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In Vivo and in Vitro Effects of Azidothymidine on Neural Stem/Progenitor Cells in Mouse

Permanent Link: http://ufdc.ufl.edu/UFE0042169/00001

Material Information

Title: In Vivo and in Vitro Effects of Azidothymidine on Neural Stem/Progenitor Cells in Mouse
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Demir, Meryem
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adult, azidothymidine, azt, prenatal, zidovudine
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: IN VIVO AND IN VITRO EFFECTS OF AZIDOTHYMIDINE ON NEURAL STEM/PROGENITOR CELLS IN MOUSE By Meryem Demir December 2010 Chair: Eric D. Laywell Major: Medical Sciences - Neuroscience Azidothymidine (3?-azido-3?-deoxythymidine; AZT) is a nucleoside reverse transcriptase inhibitor that has been used in the treatment and prevention of human immunodeficiency virus-1 (HIV-1). Even though there is no active transportation of AZT across the blood brain barrier, AZT is shown to accumulate in high levels in cerebrospinal fluid (CSF) with limited penetration into the parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system, we suggest that passive diffusion from CSF may be sufficient to expose the neurogenic cells to biologically relevant levels of AZT that may be sufficient to perturb the normal production and/or survival of newly-generated neurons. In turn, this perturbed neurogenesis contribute to, or be the basis of many of the neurological deficits seen in some cases of AIDS. In order to assess the effects of clinically relevant AZT regimens on neuronal production, we have employed in vitro and in vivo models of mouse neurogenesis. Our in vitro results show that AZT reduces the expansion potential of neural stem/progenitor cells, eventually inducing a senescent phenotype. In addition, AZT treated cells display impaired differentiation potential and increased susceptibility to apoptosis. In vivo, our AZT administration paradigm mimicking dosing regimens that administered to human patients revealed a remarkable decrease in the survival of newly formed cells in (subventricular zone)SVZ in contrast to the short term neurogenesis which is not affected significantly within the dentate gyrus and SVZ of adult mice. Finally, our analysis of in utero exposure demonstrates that AZT affects SVZ stem/ progenitor cells so their neurogenic potential is perturbed and the expansion potential of cultured neural stem/progenitor cells from treated offspring is decreased. Together, these data reveal uncharacterized negative consequences of AZT treatment on neural stem/progenitor cells. Given that HIV infection leads to the development of neurological deficits, and that human HIV (+) patients are usually treated with AZT over many years, it is important to determine to what extent AZT regimens might perturb normal levels of neurogenesis to exacerbate or contribute to these neurological problems. We suggest that in case of increased delivery of AZT into the brain, a direct exposure to the CNS would cause more dramatic changes as we have shown with in vitro cell culture systems. We expect our results will reveal new insights regarding the effect of AZT on stem/progenitor cell functioning, and the development of new treatment approaches to prevent the HIV infection in CNS.
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.
Statement of Responsibility: by Meryem Demir.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Laywell, Eric.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042169:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042169/00001

Material Information

Title: In Vivo and in Vitro Effects of Azidothymidine on Neural Stem/Progenitor Cells in Mouse
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Demir, Meryem
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adult, azidothymidine, azt, prenatal, zidovudine
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: IN VIVO AND IN VITRO EFFECTS OF AZIDOTHYMIDINE ON NEURAL STEM/PROGENITOR CELLS IN MOUSE By Meryem Demir December 2010 Chair: Eric D. Laywell Major: Medical Sciences - Neuroscience Azidothymidine (3?-azido-3?-deoxythymidine; AZT) is a nucleoside reverse transcriptase inhibitor that has been used in the treatment and prevention of human immunodeficiency virus-1 (HIV-1). Even though there is no active transportation of AZT across the blood brain barrier, AZT is shown to accumulate in high levels in cerebrospinal fluid (CSF) with limited penetration into the parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system, we suggest that passive diffusion from CSF may be sufficient to expose the neurogenic cells to biologically relevant levels of AZT that may be sufficient to perturb the normal production and/or survival of newly-generated neurons. In turn, this perturbed neurogenesis contribute to, or be the basis of many of the neurological deficits seen in some cases of AIDS. In order to assess the effects of clinically relevant AZT regimens on neuronal production, we have employed in vitro and in vivo models of mouse neurogenesis. Our in vitro results show that AZT reduces the expansion potential of neural stem/progenitor cells, eventually inducing a senescent phenotype. In addition, AZT treated cells display impaired differentiation potential and increased susceptibility to apoptosis. In vivo, our AZT administration paradigm mimicking dosing regimens that administered to human patients revealed a remarkable decrease in the survival of newly formed cells in (subventricular zone)SVZ in contrast to the short term neurogenesis which is not affected significantly within the dentate gyrus and SVZ of adult mice. Finally, our analysis of in utero exposure demonstrates that AZT affects SVZ stem/ progenitor cells so their neurogenic potential is perturbed and the expansion potential of cultured neural stem/progenitor cells from treated offspring is decreased. Together, these data reveal uncharacterized negative consequences of AZT treatment on neural stem/progenitor cells. Given that HIV infection leads to the development of neurological deficits, and that human HIV (+) patients are usually treated with AZT over many years, it is important to determine to what extent AZT regimens might perturb normal levels of neurogenesis to exacerbate or contribute to these neurological problems. We suggest that in case of increased delivery of AZT into the brain, a direct exposure to the CNS would cause more dramatic changes as we have shown with in vitro cell culture systems. We expect our results will reveal new insights regarding the effect of AZT on stem/progenitor cell functioning, and the development of new treatment approaches to prevent the HIV infection in CNS.
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.
Statement of Responsibility: by Meryem Demir.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Laywell, Eric.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042169:00001


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1 IN VIVO AND IN VITRO EFFECTS OF AZIDOTHYMIDINE ON NEURAL STEM/PROGENITOR CELLS IN MOUSE By MERYEM DEM R A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREME NTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Meryem Demir

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3 To my Mom Hacer Cakiroglu, for her endless love and support that I will forever be thankful for.

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4 ACKNOWLEDGMENTS I would like to thank my mentor, Dr Eric Laywell, for his guidance and persistent encouragement that gave me motivation throughout my study. I would like to thank members of Laywell Lab, Dr. Greg Marshall and Dr. Heather Ross, for their support. I would like to express my appreciations to Dr. Naohiro Terada, Dr. Lucia Notterpek, and Dr. Alexander Ishov, for serving on my dissertation committee and for their valuable comments. I would also like to convey my thanks to the members of Steindler and Reynolds labs, for their suggestions and colla borations. I express my gratitude to Dr. Jake Streit, for his support and motivation. I also want to thank my friends in graduate school, Ahu Demir, Daniel Silver, Florian and Dorit Siebzehnrubl and Lindsay Levkoff, for their invaluable friendship and supp ort. I am ever grateful to my mother, Hacer; my siblings Fazilet, Akif and Burak, for their love and encouragement. I would also like to thank my love Huseyin, for always being with me and for his endless love and support through this seemingly endless end eavor. Lastly, I would like to thank our son, Ilgar, for the pure love and happiness he brought to our lives.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ...... 4 LIST OF TABLES ................................ ................................ ................................ ................ 7 LIST OF FIGURES ................................ ................................ ................................ .............. 8 LIST OF ABBREVIATIONS ................................ ................................ .............................. 10 ABSTRACT ................................ ................................ ................................ ........................ 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ........ 15 Neurogenesis in the Adult Mammalian Brain ................................ ............................ 15 History ................................ ................................ ................................ .................. 15 Neurogenic Niches in the Adult Mammalian Brain ................................ ............. 16 Neural Stem Cell Functioning ................................ ................................ .............. 17 In vitro models of Neurogenesis ................................ ................................ .......... 18 Azidothymidine ................................ ................................ ................................ ............ 21 Background ................................ ................................ ................................ .......... 21 Mechanism of Action ................................ ................................ ............................ 22 Therapeutic Usage in Pregnancy ................................ ................................ ........ 23 Dosage and Administrati on ................................ ................................ ................. 25 Toxicity Mechanisms ................................ ................................ ............................ 26 Distribution of AZT in the CNS ................................ ................................ ............ 30 2 MATERIALS AND METHODS ................................ ................................ ................... 35 Generation and Expansion of Ast rocyte Monolayer Cell Cultures ............................ 35 Inducible Neurogenesis ................................ ................................ .............................. 35 Immunocytochemical Analysis ................................ ................................ ................... 36 Neurosphere Culture ................................ ................................ ................................ .. 37 Neural Colony Forming Cell (NCFC) Assay ................................ .............................. 37 In Vitro Drug Treatment ................................ ................................ .............................. 37 TUNEL Assay ................................ ................................ ................................ ............. 38 Senescence Galactosidase Labeling ................................ .................. 38 JC 1 Assay ................................ ................................ ................................ .................. 39 In vivo AZT Administration ................................ ................................ ......................... 39 In utero AZT Administration ................................ ................................ ........................ 40 Immunohistochemistry ................................ ................................ ................................ 40 Statistics ................................ ................................ ................................ ...................... 41

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6 3 IN VITRO AZIDOTHYMIDINE EXPOSURE REDUCES TH E NEUROGENIC POTENTIAL OF NAVE SVZ STEM/PROGENITOR CELLS ................................ ... 42 Background ................................ ................................ ................................ ................. 42 Results ................................ ................................ ................................ ........................ 43 Monolayer of Multipotent Astrocytic Stem Cell (MASC) Culture ........................ 43 AZT Reduces Astrocyte Monolayer Population Expansion ................................ 44 AZT Abolishes Inducible Neurogenesis from Astrocyte Monolayer Cells .......... 44 AZT Increases Apoptosis in Supplement Withdrawn MASC .............................. 45 AZT Upregulates SA B Gal Activity in Supplement Withdrawn MASC ............. 45 AZT Changes the Size and Morphology of Neurospheres ................................ 46 AZT Increases the Proportion of Senescent Neurosphere Cells ....................... 47 AZT Perturbs Formation of Neural Colonies Derived From SVZ Stem and Progenitor Cells ................................ ................................ ................................ 47 Conclusion ................................ ................................ ................................ .................. 48 4 IN VIVO AZIDOTHYMIDINE ADMINISTRATION DISTURBS NEUROGENESIS ... 69 Background ................................ ................................ ................................ ................. 69 The Effect of AZT on Adult Neurogenesis ................................ .......................... 69 The Effect of in utero AZT Exposure on Neurogenesis ................................ ...... 71 Results ................................ ................................ ................................ ........................ 74 In Vivo AZT Administration Does Not Affect Brdu (+) Cell Number in Adult Neurogenic Area ................................ ................................ ............................... 74 In Utero AZT Administration Does Not Affect the Litter Size or Pup Weight ..... 75 In Utero AZT Administration Does Not Affect the Expansion Potential of .... 76 In Utero AZT Administration Causes a Very Significant Decrease in Inducible Neurogenesis Potential of Astrocyte Monolayer Cells Derived ................................ ................................ ............... 77 In Utero AZT Administration Yields Smaller Neurospheres than Control Group ................................ ................................ ................................ ................ 77 In Utero AZT Administration Causes a Slight Decrease in Ki67 (+) Cell ................................ ................................ ........... 78 Conclusion ................................ ................................ ................................ .................. 78 5 DISCUSSION AND CONCLUSIONS ................................ ................................ ......... 90 Summary ................................ ................................ ................................ ..................... 97 LIST OF REFERENCES ................................ ................................ ................................ ... 99 BIOGRAPHICAL SKETCH ................................ ................................ ............................. 117

