibly through inc reased S phase entry ( accelerated G 1 to S phase transition ) and accelerated G 2 and M phases which is consistent with our previous in vivo gene expression results and suggests that estradiol might influence the expression of NPC cell cycle genes mostly through the action of ER transcription factors High (10 5 M) but not low (10 8 M) levels of estradiol might also increase cell differentiation by inducing neural progenitor cells to exit the cell cycle. A subs equent in vitro study of NPCs exposed to different concentrations of estradiol in culture for 1 week any significan t difference in differentiation or survival among new neurons in control or experimental conditions, suggesting estradiol does no t have a role in the previously observed increased neuronal differentiation of NPCs dividing at night versus day Estradiol and estradiol receptor levels could be modulated to induce NPC proliferation for the treatment of neurodegenerative diseases or inju ries.

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126 CHAPTER 5 GENERAL DISCUSSION Several intrinsic and environmental factors have been shown to regulate adult neurogenesis by impacting NPC proliferation and the differentiation and survival of new cells, but we are still explor ing the mechanisms regulating these processes (Ming and Song, 2011). The experiments presented in this thesis aim at uncovering the cellular and molecular regulatory candidates responsible for the circadian control of adult neural progenitor cell behavior in the adult rodent brain. The influence of the light dark cycle on the dynamics of NPC proliferation and differentiation and survival of daughter cells in the dentate gyrus as well as the substantia nigra (where the presence of neurogenesis has been controve rsial) was examined. Cell proliferation and survival were increased among cells that divided during the dark versus light phase in both the dentate gyrus and substantia nigra. Neuronal differentiation was increased among cells generated during the dark pha se in the dentate gyrus, but not new neurons were found in the substantia nigra. Using cell cycle analysis, more cells were found in G2 M and S phases, and total cell cycle and S phase lengths were decreased at night versus day. These trends correlated wit h increased levels of cell cycle and differentiation gene expression between light and dark phases. In vitro high dose estradiol exposure increased DNA replication and entry into G 0 phase of NPCs in culture and decreased total cell cycle and S phase lengt hs, while estradiol exposure did not influence the differentiation or the survival of NPCs after 1 week in culture. A general summary of our experimental results is presented first, followed by their implications in understanding neurogenesis in the contex t of circadian rhythm, and finally their potential for tumor and neuronal replacement treatments and strategies are discussed.

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127 The Circadian Cycle Influences Cell Proliferation, Cell Cycle Kinetics, and Cell Cycle Gene Expression The light dark cycle and activity influences cell proliferation in the dentate gyrus and substantia nigra of adult female mice. The experiments in chapter 2 revealed that, in female adult mice, voluntary wheel running for 1 week significantly increased the number of dividing cells in the SGZ and substantia nigra, regardless of whether cells divided at night or during the day. This finding replicated previous work showing increased cell proliferation following unrestricted voluntary wheel access (van Praag et al. 1999b; van Praag et al. 1999c) which was found to be correlated with increased growth factor levels, such as VEGF (Fabel et al. 2003) and possibly IGF (Llorens Martin et al. 2010) A novel finding of significantly increased cell proliferation in the substantia nigra was also observed following unrestricted wheel exposure; however, none of these new cells was later found to adopt a neuronal phenotype. Chapter 2 also demonstrated that cell proliferation was increased during the nighttime, but this was only observed in the group that was not exposed to a running whee l. This might be due to the ceiling effect brought about by activity with unrestricted exposure to wheel running for 1 week preceding the analysis, which resulted in the maximum rate of cell division at night and during the day and therefore, no detectable difference in new cell numbers between night and day. This finding of increased cell proliferation during the dark phase is in agreement with previous work showing increased cell division at night with similar activity levels (Goergen et al. 2002; Holmes et al. 2004) ,. NPC proliferation in the dentate gyrus SGZ is highly regulated by cell division and cell cycle in the adult mammalian brain. However, most of the studies characterizing NPC cell cycle kinetics were performed during the light (sleep) phase of

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128 the circadian cycle in nocturnal rodents, and the influence of diurnal rhythms on NPC cell cycle kinetics and cell cycle related proteins, and thus neurogenesis, had not yet been investigated. Chapter 3 reveals a novel finding about NPC cell cycle distribution and kinetics and their variations during the light dark cycle. First, the proportion of cells in S or G 2 M phase showed a clear day/night variation a few hours after BrdU injection, with a significant increase during the night, while the total number of NPCs in active phases (G 1 S, G 2 M) of the cell cycle did not differ between night and day. New cell death was not detected at night or during the day a few hours after cell division. We found that T C and T S are shorter at night versus daytime, which suggests that an accelerated entry into S or G 2 M phases is most likely responsible for the increased proportion of cells in S and G 2 M phases observed at night, and not increased S or M phase lengths. Therefore, we hypothesize that G 1 /S and G 2 /M transition of NPCs is promoted during the night, which results in an increase of cells in S and M phases at night. Increased rate of progression of S phase, and thus total cell cycle (decreased T C and T S ) was also detected at night versus day. These data suggest that progression through the cell cycle is accelerated at nighttime, leading to an overall increase in NPC proliferation during the dark phase. The daytime T S and T C data determined by cumulative BrdU labeling were comparable to previo usly reported studies for adult mice (Nowakowski et al. 1989a; Came ron & McKay, 2001a; Hayes & Nowakowski, 2002) The cell cycle is driven by the action of cyclin dependent kinases (Cdks) and their activating partners, the cyclins (Malumbres & Barbacid, 2005) These elements orchestrate the progression through the different phases of the cell cycle, leading ultimately to the segregation of the daughter ce lls. Several studies have demonstrated

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129 that most of these cell cycle regulators are redundant and not required for the survival of the organism, and that the requirement for particular regulators is rather cell type specific (Berthet et al. 2003; Kozar et al. 2004; Malumbres et al. 2004; Pagano & Jackson, 2004) Although many studies have highlighted the importance of cell cycle compon ents in the regulation of NPC proliferation during development, very few have examined their role in adult NPC proliferation and thus adult neurogenesis (Fero et al. 1996; Callaghan et al. 1999; Huard et al. 1999; Ferguson et al. 2000; Cooper Kuhn et al. 2002; Lukaszewicz et al. 2005; Lange et al. 2009) Better understanding the regulation of adult NPC proliferation by the intrinsic cell cycle machinery is crucial in developing new cell based regenerative therapies (Beukelaers et al. 2012) The experiments in chapter 3 highlights cell cycle genes that vary between day and night in adult female and male mouse hippocampus. Genes encoding cell cycle molecules Cdk 6, cyclin D2, Cdk inhibitors p21/p27, and transcription factor E2f1 have previously been found to influence adult neurogenesis in the dentate gyrus in vivo (Cooper Kuhn et al., 2002; Pechnick et al., 2008; Qiu et al., 2009; Beukelaers et al., 2011) The circadian clock has been shown to control the expression of cell cycle related genes, in particular Wee1 (G2 M transition), c Myc (G0 G1 transition), and Cyclin D1 (G1 S transition) (Fu et al. 2002; Matsuo et al. 2003; Rana & Mahmood, 2010) Our study highlights cell cycle genes that have not yet been investigated in the context of adult neurogenesis and show a circadian variation with up regulation at night, which could be linked to the increased rate of NPC division and entry into M and S phases during the dark phase. These genes include cell cycle genes encoding proteins that control entry into the S phase of DNA replication (cyclin C/D1/E1, cell division cycle

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130 25 homolog A, cyclin dependent kinase 2, CDC28 protein kinase 1b, Cohesin subunit SA 1), proteins controlling entry into G 2 /M transition and mitosis (cyclin B1/F, Cyclin dependent kinases reg ulatory subunit 1, CDC28 protein kinase 1b, cell division cycle 25 homolog A, Cohesin subunit SA 1, Adenylate kinase 1) as well as several positive regulators of the cell cycle (transcription factors E2f2, E2f3, nucleoplasmin 2, Mdm2). A decrease in genes encoding negative regulators of the cell cycle, including those involved in cell cycle checkpoint and arrest (regulatory subunit of protein phosphatase 2, serine/threonine protein kinase Chk1, breast cancer type 2 susceptibility protein BRCA2, cysteine asp artic acid protease 3) were also detected at nighttime versus daytime. Interestingly, S phase and cell cycle positive regulator genes encoding cyclins B1 and F that have not yet been associated with regulation of NPC cell cycle were increased by up to more than 4 fold at nighttime versus daytime. Interestingly, only a few genes in male adult hippocampi showed increased expression levels at night versus day, such as those encoding proteins that control entry into the S (cyclin E1) or M phase (never in mitosi s gene a related kinase 2 Nek2), and transcription factor E2F2. Cell cycle genes involved in the negative control of cell growth and division and cell cycle checkpoint and arrest were also found to be down regulated at nighttime versus daytime ( Gpr132, Slf n1, Prm1, Cdkn2a, Chek1, Brca1, Trp63 ) in male adult mouse hippocampi. Our study uncovered several new candidate genes which appear to be regulated by the circadian clock and may be associated with the increased NPC proliferation observed during the dark p hase. Furthemore, the levels of these cell cycle genes, and possibly NPC proliferation, show differential light dark variations between