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7 LIST OF TABLES Table page 1 1 November 6, 2009, the Food and Drug Administration (FDA) approved revised pediatric dosing recommen dations ............................... 34

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8 LIST OF FIGURES Figure page 1 1 Structural formula of AZT and Thymidine.. ................................ ........................... 33 3 1 Serum and mitogen withdrawal induces neurogenesis from SVZ monolayers.. ................................ ................................ ................................ ........... 56 3 2 Serum and mitogen withdrawal leads first to a period of relative quiescence followe d by a sharp increase in neurogenesis. ................................ ..................... 57 3 3 Serum and mitogen withdrawal causes a change in the monolayer cell population dynamics. ................................ ................................ ............................. 58 3 4 AZT reduces astrocyte monolayer cell population expansion.. ............................ 59 3 5 AZT abolishes inducible neurogenesis from monolayer cells. ............................. 60 3 6 A short exposure of low dose AZT perturbs inducible neurogenesis from astrocyte monolayer.. ................................ ................................ ............................. 61 3 7 AZT increases apoptosis in astrocyte mon olayers. ................................ .............. 62 3 8 AZT upregulates senescence associated mechanisms in withdrawal induced astrocyte monolayer cells. ................................ ................................ ..................... 63 3 9 AZT does not perturb mitochondrial membrane depolarization in astrocyte monolayers. ................................ ................................ ................................ ............ 64 3 10 AZT exposure changes the size and morphology of neurospheres. .................... 65 3 11 AZT exposure suppresses neurosphere forming cells. ................................ ...... 66 3 12 AZT exposure increases the proportion of senescent neurosphere cells. ........... 67 3 13 AZT severely perturbs formation of neural colonies derived from both neural stem and progenitor cells.. ................................ ................................ ..................... 68 4 1 Representative images of Dentate Gyrus and SVZ with BrdU and NeuN immunolabeling. ................................ ................................ ................................ .... 83 4 2 2 week long, low dose administration of AZT does not change the number of BrdU (+) cell number both in dentate gyrus and SVZ.. ................................ ......... 84 4 3 2 week long moderate treatment regimen caused a significant decrease in BrdU (+) cell number only in SVZ area.. ................................ ............................... 85 4 4 In utero exposure of AZT does not affect the litter size or pup weight.. ............... 86

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9 4 5 In utero exposure of AZT on MASC derived from offsprin ............ 87 4 6 In utero AZT administration yields smaller neurospheres than control group.. ... 88 4 7 In uter o exposure of AZT does not affect proliferating Ki67 (+) cell number in ................................ ................................ ................................ ... 89

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10 LIST OF ABBREVIATION S ACTG 076 AIDS Clinical Trials Group Protocol 076 ADC AIDS Dementia Complex AIDS Acquired Immune Defic iency Syndrome AZT Azidothymidine azido deoxythymidine AZT DP AZT diphosphate AZT MP AZT monophosphate AZT TP AZT triphosphate BBB Blood Brain Barrier BCSFB Blood Cerebrospinal Fluid Barrier bFGF Basic Fibroblast Growth Factor BrdU Bromodeo xyuridine 5 bromo deoxyuridine CNS Central Nervous System CPE CNS Penetration Effectiveness CS Cesarean Section CSF Cerebrospinal Fluid DAPI 4', 6 diamidino 2 phenylindole DG Dentate Gyrus dT TP Deoxythymidine Triphosphate E12 Embryonic Day 12 FBS Fetal Bovine Serum FDA Food and Drug Administration HAART Highly Active Antiretroviral Therapy HAD HIV Associated Dementia HIV Human Immunodeficiency Virus

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11 HTLV III Human T Lymphotrophic Virus Type III IC 50 50% Inhibition concentrations LAV Lymphaden opathy Associated Virus MASC Multipotent Astrocytic Stem Cell MND Mild Neurocognitive Dsorder mtDNA Mitochondrial DNA NCFC Neural Colony Forming Cell NCFCA Neural Colony Forming Cell Assay NDK Nucleoside Diphosphate Kinase NICHD National Institute of Child Health and Human Development NPC Neural Progenitor Cell NRTI Nucleoside Reverse Transcriptase Inhibitor NS Neurosphere NSC Neural Stem Cell OB Olfactory Bulb PBS Phosphate buffer solution P2 Postnatal Day 2 Pol Polymerase Gamma rhEGF Human Epidermal Growth Factor RLV Rauscher Murine Leukemia Virus Complex Senescence Galactosidase SGZ Subgranular Zone SVZ Subventricular Zone TK1 Thymidine Kinase 1 TK2 Thymidine Kinase 2

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12 TMPK Thym idylate Kinase T TP Thymidine Triphosphate TUNEL Terminal Deoxynucleotidyl Transferase mediated dUTP Nick end Labeling X Gal 5 Bromo 4 chloro 3 indolyl B Dgalactoside ZDV Zidovudine

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13 Abstract of Dissertation Presented to the Graduate School of the Univer sity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IN VIVO AND IN VITRO EFFECTS OF AZIDOTHYMIDINE ON NEURAL STEM/PROGENITOR CELLS IN MOUSE By Meryem Demir December 2010 Chair: Eric D. Laywe ll Major: Medical Sciences Neuroscience azido deoxythymidine; AZT) is a nucleoside reverse transcriptase inhibitor that has been used in the treatment and prevention of human immunodeficiency virus 1 (HIV 1). Even though there is n o active transportation of AZT across the blood brain barrier, AZT is shown to accumulate in high levels in cerebrospinal fluid (CSF) with limited penetration into the parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventri cular system, we suggest that passive diffusion from CSF may be sufficient to expose the neurogenic cells to biologically relevant levels of AZT that may be sufficient to perturb the normal production and/or survival of newly generated neurons. In turn, th is perturbed neurogenesis contribute to, or be the basis of many of the neurological deficits seen in some cases of AIDS. In order to assess the effects of clinically relevant AZT regimens on neuronal production, we have employed in vitro and in vivo mode ls of mouse neurogenesis. Our in vitro results show that AZT reduces the expansion potential of neural stem/progenitor cells, eventually inducing a senescent phenotype. In addition, AZT treated cells display impaired differentiation potential and increased susceptibility to apoptosis. In vivo our

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14 AZT administration paradigm mimicking dosing regimens that administered to human patients revealed a remarkable decrease in the survival of newly formed cells in subventricular zone ( SVZ ) in contrast to the short term neurogenesis which is not affected significantly within the dentate gyrus and SVZ of adult mice. Finally, our analysis of in utero exposure demonstrates that AZT affects SVZ stem/ progenitor cells so their neurogenic potential is perturbed and the e xpansion potential of cultured neural stem/progenitor cells from treated offspring is decreased. Together, these data reveal uncharacterized negative consequences of AZT treatment on neural stem/progenitor cells. Given that HIV infection leads to the devel opment of neurological deficits, and that human HIV (+) patients are usually treated with AZT over many years, it is important to determine to what extent AZT regimens might perturb normal levels of neurogenesis to exacerbate or contribute to these neurolo gical problems. We suggest that in case of increased delivery of AZT into the brain, a direct exposure to the CNS would cause more dramatic changes as we have shown with in vitro cell culture systems. We expect our results will reveal new insights regardi ng the effect of AZT on stem/progenitor cell functioning, and the development of new treatment approaches to prevent the HIV infection in CNS.

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15 CHAPTER 1 INTRODUCTION Neurogenesis in the Adult Mammalian Brain History ment was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be generated, It is for the science of t he future to change, if possible, this harsh in 1913, has been so influential that it took almost a century to break this long held dogma. Because most cells in the mammalian central nervous system (CNS) are in a quiescent state, neurogenesis was for many decades believed to occur only during embryonic and early postnatal development. In 1962, by using intracranial injection of thymidine H3, Altman showed the presence of proliferating glia, neurons and neuroblasts in damaged brain areas of adult r ats for the first time (Altman, 1962) More than three decades later, the discovery of neurogenesis in the dentate gyrus of adult humans had an important impact on the field (Eriksson et al., 199 8) It is now known that in the adult mammalian brain there are two main neurogenic niches, the subventricular zone (SVZ) and dentate gyrus (DG) of the hippocampus. Throughout life the neural stem cells in the adult mammalian brain are able to generate fu nctional cells regulated by signals under physiological and pathological conditions (Alvarez Buylla and Lim, 2004; Zhao et al., 2008).

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16 Neurogenic Niches in the Adult Mammalian Brain Neural stem cells (NSCs) are self renewing, multipotent cells that are located in the persistent neurogenic niches of the SVZ of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus. In the SVZ, there are three precursor cell populations: Neural stem cells compromising slowly dividing, GFAP+ SVZ astrocytes III tubulin+ migratory neuroblasts (type A). Type A neuroblasts migrate along the rostral migratory stream to reach the olfactory bulb (OB) where they then migrate radially and differentiate into granule and periglomerular cells (Laywell et al., 2000) In the developing brain, the embryonic NSCs, which are radial glia, reside in the ventricular zone directly contacting both the pial and v entricular surfaces. On the other hand, the neural stem cells (type B) in the SVZ of adult brain derived from radial glia are separated from the anterolateral walls of the adult lateral ventricle by a single layer of ependymal cells. Recently, it has been shown that the SVZ neural stem cells (type B) have an apical ending which directly contacts with the ventricle and a basal process ending on blood vessels (Mirzadeh et al., 2008) Moreover, the NSCs contacting blood vessels lack astrocyte endfeet, and pericyte coverage (Tavazoie et al., 2008) This modified blood brain barrier structure exposes SVZ stem cells to various signals from the vascular system. The SGZ niche has two precursor cell populations: Infrequently dividing, GFAP+/Sox2+ radial NSCs (type 1); and mitoticaly active and Sox2+ nonradial NSCs (type 2). A subpopulation of type 2 cells have shown to self renew and to give rise to a neuron and an astrocyte revealing their stem cell properties (Suh et al., 2007) The