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131 adult male and female mice and thus appear to be under the control of gender specific factors. Estradiol Influences the Cell Proliferation of Neural Progenitors In Vitro The differential day/night variation of cell cycle gene expression levels between female and male adult mice suggests that these genes might be under the control of steroid hormones which are themselves regulated by the light dark cycle. The experiments in Chapter 4 demonstrate that high (10 5 M), but not low (10 8 M), levels of estradiol, a steroid hormone up regulated during the dark (active) phase, increase cell division of adult female NPCs in vitro through increased S phase entry (accelerated G 1 to S phase transition) and accelerated G 2 and M phases. These findings are consistent with our previously described in vivo gene analysis results showing increased expression levels of ge nes controlling G 1 S and G 2 M transitions in female versus male adult mouse hippocampi (Chapter 3) and suggest that estradiol might influence the factors. High (10 5 M), but not low (10 8 M), levels of estradiol might also control cell proliferation by inducing NPCs to exit the cell cycle and reach the quiescent state (G 0 ). Our in vitro results are in accordance with previous findings showing that estradiol, via ERs, regulate s the production (by increasing and then decreasing progenitor cell proliferation) of new granule neurons in the rodent dentate gyrus during embryonic development and adulthood (Tanapat et al. 1999; Brannvall et al. 2002; Ormerod et al. 2003) Our study uncovers possible cellular and molecular mechanisms by which estradiol might control NPC proliferation by stimulating the expr ession of cell cycle genes controlling G 1 S and G 2

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132 levels could be modulated to induce NPC proliferation in cell based therapies for neurodegenerative diseases or injuries. The Circadian Cycle Inf luences the Differentiation and Survival of Daughter Cells and Maturation Associated Gene Expression The experiments in Chapter 2 show a significant increase in new neuron production and survival in runners injected with BrdU at night compared to runners i njected during the day. These findings suggest that the light dark cycle can influence neuron production, maturation, and survival, independently of physical activity. Neurogenic activity seems to not only be dependent on the duration or amount of activit y (Holmes et al ., 2004), but also to be under the control of the light dark cycle. The experiments in Chapter 3 suggest that exit out of the multipotential state and entry into the differentiation stage, as well as the maturation process of new granule ne urons might be accelerated among NPCs dividing at night versus day. Indeed, more BrdU/Sox2 cells were found among NPCs dividing at night, and four week old neurons generated at night had more extensive branching than those produced during the day. These f indings correlate with the increased expression of several genes related to NPC differentiation detected at night versus daytime (Chapter 3). These genes were differentially expressed between female and male mice, which suggests they might be under the con trol of steroid hormones (themselves regulated by the light dark cycle). Estradiol, a diurnal steroid hormone, did not influence the differentiation or survival of one week old daughter cells in vitro (Chapter 4) which suggests other gender specific factors might influence the differential expression of maturation related genes between male and female mice hippocampi.

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133 A Hypothetical Model of the Interactions between Circadian Timing System, Estradiol, and Cell Cyc le Machinery The circadian clockwork may orchestrate the functionality of several factors involved in the control of neural progenitor cell cycle that is fundamental for adult neurogenesis. Based on the results in this thesis and previous studies, we prop ose a model linking the circadian clock machinery, estradiol gene regulation pathways, and the components of the cell cycle ( Fig. 5 1 ). The master circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of a transcript ional and post translational feedback loop. In mammals, the positive end of the transcriptional loop are the transcription factors, CLOCK ( Circadian Locomotor Output Cycles Kaput protein) and BMAL1 (Aryl Hydrocarbon Receptor Nuclear Translocator like 1 pro tein) or NPAS2 ( Neuronal PAS domain containing protein 2) that initiate transcription of the Cryptochrome ( Cry ), Period ( Per ) genes, and other clock gene products. The transcription factors CLOCK and BMAL1 heterodimerize and activate the expression of Per and Cry genes by binding to and E box element present in the promoter region. The Cry and Per proteins in turn act as negative regulators by interacting with CLOCK and/or BMAL1 in the nucleus (Chen et al. 2009) The core transcriptional loop is also regulated by two nuclear receptors: the retinoic acid receptor D1; nuclear receptor subfamily 1, group D, member 1), which can directly regulate the expression of BMAL1, NPAS2, and CLOCK (Guillaumond et al. 2005; Crumbley et al. 2010; Crumbley & Burris, 2011) In addition to the master circadian clock in the SCN, circadian clocks in other parts of the brain and peripheral organs are also composed of a transcriptional and post translational feedback loop with many of the same molecular

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134 components but clock rela ted genes outside the SCN remain relatively unexplored (Yoo et al. 2004) Interestingly, the CLOCK/BMAL1 heterodimer activates the transcripti on of clock controlled genes that contain an E box element in their promoter, including some apoptotic and cell cycle genes. In particular, CLOCK/BMAL1 may control cell cycle progression through the repression of c Myc (involved in G 0 /G 1 transition) and p2 1 (involved in G 1 /S transition), and through the activation of Wee1 (G 2 /M transition) and tumor suppressor protein p53 (Borgs et al. 2009a; Rana & Mahmood, 2010) Transitions between different phase of the cell cycle (G 1 S, G 2 and M) are regulated by the activity of cyclins, cyclin dependent kinases (Cdks), and Cdk inhibitors, and progression through the cell cycle occurs at specific times of the light dark cycle in many orga ns, suggesting the circadian clock system plays a fundamental role in controlling this process (Scheving et al. 1978; Bjarnason & Jordan, 2000; Garcia et al. 2001; Smaaland et al. 2002) Indeed, we found that key components of the cell cycle machinery, including cyclin C, cyclin D1, cyclin E1, cyclin B1, cyclin F, CKS1B, CDC25A, Cdk 2, E2f2, E2f3, and Mdm2, exhibit variations between the light and dark phases (Chapter 3). p21 is a potent Cdk inhibitor and functions as a regulator of cell cycle progression at the G 1 /S and G 2 /M transitions. p21 has been shown to negatively regulate G 1 /S and G 2 /M progression by binding to and inhibiting the activity of cyclin E / CDK2 and cyclin D / CDK4 6 or cyclin B CDK1 complexes (Harper et al. 1993; Gartel & Radhakrishnan, 2005) whose gene expression levels have been found to be up regulated at night in our study. p21 is rhythmically expressed in several peripheral organs under the control of BMAL1 (Grechez Cassiau et al. 2008) Cyclin D1 expression is also under circadian control (Fu et al. 2005) and promoted indirectly by

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135 Per 1 and Per 2 genes through inhibiting the transcription of c Myc (Perez Roger et al. 1999) Wee1 tyrosine kinase phosphorylates CDK1, inactivating the CDK1 cyclin B complex which was found to be up regulated at night in our study (Chapter 3), thereby inhibiting G 2 /M transition (Rothblum Oviatt et al. 2001) The Wee1 gene promoter contains E boxes which are activated by the BMAL1/CLOCK heterodimer (Gekakis et al. 1998) suggesting that Wee1 expression is also under circadian control. The circadian clock genes expressed in the SCN and peripheral tissues thus seem to have a clear impact on cell cycle control and cell cycle components that showed a diurnal variation in our study (Chapter 3). Identifying the detailed mechanisms by which the circadian clock genes are related to NPC proliferation and brain tumors is crucial in developing new target therapies to overcome neurodegenerative diseaeses and brain cancers. Steroid hormones such as estradiol also seem to cause a diurnal variation in the expression of certain cell cycle genes and impact NPC proliferation in our study (Chapter 3, 4). Estradiol enters cells freely and interacts with a cytoplasmic target cell receptor o dependent transcription factors. Although ERs are expressed in different brain regions during adult life (Pfaff & Keiner, 1973; Li et al. 1997; Shughrue et al. 1997) stly found in proliferative NPCs are located), and other brain areas (Shug hrue et al. 1990) After the estrogen receptor has bound its ligand, estradiol bound to its receptor can enter the nucleus of the target cell and interact with specific regulatory sequences in DNA, the hormone responsive elements, thus regulating gene transcription, which leads to