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17 neuroblasts derived from type 2 cells migrate into the granule cell layer (GCL) and mature into neurons. Especially, the SVZ has been shown to have the largest population of proliferating cell s in the adult brain of rodents, monkeys and humans (Altman, 1963; Lois & Alvarez Buylla, 1994; Gould et al., 2001; van Praag et al., 2002; Ramirez Amaya et al., 2006; Zhao et al., 2006) In the mouse SVZ the estima ted number of cells generated bilaterally is about 30,000/day (Alvarez Buylla et al., 2002; Abrous et al., 2005) Neural Stem Cell Functioning It is now clear that neurogenic regions in the adult mammalian brain co ntinue to produce new neurons throughout life. The newly generated neurons arise from the neural progenitor cells that reside in the subventricular zone and in the subgranular zone of the dentate gyrus. Neurogenesis in dentate gyrus has been shown to be m odulated by physical activity, seizures and aging (Lugert et al.; Fabel & Kempermann, 2008) There is evidence that hippocampal neurogenesis is sensitive to environmental insults leading to impairments in synaptic p lasticity, learning and cognition (Kempermann et al., 1997; Kempermann & Gage, 1999; van Praag et al., 1999; Kronenberg et al., 2003; Steiner et al., 2008; Zhao & Overstreet Wadiche, 2008; Petrus et al., 2009) In a ddition to hippocampal neurogenesis, it is shown that an odor enriched environment enhances neurogenesis from SVZ, and improves olfactory memory without upregulating hippocampal neurogenesis (Rochefort et al., 2002; Alonso et al., 2006; Lledo et al., 2006; Alonso et al., 2008) Moreover, in neural cell adhesion molecule (NCAM) deficient mice, where the migration of OB neuron precursors is reduced, the discrimination between odors is shown to be impaired (Gheusi et al., 2000; Gheusi & Rochefort,

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18 2002) Furthermore, stroke induced neurogenesis in SVZ and SGZ has been shown both in experimental animals and human patients (Ekonomou et al.; Li u et al., 1998; Kee et al., 2001; Jin et al., 2004; Wang et al., 2005; Jin et al., 2006; Macas et al., 2006; Minger et al., 2007) In vitro models of Neurogenesis Understanding the biological features of neural stem cells and their progeny is one of the main goals in the field of neuroscience. The dogma that there are not new neurons produced in the adult mammalian brain has persisted for more than a century mainly because of the lack of methods to demonstrate neural stem cells which are capable of genera ting new cells. In 1959, Sidman et al. had introduced the [ 3 H] thymidine autoradiography method to label the DNA of dividing cells (Sidman et al. 1959) Using this technique, Smart showed the production of new neurons in the postnatal mouse brain for the first time. On the other hand, he was not able to prove the generation of new neurons in the adult brain. In 1962, Altman had published his first article showing new neuron formation in the adult rat brain (Altman, 1962) However his series of publications reporting [ 3 H] thymidine labeled cells in dentate gyrus (Altman & Das, 1965) neocortex (Altman, 1963; Altman & Das, 1966) and olfactory bulb (Altman, 1969) of adult rats had been largely unappreciated (Ming & Song, 2005) In 1977, Kaplan and Hinds showed ultrastructural evidence for new neurons in dentate gyrus and olfactory bulb of adult rats by using electron microscopy in addition to [ 3 H] th ymidine technique for the first time (Kaplan & Hinds, 1977) In 1982, the introduction of 5 bromo deoxyuridine (bromodeoxyuridine, BrdU) labeling became a very important development to prove neurogenesis in the adult brain. BrdU is synthetic thymidine analogue which

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1 9 incorporates into DNA during S phase of mitosis an d labels proliferating cells and their progeny. Since BrdU labeling can be detected by immunocytochemical methods, it makes identification of newly generated cells with their cell specific markers possible. Finally, in 1992, Reynolds and Weiss had first i solated neural stem and progenitor cells from adult rodent CNS and expanded them in culture conditions (Reynolds & Weiss, 1992) In addition, in 1999, Kukekov and colleagues showed that adult human brain also has neurogenic cells that can produce neuronal and glial progeny under certain in vitro growth conditions (Kukekov et al. 1999) The isolation and in vitro analysis of neurogenic cells from the adult brain allows characterization of mechanisms of the function and regulation of neurogenic stem/progenitor cells. A stem cell is currently defined as an undifferentiated cell that exhibits the ability to proliferate, to self renew, and to differentiate into multiple and distinct lineages. On the other hand, progenitor cells are mitotic cells with faster dividing cell cycle that maintain the a bility to proliferate and to give rise to terminally differentiated cells but are not capable of indefinite self renewal. Currently, the neural stem and progenitor systems isolated from SVZ can be cultured and maintained in vitro as monolayers of multipot ent astrocytic stem cells (MASC) and aggregates of clonal stem/progenitor cells as known as neurospheres (NS). In addition, recently developed neural colony forming cell assay (NCFCA) allows us to distinguish neural stem and progenitor cells on the basis o f their proliferative potential (Louis et al. 2008) These in vitro model systems for the isolation, expansion and differentiation of NSCs provide understanding of the biology of adult stem cells.

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20 MASCs can be isolated from dissociated SVZ tissue and form a monolayer after culture on adhesive substrate such as laminin in the presence of EGF, FGF and serum. These highly proliferative neurogenic astrocyte cultures consisting of astrocytes, neurons and microglia can be expanded for >75 population doublings (Scheffler et al. 2005; Marshall et al. 2008) Furthermore, the withdrawal of mitogens, EGF and FGF, and serum induces differentiation resulting in a rapid generation of B III tubulin expressing neuroblasts. In additio n, when cultured on nonadhesive surface in the presence of EGF and FGF, MASCs can generate multipotent neurospheres. Neurosphere culture is another in vitro cell culture system we use. Neural stem cells isolated from SVZ can be maintained in a serum free medium supplemented with mitogens EGF, FGF and heparin. The spherical aggregate of clonal cells, called neurospheres, display the stem cell features of multipotency, serial expansion and self renewal. The neurosphere assay is the most frequently adopted te chnique to isolate and investigate the biology of neural stem cells (Reynolds & Rietze, 2005) This method is shown to overestimate stem cell number since not all of the neurospheres are derived from stem cells. Indeed, it is sh own that neurospheres are heterogenous including stem cells, proliferating neural progenitor cells, postmitotic neurons and glia (Suslov et al. 2002) However another cell culture system, neural colony forming cell assay (NCFCA), allows us to distinguish stem and progenitor cells on the basis of their proliferative potential. A single cell suspension isolated from SVZ or neurospheres can be cultured on nonadhesive surface, in serum free, semi solid collagen media su pplemented with mitogens, EGF, FGF and heparin. Clonally derived colonies are formed based on the

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21 potential form the largest sized colonies (>2mm in diameter) while pro genitor cells with low proliferative potential form smaller colonies (<2mm in diameter). Altogether, these in vitro cell culture systems allow us to model postnatal/adult neurogenesis and to monitor the molecular and cellular features of stem cells. A zido thymidine Background Azidothymidine (3` azido 3` deoxythymidine (AZT); zidovudine (ZDV); Retrovir formerly BW A509U ) is a synthetic analogue of thymidine in which the 3 hydroxyl group is replaced by an azido group ( Figure 1 1 ). AZT was first synthesized in 1964 against oncoviruses however it was shown to be ineffective as an antineoplastic drug (Dube & Ostertag, 1991) Ten years later, it was sho wn that AZT inhibits virus replication in a mouse retrovirus culture system (Ostertag et al. 1974) Later in 1985, Mitsuya et al. showed that triphosphorylated AZT inhibits the reverse transcriptase of HTLV III/LAV (human T lymphotrophic virus type III/ lymphadenopathy associated virus) later HIV in H9 cells, and suggested the development of AZT as a potential treatment for HIV infection in humans (Mitsuya et al. 1985) In 1986, Ruprecht et al. showed that AZT treatment of mice with Rauscher murine leukemia virous complex (RLV) suppresses viraemia and prolongs lifespan (Ruprecht et al. 1986) Furman et al. showed that AZT triphosphate inhibits the purified HIV Reverse Transcriptase about 100 times m ore effectively than it inhibits cellular lymphocytes (H9 cells) in 1986. In addition, half inhibitory dose (IC 50 ) for cellular replication is 10,000 times higher than the IC 50 for HIV replication (Furman et al. 1986) After the first clinical trial performed in 1986 (Yarchoan et al. 1986) Fishl et al. suggested that AZT could be safely administered to HIV (+) patients, and it could

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22 prolong the life of patients with AIDS (Fischl et al. 1987) Finally, in 1987 the Food and Drug Administration approved AZT for use against HIV AIDS. Since then, AZT has been used in the treatment and prevention of human immunodeficiency virus 1 (HIV 1) infection alone or in combination wi th other antiviral agents as an integral part of the Highly Active Antiretroviral Therapy (HAART) protocols, and human T cell lymphotropic virus type I (HTLV) I associated adult T cell leukemia/ lymphoma (Falchetti e t al. 2005) In addition, AZT monotherapy has been recommended for use in pregnancy to reduce vertical transmission of HIV 1 from mother to fetus (Olivero, 2007) even though it is classified in Food and Drug Admin istration (FDA) Pregnancy Category C which states that safety of AZT usage in human pregnancy has not been determined, and it should not be used unless the potential benefit outweighs the potential risk to the fetus. Mechanism of Action As a thymidine an alog, AZT interacts with the same metabolic enzymes as thymidine. The therapeutic activity of AZT depends on the level of its conversion to the active form, AZT triphosphate (AZT TP), by a three step cascade of phosphorylation which can be catalyzed in eit her cytoplasm or the mitochondrion. AZT is first phosphorylated to AZT monophosphate (AZT MP) in a reaction catalyzed by either cytosolic thymidine kinase 1 (TK1) or mitochondrial thymidine kinase 2 (TK2). TK1 is more abundant in cells with rapid mitotic t urnover and its concentration is increased during the S phase (Lewis et al 2003) The phosphorylation of AZT MP to AZT diphosphate (AZT DP) is catalyzed by thymidylate kinase (TMPK). Finally, the phosphorylation of AZT DP to the active form AZT TP is catalyzed by nucleoside diphosphate kinase (NDK). TMPK plays a rate determin ing role in the conversion of

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23 AZT MP to AZT TP. Since the P azido group of AZT MP from adopting the necessary conformational change, AZT MP accumulates in cells exposed to AZT (Lavie et al. 1997; Ostermann et al. 2000) This leads a decrease in the catalytic efficacy of the enzyme and depletion of thymidine triphosphate (T TP) levels, giving AZT TP a competitive advantage over T TP for the incorporation into the growing HI V DNA by HIV reverse transcriptase (Bradshaw et al. 2005) After incorporation into the proviral DNA, the AZT TP terminates the formation of DNA chain, and reverse transcription starts from the beginning following the release of incomplete DNA. Therapeutic Usage in Pregn ancy At the end of 2008, 33.4 million people worldwide were estimated to be living with HIV infection. About 2.7 million of adults and children are newly infected and 2 million adults and children died due to AIDS. At the end of 2009 there are an estimate d 2.1 million children living with HIV, most of who were infected by their mothers. The current case number of HIV 1/AIDS infected pregnant women a year in United State is about 7000 (Olivero, 2008) It is estimated that approximately 25% to 48% of infants born to HIV infected mothers may become HIV 1 infected through breastfeeding (Mbori Ngacha et al. 2001; Thior et al. 2006) In 1994, the AIDS Clinical Trials Group Protocol 076 (ACTG 076) study established that AZT monotherapy plays a beneficial role in reducing mother to child transmission of HIV (Connor et al. 1994) In a nonbreastfeeding population, AZT monotherapy was administered at 14 34 weeks of gestation (600mg/day), continuous intravenous infusion during labor (2mg/kg loading dose followed by 1/mg/kg/h) and 6 weeks of oral dosing to the newborn reduced the mother to child viral transmission rate from 25.5% to 8.3%. Since then, AZT has been used in