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136 formation of messenger RNA. The mRNA interacts with ribosomes to produce specific proteins that express the effect of estradiol upon the target cell. Estradiol indu ces proliferation of ER positive cells by stimulating G 1 /S transition with increased expression of cyclins, activation of Cdks, and phosphorylation of the tumor suppressor Rb (Retinoblastoma protein) in vitro in MCF 7 breast cancer cells (Foster et al. 2001) This is indirectly achieved by the downregulation of the Cdk inhibitors p21(CIP1) and p27(KIP1) which act to keep the cell cycle from progressing until all repairs to da maged DNA have been completed. Cyclin A and Cyclin D1 have been identified as ER activators. ER bound to its ligand activates c Myc and Cyclin D1 expression in early G 1 phase, facilitating Cyclin E CDK2 activation in mid to late G 1 and S phase entry. CDK4 is activated by binding to Cyclin D1 and acts early in G 1 phase, while CDK2 kinase functions in conjunction with Cyclin E and Cyclin A and is necessary for progression through late G 1 and entry into S phase. c Myc participates in Cyclin E CDK2 activation b y stimulating CDC25A (Cell Division Cycle 25A) expression; CDC25A expression is required for S phase entry and is induced in G 1 by c Myc and E2f. CDC25A is active from mid G 1 through S phase and also participates in activation of CDK2. Ultimately, active C yclin E CDK2 elicits S phase entry both through contribution to pocket protein phosphorylation and E2f release and through phosphorylation of additional mediators of S phase entry (Foster et al. 2001) Cyclin D, cyclin E, CDK2, CDC25A, Rb, and E2f gene expression levels were found to be differentia lly regulated in male and female mouse hippocampi during the light dark cycle (Chapter 3), suggesting that they might be involved in the increased NPC proliferation observed at night in female adult mice in vivo (Chapter 2) and in culture with a high estra diol dose in vitro (Chapter 4). Moreover,

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137 estradiol was shown to modulate the expression of clock genes, such as Per1 and Per2 in reproductive as well as non reproductive tissues (Nakamura et al. 2005; Nakamura et al. 2008; Nakamura et al. 2010) Clock genes themselves regulate the expression of estrogen and estrogen receptors in various tissues (Gery et al. 2007; Cai et al. 2008 a; Damdimopoulou et al. 2011; Shimizu et al. 2011) This suggests intricate relationships between the circadian clock machinery, estradiol gene regulation pathways, and the components of the cell cycle ( Fig 5 1).

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138 Figure 5 1. A Hypothetical Model of the Interactions between the C ircadian Timing System, Estradiol Gene Regulation Pathways, and the Cell Cycle Machinery. Red lines indicate an inhibitory relationship; blue arrows indicate a stimulating effect.

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139 Functional Implications of Adult Neurogenesis Correlation, ablation, and c omputational modeling studies have uncovered some function of SGZ neurogenesis in learning and memory. A correlation between hippocampal neurogenesis and learning in spatial memory task (using the Morris water maze) was observed in mice of different strain s (Kempermann et al ., 1997; Kempermann & Gage, 2002; Deng et al ., 2010), and mutant mice with decreased SGZ neurogenesis have decreased performance on hippocampus dependent learning tasks (Zhao et al ., 2003b; Shimazu et al ., 2006; Zhao et al. 2007). Manipulations that attenuate neurogenesis chronically are associated with impaired learning and memory (Madsen et al ., 2003; Raber et al., 2004; Rola et al., 2004; Winocur et al., 2006) while those that increase neurogenesis are associated with be tter performances on hippocampus depend ent tasks (Ormerod et al., 2004 ; van Praag et al., 2005). S ome forms o f hippocampus dependent l earning require the presence o f young granule neurons (Shors et al. 2001; Shors e t al. 2002) and increases the survival of young granule neurons (Gould et al. 1999; Ambrogini et al. 2000) Neurogenesis was also correlated with learning abilities in anti mitotic drug treated rats that failed to form con ditioned responses in trace eyeblink or trace fear conditioning (Shors et al., 2001) and in irradiated or ganciclovir treated rats or mice (Saxe et al ., 2006; Winocur et al., 2006). Studies correlating neurogenesis with spatial learning and memory remain c ontradictory, with some showing impaired spatial learning and memory in the Barnes maze but not the Morris maze, and others the opposite (Raber et al., 2004; Rola et al., 2004). Place and object recognition memories were also impacted by ablated neurogenesis in irradiated rats (Winocur et al., 2006) and in anti mitotic drug treated animals (Bruel Jungerman et al., 2005) in enriched environments. Collectively, these

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140 studies suggest the significant contribution of adult neurogenesis in learning and memory although the exact mechanisms of this function still remain unknown. Neurogenesis has also been implicated in mood regulation in many studies, parti cularly by Gould and c olleagues (Gould et al. 1992; Cameron & Gould, 1994; Gould et al. 1997; Tanapat et al. 1998) who showed that SGZ progenitor cell division is suppressed by corticosteroids, which are elevated in depressed and stressed patients. SGZ neurogenesis might also be necessary for various antidepressant functions, as demonstrated by Santarelli and colleagues (Santarelli et al., 2003) who showed that disrupting antidepressant induced neurogenesis block ed behavioral responses to antidepressants Therefore g reater understanding of the physiological and molecular mechanisms regulating in vivo neurogenesis is crucial in restoring normal neurological functions in cases of functional loss accompanying aging and neurodegenerative diseases or brain injuries. Circadian Rhythms and Tumor Growth As metabolism, hormone secretion, and cell cycle are all under rhythmic control and regulate cell proliferation, the connection between cancer and circadian rhythms has been an important recent focus of research (Sahar & Sassone Corsi, 2009; Wood et al. 2012) Lack of rhythmic control leads to uncontrolled proliferation and cancer (Lee et al. 2010; Yu & Weaver, 2011) particularly in the breast and colon of rotating shift workers in humans (Davis & Mirick, 2006; Haus & Smolensky, 2006) Circadian disruption by dim light at night or chronic jet lag conditions accelerates tumor growth in affected animals (Filipski et al. 2004; Cos et al. 2006) through variations of circadian controlled factors such as insulin/IGF 1, glucocorticoids, catecholamines, and melatonin. In turn, core clock genes, such as Per1 and Per2 seem to function as tu mor

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141 suppressors in mice (Fu et al. 2002; Gery et al. 2006) Research indicates that the circadian clock controls the temporal behavior of cells in many tissues, and that its perturbation affects homeostasis and the predisposition to tumorigenesis (Mormont & Levi, 1997; Fu & Lee, 2003; Takahashi et al. 2008) possibly in the brains of affected subjects. Therefore, a clo se correlation exists between circadian rhythms and tumor growth, and the understanding of circadian controlled mechanisms regulating adult NPC proliferation might help improve the prevention and treatment of tumor development and growth in the adult b rain Implications and Approaches for Gene Delivery and S tem Cell Based Strategies for Neuroregenerative Therapies Our study confirmed that NPC behavior and neurogenesis are under circadian control that regulates most physiological processes. Clinically, the timing of stem cell transplant or growth factor infusion may i nfluence the engraftment or impact of those treatments and may result in better therapeutic outcomes, a s timing is a variable that can be readily manipulated. Indeed, as cell based therapies tha t use exogenous, culture expanded neural stem cells or target endogenous stem cells and their associated microenvironment are developed, it is crucial to optimize their effectiveness and maximize the returns to patients. Studies of the circadian clock in t he context of stem cell biology will allow us to optimize the time when stem cell based treatments are administered (Gimble et al. 2009) Moreover, the circadian clock regulatory proteins might provide a new regulatory pathway for potential direct interv entions. For instance, these small molecules capable of modulating the circadian clock such as CLOCK, BMAL1, Period, or Cryptochrome proteins, could be used as drug discovery targets to manipulate adult neural progenitor