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24 the prevention of mother to child transmission of HIV 1 and it remains the only licensed antiretroviral for use during pr egnancy even AZT is categorized in FDA Pregnancy C (Walker et al. 2007; Durand Gasselin et al. 2008; Read et al. 2008; Foster et al. 2009) In the absence of prenatal treatment, 5 10% of infants born to HIV infe cted women are infected across the placenta 10 20% of infants are infected from exposure at the time of delivery and 5 20% of infants are infected through breast feeding which is responsible of 50% of HIV infections in children in Africa (Wade et al. 1998; De Cock et al. 2000) AZT is metabolized by the liver through glucuronidation resulting in bioavailability about 63%. Its plasma peak concentration is achieved in 1h and its half life is about 1.1 h in non preg nant adults. It is shown that AZT crosses the placenta rapidly with similar concentrations in maternal plasma amniotic fluid and cord blood plasma .The AZT monotherapy regimen is shown to result in an average plasma concentration of 0.82 (Capparelli et al. 2005) Busidan et al. have shown that after a single dose of 150 mg/kg AZT administration to E20 pregnant rats and P20 pups, the distribution of AZT in fetus brain is heterogeneous with relatively greater amounts of AZT in the periventricular area. On the other hand, there was less exposure of the brain in t he P20 pups than at E20 fetuses. They hypothesized that in the E20 fetuses, the efflux transporter of AZT has either not developed or is not yet efficient at removing AZT from the brain (Busidan et al. 2001) In addition to the perinatal genotoxicity fi ndings, there have been neurobehavioral studies focusing on the possible toxic effects of AZT administration during development.

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25 Prenatal AZT exposure has been shown to interfere with CNS development and have a long term neurobehavioral consequences such a s impaired locomotor activity, deficit in learning and spatial tasks, deficits in social, agonistic and investigative behavior, and long term functional alterations within sensorimotor reflexes (Calamandrei et al. 1999b; Rondinini et al. 1999; Venerosi et al. 2000; Ricceri et al. 2001; Calamandrei et al. 2002b; Venerosi et al. 2003; Levin et al. 2004; Melnick et al. 2005; Venerosi et al. 2005) Since AZT has been shown to distribute to the CNS of a develop ing brain, it is important to examine neurogenic potential of SVZ and dentate gyrus stem/progenitor cells which may be particularly vulnerable to the toxic effects of AZT due to these regions anatomical proximity to the ventricular surfaces. Dosage and Adm inistration In the present day, for human adults the total recommended daily amount of AZT is 600 mg, which is about 10mg/kg, resulting in a steady state serum AZT concentration of 0.8M (Fletcher et al. 2002) Wh 3 hours, its active TP) intracellular half life is 7 hours. The levels of AZT in brain tissue have been shown to be IC 50 (0.003 (Cook et al. 2005) Mo reover, AZT monotherapy at 600mg/day dose is administered at 14 34 weeks of gestation, continuous intravenous infusion during labor (2mg/kg loading dose followed by 1/mg/kg/h) and 6 weeks of oral dosing to the newborn results in an average plasma concentr ation of 0.82 (Capparelli et al. 2005) For human neonate, the recommended dose o f AZT is 2 mg/kg every 6 hr (8 mg/kg/day) (Wi tt et al. 2004) AZT is between 24 mg/kg/day and 600mg/kg/day as shown in Table 1 Alternatively,

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26 dosing for AZT can be based on body surface area for pediatric patients. The recommended ora l dose is 480mg/m 2 /day. In order to equate mouse and human doses, we used mg/m 2 conversion factors AZT/day, administered to human with 1.1710m 2 body surface area would be equal to 133 mg/kg dose administered to mouse with 0.007m 2 body surface area. In addition, AZT concentrations at 500 to 1500 mg/day correspond to 20 to 60 M of AZT (Brown et al. 2003) Toxicity Mechanisms is proven in controlling viral infection in adult patients and in reducing vertical viral transmission, several studies showed adverse effects of AZT such as bone marrow suppression, pancytopenia, anemia, macrocytosis, cardiomyopathy, hepatic steatosis, f atal lactic acidosis, myopathy, peripheral neuropathy, distal symmetrical neuropathy, and carcinogenicity (Ayers et al. 1996; Chow et al. 1997; Zhang et al. 1998; Diwan et al. 1999; Anderson et al. 2003; Lee et al. 2003; Lewis et al. 2003; Lai et al. 2004; Lewis et al. 2004; Torres et al. 2007) It is difficult to distinguish the toxicity of AZT from that due to HIV infection in patients receiving HAART. Ippolito et al. examined the short term toxicity of AZT given as prophylaxis to HIV exposed healthcare workers. They showed that 49% of 674 healthcare workers given 300 to 3000mg/day AZT had at least one adverse effect, and 20% of these discontinued prophylaxis because of side effects. In addition, they fou nd that all side effects were frequent, mild, dose related and reversible after the prophylaxis was stopped (Ippolito & Puro, 1997) Finally, it was shown that pathologies su ch as myopathies, cardiomyopathy, hepatotoxicity usually resolve when AZT is removed from

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27 symptoms of AIDS (Lynx & McKee, 2006) The exact mechanism by which AZT causes toxicity is not know n. The inhibition of mitochondrial DNA (mtDNA) polymerase gamma (pol mechanism for AZT related adverse effects due to the fact that the strongest interaction of AZT with the host polymerases is with mtDNA pol suggesting that the inhibition of mtDNA pol mitochondrial dysfunction, the mitochondrial dysfunction hypothesis includes the p athophysiological results of mtDNA mutations and mitochondrial stress. AZT has been shown to decrease mtDNA levels both in vivo in experimental animals and clinically in humans (Lewis et al. 1992) AZT MP accumulates at high concentrations intracellularly because of the fact that AZT MP prevents th e conformational change in the TMPK which also catalyzes TMP. As a result, compared to the phosphorylation rate of TMP, the phosphorylation rate of AZT MP to AZT DP by TMPK is about 60 fold low (Papadopulos Eleopulos et al. 1995; Brundiers et al. 1999; Ostermann et al. 2000) While 94% of AZT metabolites are AZT MP, the concentration of active AZT TP and AZT DP consists only 6% of the metabolites (Lavie & Konrad, 2004; von K leist & Huisinga, 2009) The depletion of TTP levels caused by AZT MP and competitive inhibition of TTP by AZT TP are generally accepted factors leading to toxicity (Samuels, 2006; von Kleist & Huisinga, 2009) Ho wever, studies have shown that adverse events are likely caused by mechanisms other than the inhibition of mtDNA pol TP is a poor substrate for mtDNA pol

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28 AZT TP has never been detected at a concentration high enoug h to inhibit mtDNA pol 50 of AZT TP was found over 100M, AZT was shown to inhibit thymidine phosphorylation with IC 50 ranging from 4.4 to 21.9M in vitro (Lynx et al. 2006; Lynx et al. 2008) This sug gests that AZT is a more potent inhibitor of thymidine phosphorylation than AZT TP is of mtDNA pol inhibition of phosphorylating enzymes, resulting in the reduction of the intracellular TTP pools and indirect inhibition of mtDNA causing mitochondrial toxicity (Cihlar & Ray; Scruggs & Dirks Naylor, 2008) Moreover, AZT MP has been shown to inhibit human mtDNA pol activity in vitro Free AZT MP or terminally incorporate d AZT binds readily in the exonuclease active site, preventing the efficient catalysis. Inhibition of mtDNA pol proofreading by monophosphates and inactivation of exonuclease activity results in defective mitochondrial DNA replication, increase in mutati ons within mtDNA and altered mitochondrial ultrastructure (Lewis et al. 2 003) In addition, it is suggested that the indirect depletion of mtDNA leads to impaired function of the electron transport chain causing an increase in production of reactive oxygen species (ROS) and oxidative damage (Modica Napolitano, 1993; Cazzalini et al. 2001; Yamaguchi et al. 2002; Scruggs & Dirks Naylor, 2008) In addition, it has been shown that AZT is selectively incorporated into telomeres, acting as a telomerase inhibitor and causes telomere shortenin g (Olivero & Poirier, 1993; Olivero et al. 1997; Gomez et al. 1998; Olivero et al. 2002; Caporaso et al. 2003; Liu et al. 2007; Zhou et al. 2007) Long term AZT treatment is shown to induce shortening of the t elomeres in HeLa cell line, in mice exposed in utero and inhibits cell

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29 proliferation with senescent like phenotype induction in cultured mouse fibroblasts. Inhibition of telomerase has been proposed as the mechanism underlying AZT induced telomeric short ening (Strahl & Blackburn, 1994; 1996; Falchetti et al. 2005) It was shown that AZT leads to intracellular accumulation of AZT MP and inhibition of telomerase, which correlate with inhibition of cell proliferatio n and with AZT induced apoptotic cell death in vitro (Falchetti et al. 2005) Finally, Haik et al. showed that AZT treatment markedly resulted in a dose dependent inhibition of FGF2 induced NPC proliferation associated with a decrease of telomerase activity (Haik et al. 2000) It is established that AZT becomes incorporated into nuclear DNA and mtDNA in place of thymidine and induces cell cycle arrest with accumulation of cells in S phase (Chandrasekaran et al. 1995; Olivero et al. 2005 ; Escobar et al. 2007; Olivero et al. 2008) Mechanisms involved in AZT induced cell cycle arrest could be related to the potential of AZT to inhibit cell polymerases or to directly target proteins controlling the cell cycle and DNA repair mechanisms (Sussman et al. 1999; Escobar et al. 2007; Olivero, 2007) Indeed, it is shown that AZT induces cell cycle delay and decrease in cell proliferation in vitro with upregulation of Cyclin D1, accompanied by down regula tion of Cyclin D1 associated inhibitors P18, P57, G1 S checkpoint gene P21. Moreover, Cyclin A2 was shown to downregulated in cells exposed to AZT, suggesting a block in S G2 M progression which is consistent with the accumulation of cells in S phase (Olivero, 2007) The ability of AZT to incorporate preferentially into DNA increases the potential for genomic instability which may lead to formation of chromatin bridges and micronuclei (Oliv ero, 2007) Indeed, AZT has been shown to induce mutations, micronuclei