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142 cell proliferation, lineage differ entiation and function. As neural progenitor cell number and metabolic activity are found to be highly correlated to circadian oscillations, directing increased focus to these circadian mechanisms has the potential to improve our understanding of stem cel l biology and, subsequently, our ability to modulate stem cell proliferation, differentiation, and function. The outcomes of such studies will directly impact our future ability to manipulate neural stem cell physiology and prevent stem cell pathology (Gimble et al. 2009) In the present study, we uncovered several cell cycle genes that are up regulated at night, including cyclin C/B1/D1/E1/F, Cdc25A, Cdk2, Cks1b, E2f2, E2f3, Mdm2 when adult NPC proliferation is increased. We also uncovered some candidate genes associated with neuronal maturation processes which were up regulated at night versus day in one week old mouse hippocampi. These genes have not yet been investigated in the context of adult neurogenesis, and future s tudies could assess their individual impact on adult NPC proliferation using knockout mouse mutants. We also found a correlation between diurnal estradiol hormone and in vitro which confirms previous studies h ighlighting the regulatory role of estradiol on adult neurogenesis. Two predominant strategies could be used to restore neurogenesis in cases of neuronal loss and function (such as in age related decline within neurogenic niches): replacement of missing c ell via transfer of stem cells and transfer of genes responsible for neurogenesis or a combination of both Stem cells could be used therapeutically through a variety of approaches (Ormerod et al. 2008) Exogenous stem cells may be isolated and expanded in vitro in preparation for grafting into the brain by stereotax ic injection ; they could then integrate into the existing neurocircuitry and replace missing

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143 neurons. Cultured stem cells could also be genetically modified ex vivo prior to stereotaxic injection into the brain. The ex vivo genetic approach could be useful for modifying stem cells to express a therapeutic transgene, thus using the stem cell as delivery vectors. Neural stem cells (NSCs) have several advantages which make them suitable as delivery vehicles in gene therapy ; NSCs can potentially integrate into the brain without disruption of normal function and they can be propagated for long lengths of time in vitro allowing for genetic manipulation (Muller et a l. 2006) Gene expression vectors are under investigation for use in various diseases caused by deficiency in some crucial factor, as these vectors have the potential to restore the missing factor. They could also be use d to stimulate the express ion o f particular cell cycle or maturation related genes, such as those uncover ed in our study or up regulate the synthesis of certain hormones or receptors, such as estradiol and ER in order to increase endogenous NPC proliferation and daughter cell differentiation, thus restoring normal neurogenesis. In terms of the genetic modification, neural stem cells can be genetically transduced in vitro or in vivo. At present, the most common and efficient manner of introducing genes into NSCs is by use of vir al vectors in vitro It is very challenging to introduce DNA into cells without causing harm, however, by using viruses which have a natural capacity for introducing genes into the cells of the host, this can be accomplished efficiently (Edry et al. 2011) The use of viral vectors for transducing stem cells has expanded in recent years, and this technology is being continuously developed to increase its safety and practicality. Through genetic engineering, genes involved in pathogenesis and replication are deleted and specific transgenes are inserted, enabling viruses to ser ve as effecti ve tools for gene transfer.

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144 These viral vectors can introduc e the transgenes into NSCs but do not replicate. Viral vectors have several characteristics which make them particularly attractive for use in manipulation of cells of the brain, such as specificity of target cell type and capability of reporter gene co expression. Tropism towards, and attachment and infection of, specific NSCs is mediated by the particular proteins present on the viral envelope or capsid (Edry et al. 2011) Viral vectors derived from adenovirus, adeno associated virus, herpes simplex v irus type 1 and most commonly lentivirus (such as HIV 1), have been found useful for NSC transduction for gen e therapy in the brain. After years of preclinical progress, several gene therapies using viral vectors are now in Phase I and Phase II clinical t rials, showi ng some encouraging results ( Nobre & Almeida, 2011) Many of the viral vectors in clinical use maintain lifetime expression in the brain, and currently research is being done to try and find a mechanism that could control the expression of the transgene product through the applicat ion of a prodrug; this would be valuable if the continued protein expression were to become problematic long term (Manfredsson et al. 2012) resulting in cancerous cells in the case of long term up regulated cell cycle gene expression, for instance. Gene s could be directly delivered into the CNS by stereotaxic injection to encode infected cells to express signals/factors necessary to sustain a neurogenic niche for grafted stem cells. Alternatively, in vivo gene delivery could result in the expression of appropriate transgenes for the local recruitment of endogenous stem cells. Such therapeutic niche engineering could be used for reversing age related decline within neurogenic niches or for generating a neurogenic niche in regions that otherwise did not express the necessary signals/factors (Williams & Lavik, 2009; E dalat et al. 2012) As