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30 formation, chromosomal aberrations, sister chromatid exchange and telomeric attrition in vivo and in vitro (Gonzalez Cid & Larripa, 1994; Olive ro et al. 1994; Stern et al. 1994; Ayers et al. 1996; Dertinger et al. 1996; Agarwal & Olivero, 1997; Olivero et al. 1997; Diwan et al. 1999; Sussman et al. 1999; Meng et al. 2000; Olivero et al. 2002; Poirier et al. 2003; Von Tungeln et al. 200 4; Olivero, 2007) Moreover, it is reported that AZT acts as a centrosome disruptor with additional abnormalities in tubulin polymerization (Borojerdi et al. 2009) Furthermore, i n short term in vitro incubations, AZT has been demonstrated to have anti proliferation effects in mammalian cells (Olivero et al. 2005; Fang et al. 2009) The inhibitory effect of AZT on cell growth is shown to be associated with a combination of factors, including the induction of apoptosis, the inhibition of telomerase activity, and S and G2 M phase cell cycle arrest (Fang & Beland, 2009) I t is possible that synergistic combinations of these mechanisms m ight exert the clinical side effects of AZT therapy. Distribution of AZT in the CNS endothelial cells creating a barrier between the blood and the brain parenchyma with tight junctio ns and blood cerebrospinal fluid (CSF) barrier (BCSFB), formed by blood vessels within choroid plexuses located in the lateral, third and fourth ventricles play an important role in distribution of AZT to the brain. It has been shown that AZT can pass thro ugh the BBB and BCSFB (Thomas & Segal, 1997; Kearney & Aweeka, 1999; Cysique et al., 2004; Evers et al., 2004; Letendre et al., 2004) penetration to the brain is limited with passive diffusio n (Thomas & Segal, 1997) even though it has a small, lipophilic structure. In addition, AZT is shown to be removed from

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31 the bra in via an active probenecid sensitive transport efflux (Dykstra et al., 1993; Takasawa et al., 1997a; Takasawa et al., 1997b) Hence, compared to the other systems the level of AZT in the CNS is low (Wu et al., 1998; Im et al., 2009) CSF to plasma concentration ratios of AZT after iv infusion have been reported in rats (0.15) (Galinsky et al., 199 0) rabbits (0.26) (Wong et al., 1993) and humans 0.5 (Blum et al., 1988; Hong et al., 2001) Furthermore, Busidan et al .have shown that a single dose of radiolabeled AZT penetrated the brain very poorly, except for a notably conspicuous region of periventricular incorporation (Busidan et al., 2001) Besides, Letendre et al. classified AZT into high rank of CNS penetration effectiveness (CPE) (rank 1) based on the chemical features, measured CSF concentrations and effectiveness of AZT in the CNS (Letendre et al., 2008) HIV infection is associated with the disruption of BBB with an increase in the diameter of blood vessels d ue to inflammation of the vessel walls, alterations in the basal lamina, loss of glycoproteins in endothelial cells, endothelial cell apoptosis, and tight junction disruption (Toborek et al., 2005; Guillevin, 2008) As BBB disruption leads the HIV infected cells to enter the brain, it also facilitates the entry of drugs into the CNS (Varatharajan & Thomas, 2009) HIV infection in the CNS leads to the development of asymptomatic neurocognitive impairment, HIV associated mild neurocognitive disorder (MND), and AIDS dementia complex (ADC) or HIV associated dementia (HAD) with impairment in cognitive activity, memory, attention, and motor and behavioral functioning (Anti nori et al., 2007) .Therefore prevention of HIV infection in the CNS is one of the major goals in the field. In order to enhance levels of antiretroviral drugs including AZT in CNS and to

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32 make them more efficient, researchers focus on developing new strate gies such as developing BBB permeable derivatives of antiretroviral drugs and efflux inhibitors, and modulating transporters (Li et al.; Saiyed et al.; Zhivkova & Stankova, 2000; Eilers et al., 2008; Miller et al., 2 008; Quevedo et al., 2008; Im et al., 2009) On the other hand, the potential effects of direct exposure of excessive AZT concentrations and immune response to the toxicity on the CNS are not known. In this study, we aimed to determine whether clinically relevant AZT regimens perturb normal levels of neurogenesis in mouse brain. We suggest that t he relatively superficial location of neural stem cell niches of both SVZ and dentate gyrus with respect to the ventricles might expose these neurogenic niches to harmful levels of AZT from CSF. In addition, t he cellular architecture of SVZ might expose stem cells to various signals including AZT from vascular system. Indeed, perturbed neurogenesis might play a contributing or enhancing role in neurological deficit s seen in HIV (+) patients. In case of BBB permeable AZT administration, we claim that neurogenic niches would be exposed to toxic levels of AZT leading to severe disruption of prenatal and postnatal neurogenesis.

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33 C 10 H 13 N 5 O 4 C 10 H 14 N 2 O 5 Figure 1 1 Structural formula of AZT and Thymidine. 3` azido 3` deoxythymidine (AZT; C 10 H 13 N 5 O 4 ) is a synthetic thymidine ( C 10 H 14 N 2 O 5 ) analogue in which the 3 hydroxyl group is replaced by an azido group (A). A B

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34 Table 1 1 November 6, 2009, the Food and Drug Administration (FDA) approved revised pediatric dosing recommendations Body weight (kg) Total Daily Dose 4 to <9 24 mg/kg/day 18 mg/kg/day 600 mg/day

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35 CHAPTER 2 MATERIALS AND M ETHODS Generation and Expansion of Astrocyte Monolayer Cell Cultures Following decapitation, the SVZ tissue surrounding the lateral ventricles was dissected from C57BL/6 neonatal ( P ostnatal D ay 2 4) mice brains using a sterile razor blade. The tissue was then minced and placed in ice cold DMEM/F12 medium with N2 supplements (Gibco BRL, 17502 048), (N2 Media), containing 1X antibiotic antimycotic (100X, Invitrogen, 15240 062) for 15 minutes. After centrifugation at 400xg for 5 minutes, the tissu e was incubated in 0.25% Trypsin/EDTA solution (Atlanta Biologicals; B81310) for 5 7 minutes in 37 0 C water bath. Trypsin activity was inhibited by addition of N2 media containing 5% fetal bovine serum (FBS; Atlanta Biologicals). The tissue was triturated t o single cell slurry by using fire polished Pasteur pipettes. Cells were washed in N2 media and pelleted by centrifugation at 400xg for 5 minutes, and re suspended in neural growth medium consisting of N2 media containing 5% FBS, recombinant human epiderma l growth factor at a concentration of 20ng/ml (rhEGF, Sigma Aldrich, St. Louis, MO; E9644), and basic fibroblast growth factor at a concentration of 10ng/ml (bFGF, Sigma Aldrich, F0291). The single cell suspension was plated onto tissue culture T 25 flask s and incubated at 37C in 5% CO2. After two days of incubation, the neural growth medium was refreshed, and cells were supplemented every other day with EGF and FGF until the primary passage of monolayer cell culture reached confluence. Inducible Neuroge nesis Confluent primary astrocyte monolayer cells were passaged at a density of 17,500 cells/ cm 2 and supplemented every other day with EGF and bFGF as above for 7 10 days until confluency was established. To induce differentiation of Passage 1 astrocyte

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36 m onolayer culture, cells at a density of 17,500 cells/ cm 2 were plated onto poly L ornithine (10g/ml, Sigma, P4957) coated glass coverslips in 12 well plastic plates in neural growth medium (1ml in volume), and were supplemented every other day with 20ng/m l EGF and 10ng/ml bFGF. Four days later, the growth medium was withdrawn and replaced with an equivalent volume of N2 medium (without serum or growth factors) to induce formation of neuroblast cells. 48 hours after withdrawal of neural growth medium, cells were either fixed with 4% paraformaldehyde for subsequent immunocytochemical analysis, or quantified with a Z2 Coulter Counter (Beckman Coulter, Fullerton, CA). Immunocytochemical Analysis Fixed cells were prepared for immunocytochemistry by washing with phosphate buffered saline (PBS) and blocking at room temperature for 30 60 minutes in PBS containing 0.01% Triton X 100 (PBSt) and 10% FBS. Primary antibodies were applied overnight in PBSt containing 10% (how many times) FBS with moderate agitation at 4 C. Residual primary antibody was removed by washing with PBS (how many times) and secondary antibodies were applied at room temperature for one hour in PBSt containing 10% FBS. Residual secondary antibodies were removed by washing with PBS. For nuclear st aining, the coverslips were mounted onto glass slides and layered with Vectashield mounting medium containing 4', 6 diamidino 2 phenylindole (DAPI) (H 1200) prior to cover slipping. Coverslips were analyzed and photographed by using a Leica DMLB upright ep ifluorescence microscope (Leica Microsystems AG, Wetzlar, Germany) with a Spot RT color CCD camera (Diagnostic Instruments). For quantification of stained cells, a minimum of 10 randomized fields were selected at 20X magnification.

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37 Neurosphere Culture Sin gle cell dissociates from SVZ ( P ostnatal D ay 2 4) were cultured in non adherent flasks at clonal density (10,000 cells/cm 2 ) in NeuroCult NSC Proliferation Medium (Mouse), consisting of NeuroCult NSC Basal Medium and NeuroCult NSC Proliferat ion Supplement (Stem Cell Technologies, 05700 and 05701) supplemented with growth factors EGF and FGF at 20g/ml and 10g/ml respectively and heparin (2g/ml, Stem Cell Technologies, 07980). After 7 10 days, the number and size of neurospheres (NS) were as sessed and classified based on diameter (40m, 40 80m, >80m) using Spot Advanced digital capture software. Neural Colony Forming Cell (NCFC) Assay A single cell suspension from Postnatal Day 2 4 SVZ (obtained as described above for neurosp here cultures) was plated in 35mm culture dishes at low density in a serum free, semi solid collagen media containing NeuroCult NCFC serum free medium without cytokines (Stem Cell Technologies, 05720), NeuroCult Proliferation Supplement, hEGF (20g/ml) hbFGF (10g/ml), heparin (2g/l) for 3 weeks. Cultures were added with Complete Replenishment Medium consisting of NSC Basal Medium, NSC Proliferation Medium, hEGF (20g/ml), hbFGF (10g/ml), heparin (2g/l) once a week. By day 21 28 colonies were classifi ed into four categories based on diamete r (<0.5mm, 0.5 mm, 1 magnification. In Vitro Drug Treatment AZT (TCI America, A2052) was dissolved in N2 medium, filtered through 0.22m mesh, and stored in ready to use aliquots at 200C. AZT was added to the groups of cultures at 0 60M concentration which corresponds to the range of doses administered

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38 in human patients. Exposure times ranged from 2 hours to 48 hours, after which the medium was replaced with fresh medium without AZT. Control and treate d cultures received the same number of medium changes. TUNEL Assay Apoptotic cells were labeled by using fluorimetric terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay (DeadEnd Fluoremetric TUNEL System; Promega, G3250) a ccording to the manufacturer's recommendations. This assay measures the fragmented DNA of apoptotic cells by incorporating fluorescein OH ends of DNA strands. Briefly, astrocyte monolayer cells attached onto the coverslips were fixe d with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed 2x5 minutes with PBS and permeabilized by using 0.2% Triton X 100 solution in PBS for 5 minutes. After cells treatment with equilibration buffer for 10 minutes, cells were inc ubated within rTdT incubation buffer at 370C for 60 minutes in a humidified chamber. Reactions were terminated by using 2X SSC for 15 minutes. After being washed 3x5 minutes with PBS, cells were counterstained with Vectashield + DAPI (H1200, Vector) and pe rcentage of TUNEL+ apoptotic cells was calculated by assessing 10 random fields of triplicate samples. Senescence Galactosidase Labeling X Gal cytochemical staining at pH 6.0 was performed as described (Dimri et al., 1995). Briefly, cells were fixed for 5 minutes in 0.2% glutaraldehyde in PBS. After two washes with PBS, cells were incubated in 5 bromo 4 chloro 3 indolyl B Dgalactoside (X Gal), 40mM sodium citrate pH 6.0, 5% dimethylformamide, 5% potassium ferrocyanide, 5 mM ferricyanide, 150mM sodium

PAGE 39

39 chloride and 2 mM magnesium chloride for 6 hours at 37C. Cells were washed with PBS, and counterstained with Vectashield + DAPI. The percentage of positive blue JC 1 Assay The integrity of the inner mitochondrial membrane was determ ined by measuring the potential gradient across the mitochondrial membrane using the fluorescent stain, JC To determine whether AZT exposure causes changes in mitochondrial membrane potential, astrocyte monolayer cells received a 48 hour pulse of 0 60M AZT at the time of serum and mitogen withdrawal, and mitochondrial membrane physiology was assessed via the JC 1 potentiometric dye. Control and AZT treated astrocyte monolayer cells were suspended in warm medium at 1x10 6 cells/ml. Control cells were treated with CCCP, mitochondrial membrane disrupter, at 37 0 C for 5 minutes. All groups received 2M JC 1 and were incubated at 37 0 C, 5% CO 2 for 30 minutes. Cells were washed and resuspended in 500ul PBS. Samples were t hen analyzed on a flow cytometer with 488nm excitation using the appropriate emission filter for Alexa Fluor 488 dye and R phycoerythrin. Mitochondrial membrane depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. In viv o AZT Administration Adult, male, C57 BL/6 mice (n= 4) received daily i.p. injections of 200ul of 0.9% saline containing AZT at 0, 1, 10, 20 and 100mg/kg/day for two weeks. On the day following the last AZT injection, all animals received three BrdU injec tions (100mg/kg) every two hours. In order to assess immediate neurogenesis versus survival of new neurons, animals were sacrificed after two post BrdU survival times, 1 week and 4

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40 weeks. The brains were processed for combined BrdU/NeuN immunolabeling on a 1 in 6 series of sagittal sections, and BrdU+ cells within the dentate gyrus and SVZ were counted in a single focal plane. In utero AZT Administration C57BL/6 pregnant mice (n=4) received two subcutaneous injections of 200ul of 0.9% saline containing 0 and 250mg/kg/day AZT during the last 7 days of gestation (E12 E18; final 37% of gestation period). Pups were exposed to AZT via nursing for 3 days after birth. Astrocyte monolayers and neurospheres were generated from the SVZ of litters exposed to AZT. I n addition, P3 brains were processed for paraffin embedding. Briefly brains were fixed with % paraformaldehyde and placed in embedding cassettes. After the dehydration process, the tissue was cut and mounted on glass slides to be analyzed for Ki67 immunola beling. Immunohistochemistry The adult animals were transcardially perfused with 4% paraformaldehyde. Fixed brains were immersed in 30% sucrose for 24 hours. Using a freezing microtome, the hemispheres were cut through the sagittal plane at 40m, and store d at 20 0 C in a cryoprotectant solution consisting of glycerol and polyethylene glycol. BrdU immunolabeling was performed as previously described (Laywell et al. 2005) Briefly, fixed brain sections were washed i n PBS and incubated in 2xSSC:formamide (1:1) at 65 o C for 2 hours. After a wash in 2xSSC, sections were incubated in 2N HCl at 37 o C for 30 minutes. Finally, sections were rinsed in 0.1M borate buffer at room temperature for 10 minutes and processed for the standard immunofluorescence detection of BrdU with a rat anti BrdU antibody (Abcam, Cambridge, MA, ab6326).

PAGE 41

41 Statistics All analyses were performed with GraphPad Prism 4.02 (San Diego, CA). Data subjected to One way ANOVA with Tukey ple Comparison Test for multiple group comparisons and unpaired T test for two group comparisons. An asterisk (*) indicates significance (p<0.05, **p<0.01, ***p<0.001).

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42 CHAPTER 3 IN VITRO AZIDOTHYMIDINE EXPOSURE REDUCES THE NEUROGENIC POTENTIAL OF NA VE SVZ STEM/PROGENI TOR CELLS Background azido deoxythymidine; AZT) is a synthetic thymidine analog hydroxyl group is replaced with an azido group. Intracellulary, AZT is converted to its active form AZT triphosphate (AZT PPP) which competes with the natural substrate deoxythymidine triphosphate (dTTP) for incorporation by the reverse transcriptase of HIV. Once added to the growing DNA chain, AZT prevents further addition of nucleotides into the replicating strand of D azido group. It has been shown that AZT inhibits HIV reverse transcriptase 100 times more effectively than it inhibits cellular DNA polymerase (Furman et al., 1986) AZT, as a nucleoside reverse transcriptase inhibitor, has been used in the treatment and prevention of human immunodeficiency virus 1 (HIV 1) infection alone or in combination with other antiviral agents as a part of Highly Active Antiviral T herapy (HAART). In addition, AZT monotherapy has been used in pregnancy to reduce vertical transmission of HIV 1 from mother to infant since 1994. reducing vertical vi ral transmission, several studies showed adverse effects of AZT such as bone morrow suppression, cardiomyopathy, hepatotoxicity, neuropathy and mitochondrial damage. Even there is extensive literature on AZT toxicity on different cell types, the possible effects of AZT administration on the neural stem and progenitor cell functioning has not been examined. Here we investigated the hypothesis that AZT exposure perturbs neural stem and progenitor cells in vitro We show that AZT has a strong antiproliferati ve effect on

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43 cultured stem and progenitor cells. Reduced proliferation is concurrent with the onset of a senescent phenotype in AZT treated cells that alters cell morphology, differentiation potential and susceptibility to apoptosis. Results Monolayer of Multipotent Astrocytic Stem Cell (MASC) Culture P rimary MASC was generated from the SVZ tissue surrounding the lateral ventricles of C57BL/6 neonatal ( Postnatal Day 2 4) mice. Confluent cell layers were then dissociated as a single cell suspe nsion and re plated for another series of passage to eliminate postmitotic neurons. The first passage of MASC was dissociated and plated onto poly L ornithine coated glass coverslips in neural growth medium, and was supplemented every other day with mitoge ns (EGF and FGF). Four days later, the growth medium was withdrawn and replaced with an equivalent volume of N2 medium without serum and mitogens to induce formation of neuroblast cells. 48 hours after withdrawal of neural growth medium, cells were fixed w ith 4% paraformaldehyde for immunofluorescence analysis with neuronal B III tubulin marker protein. Here we systematically examined the time course of the growth supplement withdrawal induced neurogenic period. In cultures which are non withdrawn the adhe rent monolayer cells have a homogenous distribution ( Figure 3 1 A, C). Following the first 24 hours of withdrawal of growth medium, a rapid change in monolayer cell pattern, a rosette formation consisting of B III tubulin (+) neuroblast cells, occurs ( Figure 3 1 B, D). While non withdrawn control groups do not show any change in the number of B III tubulin (+) neuroblasts, withdrawn monolayer has a sharp increase of an extens ive production of B III tubulin (+) neuroblast following the first 24 hours ( Figure 3 2 A D). We show that serum and mitogen withdrawal from second passage of monolayers leads

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44 first to a period of relative quiescen ce, with little change in total cell number ( Figure 3 2 C) due to both proliferation and apoptotic cell death ( Figure 3 3 A C ). In addition to the baseline level of SA B Ga l (+) cells in the control groups, serum and mitogen withdrawal causes an increase in the number of SA B Gal (+) cells ( Figure 3 3 D). AZT Reduces Astrocyte Monolayer Population Expansion MASC culture enables us to investigate the chain of events involved in proliferation and differentiation of neurogenic stem/progenitor cells. In order to investigate the possible adverse effects of AZT on neurogenic stem/progenitor cell expansion, primary MASC were treated with 30 M AZT and analyzed for the next three passages. We show that single 7 day pulse AZT exposure of primary astrocyte monolayer cells cause a significant decrease in the expansion potential of the progeny of the next two passages. Third passage of AZT treated primary cells was able to recover its expansion potential ( Figure 3 4 A). On the other hand, a single 7 day pulse of 30M AZT exposed on the nave first, second and third passages of MASC caused a very significant decrease in population expansion of cells. Interestingly, late passages of cells were more vulnerable to be affected by AZT ( Figure 3 4 B). AZT Abolishes Inducible Neurogenesis from Astrocyte Monolayer Cells As ind icated above MASC can be induced to generate large numbers of neuroblasts upon withdrawal of serum and mitogens. To assess the effect of AZT on the model of inducible neurogenesis from MASC, we exposed monolayer with AZT at a clinically relevant dose range at the time of serum and mitogen withdrawal. We show that single 48 hour pulse of 0.3 60M AZT exposed to MASC at the time of withdrawal causes a significant decrease in both total number and %B III tubulin (+) neuroblasts however that effect was more sev ere on B III tubulin (+) cells ( Figure 3 5 A). In order to

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45 examine the minimum duration and dosage of AZT exposure abolishing the inducible neurogenesis, MASC was treated with 24, 8 and 2 hour pulse of 0.03 3M AZT at the time of serum and mitogen withdrawal. We show that even the lowest concentration of AZT, 0.03M, exposed to cells even for only 2 hours at the time of serum and mitogen withdrawal causes a very significant decrease in the % of B III tubulin (+) neu roblast formation. On the other hand, MASC treated with the same concentration range of AZT for three days before supplement withdrawal did not show such a dramatic response ( Figure 3 6 ). AZT Increases Apoptosis in Supplement Withdrawn MASC In order to investigate the possible mechanism causing AZT to abolish inducible neurogenesis, we assessed the activation of cleaved caspase 3 as an early marker in cellular apoptosis and the Terminal Uridine Deoxynucleotidyl T ransferase dUTP Nick End Labeling (TUNEL) assay, which detects DNA fragmentation of late stage apoptotic cells, on MASC exposed to AZT for 48 hours applied at the time of withdrawal. We show that there is a baseline level of cells expressing caspase 3 in w ithdrawn control group while 30M AZT causes a significant increase in the percentage of cells expressing caspase 3 ( Figure 3 7 A). In addition AZT exposure causes a significant increase in the percentage of TUNEL (+) cells compared to the control group ( Figure 3 7 B). Both TUNEL assay and detection of cleaved caspase 3 indicate that AZT exposure causes an slight increase in early and late apoptotic events in serum and mito gen withdrawal induced astrocyte monolayers ( Figure 3 7 ). AZT Upregulates SA B Gal Activity in Supplement Withdrawn MASC Stress induced senescence, which is permanent arrest of cell division, can be induced by ex posure to a variety of factors, such as UV and gamma radiation,