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145 suitable signals/factors supporting neural stem cells or recruiting endogenous neural stem ce lls are identified, it may be possible to move beyond cell or gene delivery mediated therapies and systemically and safely deliver drugs or small molecules to the brain instead. Implications for Cancer Therapy The cellular circadian clocks are coordinated by endogenous physiological rhythms, as are the host tissues targeted by anticancer agents. As a result, circadian timing was found to increase 2 to 10 times the tolerability of anticancer medications in cancer patients (Eriguchi et al. 2003; Mormont & Levi, 2003; Levi et al. 2010) Improved efficacy is also seen when drugs are given near their times of best tolerability and when anticancer drugs are administered at their most toxic time. Gender, circadian ph ysiology, clock genes, and cell cycle critically affect outcome in cancer chronotherapeutics (for instance, medical treatment administered according to a schedule that corresponds to a person's biological clock in order to maximize the health benefits and minimize adverse effects), and cancer therapeutic approaches should focus on develop ing technological tools in order to further optimize and personalize the circadian administration of cancer treatments, including brain tumor therapies (Eriguchi et al. 2003; Mormont & Levi, 2003; Levi et al. 2010) as some brain tumors have been hypothesized to originate from neural stem cells (Vescovi et al. 2006) and our study revealed some circadian controlled aspects of neural progenitor cell proliferation. Summary In the present study, we uncovered extrinsic and endogenous factors, along with genetic regu lators of cellular activity linked to the circadian clock, in the scope of better understanding the mechanisms regulating adult neurogenesis. We shed light on some

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146 new signaling pathways (genetic, hormonal) activated by circadian regulators, as these path ways hold the potential for the development of new strategies for neural stem cell based therapies as well as cancer prevention and treatment. Novel approaches to neuroregeneration will, to be most effective, make use of the circadian related effects on ne uronal progenitor cell behavior in the adult brain to activate these progenitor cells in a targeted manner in order to enhance or repair brain function. Further studies are needed to determine which individual components of the clock are involved in the re gulation of NPC behavior. Future work might examine, for instance, the possible presence of BMAL1/CLOCK binding sites in the promoters of proliferation or differentiation genes found to be up regulated at night when progenitor cell proliferation and daught er cell differentiation is stimulated. The precise mechanism of action of hormones such as estradiol which has been shown to regulate NPC proliferation in vitro in our study, as well as in vivo in previous studies, and survival in vivo still remains to b e elucidated Both the circadian clock and neurogenesis are also related to psychological or neurological disorders such as depression and brain infarction, and our findings may help in better understanding these diseases and developing new therapies. Neural progenitor cells that are found in the subventricular zone and dentate gyrus subgranular zone of the adult brain could be useful in cell replacement therapies disease, epilepsy, aging, and other neurodegenerative diseases and brain trauma could involve administering factors (orally, intravenously or locally through gene delivery or stem cell transplant ) that would stimulate endogenous neurogenesis for the

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147 replacement of degenerating neurons. Once the variations and mechanisms of the transcriptional clock and gene expression are fully understood, neural stem cell research can be directed toward altering the gene clock in ways that could implement the favorable regulation of normal neural progenitor cell activity. Indeed, the development of treatments and/or cures that would increase production of new neurons requires the identification of endogenous or natural molecular regulators of adult neurogenesis. Robust circadian cy cle induced changes in neurogenesis are a powerful means for exploring the mechanisms that regulate cell cycle behavior and neural progenitor cell proliferation, without submitting the brain to injury or other external manipulati on.

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181 BIOGRAPHICAL SKETCH Lan Hoang Minh was born and grew up in Paris, France. After graduating from high school in Paris with a baccalaureate in Science with high honors, she came to the United States in order to attend college at the University of Florida. During her undergraduate studies she gained valuable research experience as a n u ndergraduate r esearch s cholar in the Clinical M icrobiology L aboratory directed by Dr. Kenneth Rand at Shands Hospital at U.F., d etermining the s usceptibility of Pseudomonas aeruginosa to the antibiotic Cefepime Lan earned her Bachelor of Science in M icrobiology and Cell Science and was accepted in the U.F. College of Medicine S he transferred to the Biomedical Engineering Department at U.F. in 2005, which she thought was more suitable to her scientific interests. She joined the laboratory of Dr. Thomas Mareci, in col laboration with Dr. Paul Carney, in studying temporal lobe evolution into epilepsy using magnetic resonance imaging and histology and earned her degree with thesis in 2007 She then Laboratory of Stem Cell Research in which she examined various aspects of adult neurogenesis, particularly the circadian control of hippocampal neurogenesis and associated molecular and genetic regulators.