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46 pharmacological agents, and oxidative stress; and is characterized by DNA damage. Senescent cells express high levels of lysosomal B galactosidase enzyme at pH 6.0. d B B Gal activity has been used as a biomarker to detect senescent cells in vitro and in vivo In order to examine if AZT, as a genotoxic agent, is causing any increase in senescent associated B galactosidase activity, we exposed MASC wi th single pulse of 0.3M and 30M AZT for 48 hours starting at the time of serum and mitogen withdrawal. Our data show that there is a baseline level of senescent cells in controls withdrawn MASC. In addition, AZT exposure causes an increase the number of SA B Gal (+) cells in a concentration and exposure time dependent manner ( Figure 3 8 ). In order to understand whether AZT induced SA B Gal activity is due to mitochondrial damage, we performed JC 1 assay. MASCs w ere exposed to single pulse of 3M and 60M AZT following serum and mitogen withdrawal. The control cells were treated with CCCP (carbonyl cyanide 3 chlorophenylhydrazone), a mitochondrial membrane disrupter, as a positive control for depolarization. 48 ho urs later, mitochondrial membrane physiology was assessed via the JC 1 potentiometric cationic dye which exhibits membrane potential dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529nm) to red (~590nm). Our data indicates that AZT exposure does not perturb mitochondrial membrane polarization of serum and mitogen withdrawn MASC ( Figure 3 9 ). AZT Changes the Size and Morphology of Neurospheres Neurosphere assay is anot her in vitro cell culture system that enables us to investigate the possible effects of AZT on neurogenic stem and progenitor cells. Single cell suspensions from primary postnatal SVZ regions plated in the serum free media

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47 containing EGF, FGF and heparin g ive rise to neurospheres within 7 10 days. In order to examine whether AZT affects the expansion potential of neurosphere forming cells, one day following the single cell suspension incubation, cells are treated with a pulse of 0.3M and 30M AZT for 1, 3 and 10 days. At the end of exposure time, cell culture media is refreshed, and the neurosphere size and number is quantified on day 10. We show that AZT does not inhibit neurosphere formation but perturbs the formation of neurospheres Figure 3 10 ). Our data reveal that 1, 3, and 10 day long AZT exposure of neurosphere forming cells causes a significant reduction in neurosphere size both in concentration and exposure time dependent manner while there is no difference in total number of neurospheres between control and AZT treated groups ( Figure 3 11 ). AZT Increases the Proportion of Senescent Neurosphere Cells To determine whether AZT exposure causes an increase in senescenc e associated SA B Gal activity in neurosphere forming cells, we treated neurosphere forming cells with a single pulse of 30M AZT following the day of single cell suspension incubation. Neurospheres grown in 30M AZT were dissociated 7 days after initial c ell culture and cytospinned onto glass slides and stained to detect SA B Gal activity. Quantification of SA B Gal (+) neurosphere cells reveal that AZT exposure increases the number of SA B Gal stained cells compared to the control group as in MASC culture ( Figure 3 12 ). AZT Perturbs Formation of Neural Colonies Derived From SVZ Stem and Progenitor Cells Neural Colony Forming Cell Assay allows us to quantify Neural Stem Cell (NSC) and Neural Progenitor Cell (NPC) f requency in certain cell culture conditions. With high

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48 progenitor cells which lack self renewal ability and multipotency form colonies < 2 mm in diameter. In order to examine whether AZT affects either stem or progenitor cells or both, we exposed dissociated primary neurosphere cells, which give rise to neural colonies in certain cell culture conditions, to single pulse of 0.3M and 30M AZT. After 21 days in culture, colonies were classified into four categories based on the diameter. Our data show that AZT exposure causes a concentration dependent decrease in formation of neural colonies <2mm in diameter, derived from progenitor cells but not of colonies derived from stem ce lls, >2mm in diameter (A D). Conclusion In order to investigate possible toxic effects of AZT on neurogenic stem/progenitor cells, we employed different in vitro cell culture systems. First, we examined the effect of AZT on stem/progenitor cells of monolay ers consisting of MASC. To do it, we designed an experimental paradigm which gives rise to a monolayer consisting mostly of GFP positive astrocytes but very low number of neurons and microglia. In order to reach the most homogenous monolayer, we tested dif ferent number of cells for initial seeding density of cell culture. The SVZ tissue surrounding the lateral ventricles was isolated and dissociated into single cell suspension and cultured at 17500 cells per cm 2 density and expanded for 7 days. By passaging the primary monolayer of MASC twice, we are able to eliminate postmitotic neurons and create more homogenous cell culture. Consistently, we always used 2 nd on MASC expansion and differentiation potential. Moreover, we modified the protocol of induction of neurogenesis from monolayers of MASC. In our experimental design at the time MASCs reach 60% confluency, the growth supplements FBS, FGF and EGF are withdrawn for 48 hours to induce neurogenic differenti ation from monolayers of MASC.

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49 Here we systematically examined the time course of the growth supplement withdrawal induced neurogenic period. In cultures which are nonwithdrawn, the adherent monolayer cells have a homogenous distribution. While the nonwith drawn MASC has almost a linearly increasing growth curve over 7 days in culture conditions, the level of newly generated B III tubulin (+) neuroblasts within the monolayer is always below 5% of total population. Following the first 24 hours of withdrawal of growth supplements, a rapid change in homogenous monolayer pattern occurs. Rosette like cell clusters become more apparent 48 hours following the withdrawal. By immunolabeling the CD45(+) microglia, GFAP (+) astrocyte, B III tubulin (+) neuroblast, we s how that the rosette like clusters consist of B III tubulin (+) neuroblasts surrounding CD45 (+) microglia (data not shown). In our induction of neurogenesis from monolayer of MASC paradigm, the level of B III tubulin (+) neuroblasts increases up to 45% of total population upon withdrawal of growth supplements. In contrast to the lack of growth supplements, the level of Ki 67 expressing dividing cells increases significantly within the same rosette formation window. In addition to the cell division, the lev els of TUNEL and caspase 3 (+) apoptotic cells and SA B Gal labeled senescent cells increase significantly within the first 48 hour of growth supplement withdrawal. Obviously, the serum and mitogen withdrawal causes significant changes in the monolayer cel l population dynamics. Cell proliferation in addition to the cell death creates a turnover within the monolayer of MASC population. Different cell types seem to respond to the starvation insult in a different way however the downstream signaling mechanisms that are activated upon growth supplement withdrawal are unknown.

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50 In order to examine if AZT affects the expansion potential of monolayer of MASCs, we performed two separate experiments. First, the single cell suspension isolated from SVZ was seeded to g ive rise primary monolayer of MASC. Two days after the initial seeding, the primary MASC was exposed to single pulse AZT at 30uM concentration for 7 days. In order to examine whether AZT has a long term antiproliferative effect on MASC expansion, the expa nsion potential of 1 st 2 nd and 3 rd passages of MASC was examined. Our results show an initial decrease in expansion potential of monolayer of MASC population at 1 st and 2 nd passages however 3 rd passage of monolayer of MASC show recovery. As a result, AZT expansion potential however this effect is temporary. In the second set of experiments, we exposed 1 st 2 nd and 3 rd passages of untreated, nave monolayer of MASCs to the same concentration of AZT. In co ntrast to the 1 st experiment results, we show a dramatic decrease in monolayer of MASC expansion potential which reduces substantially in further passages. The stem/progenitor cells residing in a quiescent state within the primary cell culture might not be affected by AZT because of the fact that increases the number of stem/progenitor cells might decrease within the monolayer so the recovery potential of the cell populati on might also be reduced. In addition, it is a known fact that as the cell culture duration increases, the proliferation and survival potential of cultured cells decreases, they become senescent. Our data showing the baseline level of senescence in nonwith drawn monolayer of MASC supports this notion. In order to examine the potential toxic effect of AZT on the differentiation of MASC, we used our growth supplement withdrawal paradigm to induce neurogenesis. At the

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51 time of 60% confluency of 2 nd passage of m onolayer of MASC the growth supplements were withdrawn and AZT is administered to the cell culture. Following 48 hours of the withdrawal and AZT exposure, the total number of cells and B III tubulin expressing cells is quantified in order to represent the effect of AZT on expansion and differentiation potential of monolayer of MASC. Our data show that AZT dramatically decreases both total cell number and also B III tubulin expressing cell number within the monolayer. However, this effect is more severe on B III tubulin expressing cells. It is expected that in addition to the supplement withdrawal as a starvation insult, AZT exposure would cause a crisis within the cell culture. Even in nonwithdrawn conditions AZT causes a significant decrease in cell populat ion expansion. Here the important III tubulin expressing cells could not be formed. To prove the hypothesis that AZT is selectively toxic to neuronal precursors giving rise to the B III tubulin expressing cells, we analyzed the minimum duration and concentration of AZT leading to the same effect. We examined the clinically relevant AZT concentrations first which is between 20 to 60UM; then we decreased the dosage up to 0.03uM. When one pulse AZT is exposed on monolayer of MASC at the time of growth supplement withdrawal for 8 hours, we show a statistically very significant reduction in the level of B III tubulin expressing cells within the monolayer population caused by the lowe st concentration, 0.03uM, of AZT. Besides, monolayer of MASC exposed to 3uM AZT during the first 2 hours of withdrawal, results in the same response. Given the fact that the total population is not affected by the toxicity of AZT as B III tubulin expressin g neuroblasts is, we claim that AZT specifically disturbs neurogenic cells. The decrease in total cell number of the monolayer could be

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52 explained by disruptions of expansion potential of both neurogenic and non neurogenic cells present in MASC population. In our previous experiments, we showed that AZT exposure results in an increase in apoptotic cell death and senescence in control and growth supplement withdrawn monolayer of MASC. In order to investigate whether AZT exposure leads to a substantial increa se in apoptotic cell death and senescent associated events within the growth supplement withdrawal induced MASC monolayer, we exposed MASC to AZT during the first 48 hours withdrawal induced differentiation. Our results show that, AZT leads a statistically significant increase in capsase 3 (+) and TUNEL (+) apoptotic cell number when exposed at relatively high concentration, 30uM. In addition, we show a statistically very significant increase in the level of SA B Gal labeled cells exposed to a single pulse of 0.3uM and 30uM AZT. Interestingly, compared to the control groups the level of SA B Gal (+) cells within the monolayer is stable within the first 24 hours of withdrawal. At the time we see a sharp increase in the B III tubulin expressing cell level in c ontrol conditions, AZT leads to a sharp increase in the level of SA B Gal labeled cells. It seems like during the first 24 hours following the withdrawal of growth supplements, neurogenic cells within the monolayer of MASC undergo a cell fate decision proc ess during which cells become more vulnerable to the insults like AZT toxicity. In addition to the monolayers of MASC, we examined the effect of AZT on stem/progenitor cells of neurosphere cell culture system. Neurospheres, the spherical aggregates of clo nal stem/progenitor cells, are generated from single cell dissociates of SVZ isolated from neonatal mouse brain when cultured in a serum free media

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53 containing mitogens EGF, FGF and heparin. 10 days following the initial seeding, the individual neurospheres can be identified with smooth and well defined border. When AZT is exposed to the neurosphere forming cell culture, we see a dramatic change in size and morphology of neurospheres. Besides, our data show that AZT exposure leads to a dramatic decrease in s ize of neurospheres in concentration and time dependent manner. On the other hand, this dramatic effect is not seen in neurosphere yield. Together, AZT exposure does affect the expansion potential of neurosphere forming cells however it does not kill them so that the yield stays at the same level compared to the control groups. This antiproliferative effect of AZT seems consistent with a senescence profile. In order to test if AZT upregulates senescence associated events within the neurosphere forming cell culture as seen in monolayers of MASC, we exposed neurosphere forming cells to 30uM AZT and performed SA B Gal labeling. Our data show that AZT significantly increases the level of SA B Gal labeled neurosphere forming cells consistent with monolayers of M ASC data. Moreover, we examined another in vitro cell culture system, the neural colony forming cell assay (NCFCA), in order to investigate if AZT shows a specific toxicity on stem and/or progenitor cells. NCFCA is new cell culture method which enables us to distinguish neural colonies which are derived from neural progenitor or stem cells based on their sizes. The single dissociate of primary neurospheres formed from SVZ dissociates cultured in a semi solid collagen media including the mitogens, EGF, FGF, and heparin. In order to examine if AZT affects primary stem and/or progenitor cells to give rise neural colonies, we exposed the primary neurospheres to a single pulse of 0.3uM and 30uM AZT. AZT treated primary neurospheres were then dissociated and

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54 cult ured in neural colony forming conditions. Our results demonstrate that 30uM AZT exposure of primary cells causes a very significant decrease in the frequency of neural colonies which are derived from neural progenitor cells but not from stem cells. Further more, a direct exposure of single pulse AZT at 0.3uM and 30uM concentrations leads to a very significant decrease in frequency of neural colonies which are derived from neural progenitor cells but not from stem cells. Consistent with our previous data with colonies derived from relatively high dose AZT exposed cells show a reduction however the direct exposure of AZT at low concentration also causes a dramatic effect in colony for ming potential of progenitor cells. Interestingly, AZT has disturbed the formation of colonies derived from neural progenitors only. Altogether, single pulse of AZT at concentrations below the clinically relevant doses leads to a dramatic decrease in expa nsion potential of monolayers of MASC, neurosphere forming cells, and neural colony forming cells. This effect was more severe when AZT is directly exposed to the cells. The primary cells treated with AZT show disruption in expansion potential of further p rogeny only when cells are exposed to relatively higher concentrations of AZT. In addition to the reduced expansion potential, our data show that AZT abolishes neural differentiation. Concurrently, we show an increase in SA B Gal labeling within the AZT ex posed neurogenic cells. Together, we conclude that AZT induces a senescence profile in neural stem/progenitor cells in vitro We already know that AZT is a telomerase inhibitor, causing telomere shortening. There are numerous studies showing that telomere shortening leads cells to enter a replicative senescence. However we should take the short duration of AZT exposure leading a

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55 strong antiproliferative effect on cell expansion and abolishing effect on neural differentiation into consideration. So we claim that telomerase inhibition by AZT exposure is not the reason of the senescent profile. There is evidence that cellular senescence could be induced by physiological stressor factors which can lead to senescence more rapidly independent from telomerase inhi bition and/or telomere shortening. The DNA damage also could lead cells to enter senescence. We know that AZT can incorporate into cellular DNA especially into mitochondrial DNA causing mitochondrial dysfunction. In order to investigate whether AZT causes mitochondrial dysfunction leading neurogenic cells to enter senescence, we performed JC 1 assay however our results show that AZT does not perturb mitochondrial functioning. Moreover, AZT is shown to lead to oxidative stress, cell cycle arrest, impairment in DNA repair mechanisms, which are also senescent leading factors. The synergistic combinations of these mechanisms might exert the senescent profile we observed in our study. Future study is required to analyze the role of these mechanisms on AZT leaded toxicity on neurogenic stem/progenitor cells in vitro Together, these data reveal uncharacterized effects of AZT treatment on stem and progenitor cells. Given the fact that most human HIV (+) patients are treated with AZT over many years, AZT regimens mi ght perturb normal levels of neurogenesis in vivo

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56 Figure 3 1 Serum and mitogen withdrawal induces neurogenesis from SVZ monolayers. Multipotent astrocytic stem cells (MASC) isolated from subventricular z one (SVZ) were plated on an adhesive surface in the presence of serum and mitogens EGF and FGF. Within 7 10 days cell density become confluent (A, D). Upon withdrawal of serum and mitogens, monolayers of MASC were induced to generate large numbers of neuro blasts. 24 hours following induction by serum and mitogen withdrawal, the monolayer generated rosette like cell clusters consisting of B III tubulin expressing neuroblasts (B, D). Representative phase contrast (A, B) and immunofluorescence (C, D) images ar e showing control (A, C) and induced (B, D) astrocyte monolayers. C, D: B III tubulin, red; DAPI, blue. A B C D

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57 Figure 3 2 Serum and mitogen withdrawal leads first to a period of relative quiescence followed by a sharp increa se in neurogenesis. Multipotent astrocytic stem cells (MASC) isolated from subventricular zone (SVZ) were plated on an adhesive surface in the presence of serum and mitogens EGF and FGF. 7 days late r monolayer reached confluency Panel A shows the graphic al representation of MASC expansion for 7 days. At the time of 60 % confluency, on day 5, serum and mitogens were withdrawn from monolayer. Quantification of immunofluorescent staining showed that 48 hours following withdrawal, the % of B III tubulin expres sing neuroblasts within the MASC population increase s dramatically (p<0.001) ( B ). One way Anova, Tukey's Multiple Comparison Test of significance ; N =3 for all groups Error bars represent standard deviation. B A

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58 Figure 3 3 Serum and mitogen withdrawal causes a change in the monolayer cell population dynamics. At the time of 60 % monolayer confluency, serum and mitogens were withdrawn from MASC culture. (A) Quantification of immunofluorescent staining showed that 48 hours following withdrawal (wd), the % of Ki 67(+) cells increased dramatically compared to the control nonwithdrawn (nwd) cells (p<0.001) The level o f TUNEL (+) and Caspase 3 (+) apoptotic cells i s also increased significantly (p<0.001) (B, C) SA B Gal staining showed that withdrawal of serum and mitogens increase s the baseline level of senescence associated events compared to the nonwithdrawn control group (D). Unpaired t test significance; N=3 for all groups ; *p<0.05; ***p<0.001 ; NS: non significant E rror bars represent standard deviation. A B C D

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59 Figure 3 4 AZT reduces astrocyte monolayer cell population expansion. (A) Primary MASCs were treated with 30 M AZT and analyzed for the next three passages. A single 7 day p ulse AZT exposure of primary MASCs results in a significant decrease in the expansion potential of the progeny of the next two passages Third passage of AZT treated primary cells i s able to recover its expansion potential. (B) A single 7 day pulse of 30M AZT exposed on nave first (1 st P), second (2 nd P) and third (3 rd P) passages of MASC cause s a very significant decrease in population expansion. Late passages exposed to AZT a re more vulnerable to be affected by AZT (B). U npaired t test of significance; N=3 for all groups ; *p<0.05; **p<0.01; ***p<0.001. Error bars represent standard deviation. A B

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60 Figure 3 5 AZT abolishes inducible neurogenesis from monolayer cells. Astrocyte monolayer cells treated with AZT at a clinic ally relevant dose range at the time of serum and mitogen withdrawal for 48 hours. A single 48 hour pulse of 0.3 60M AZT exposed to MASC at the time of withdrawal causes a significant decrease in both total number and %B III tubulin (+) neuroblasts (A) ho wever that effect i s more severe on B III tubulin (+) cells (B). 1way ANOVA, Dunnet's Multiple C omparison Test of significance; N=3 for all groups ; **p<0.01. Error bars represent standard deviation. A B

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61 Figure 3 6 A short exposure of low dose AZT perturbs inducible neurogenesis from astrocyte monolayer. MASC s w ere treated with 24, 8 and 2 hour pulse of 0.03 3M AZT at the time of serum and mitogen withdrawal (A, B, C respectively). We show that even the lowest co ncentration of AZT, 0.03M, exposed to cells even for only 2 hours at the time of serum and mitogen withdrawal significantly decrease s the % of B III tubulin (+) neuroblast formation compared to the control group (C). On the other hand, MASC treated with t he same concentration range of AZT for three days before supplement withdrawal d oes not show such a dramatic response (D). 1way ANOVA, Dunnet's Multiple Comparison Test of significance; N=3 for all groups ; *p<0.05; **p<0.01. Error bars represent standard d eviation. A B C D

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62 Figure 3 7 AZT increases apoptosis in astrocyte monolayers. MASCs were exposed to AZT for 48 hours applied at the time of withdrawal. In addition to t he baseline level of caspase 3 (+) and TUNEL (+) cells in withdrawn control (A, B respectively) 30M AZT exposure increase s the percentage of caspase 3 (+) and TUNEL (+) cells significantly (A B ). One way ANOVA, Dunnett's Multiple Comparison Test of sign ificance; N=3 for all groups; p<0.05. NS: non significant. Error bars represent standard deviation. A B

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63 Figure 3 8 AZT upregulates senescence associated mechanisms in withdrawal induced astrocyte monolayer cells. MASC s w ere exposed to a single pulse of 0.3M and 30M AZT for 48 hours starting at the time of serum and mitogen withdrawal. Representative images show senescence and DAPI stained nuclei (pseudocolored magenta) showed that compared to the control su pplement withdrawn cells AZT incr ease s concentration and exposure time dependent manner (C). The baseline level of senescent cells in withdrawn control group increase s markedly within the first 24 and 48 hours of AZT exposure (C). One way ANOVA, Dunnet t's Multiple Comparison Test of significance; N=3 for all groups ; **p<0.01. Error bars represent standard deviation. B C A

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64 Figure 3 9 AZT does not perturb mitochondrial membrane depolarization in astrocyte monolayers MASCs were exposed to single pulse of 3M and 60M AZT following serum and mitogen withdrawal. The control cells were treated with CCCP (carbonyl cyanide 3 chlorophenylhydrazone), a mitochondrial membrane disrupter, as a positive control for depolarizati on. 48 hours later, mitochondrial membrane physiology was assessed via the JC 1 potentiometric cationic dye which exhibits membrane potential dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529nm) to red (~59 0nm). Compared to the non treated supplement withdrawn cells (A) and CCCP treated positive control groups (D), 48 hour long exposure of 3M (B) and 60M (C) AZT d oes not affect mitochondrial membrane physiology. A B C D

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65 Figure 3 10 AZT exposure changes the size and morphology of neurospheres. Single cell suspensions from postnatal SVZ plated in a serum free media containing EGF, FGF and heparin give rise to neurospheres within 10 days. 30M AZT was added to the cell culture t he day following the primary cells were plated. Representative phase contrast images showing differences in size and morphology of control (A) and 30M AZT treated neurospheres (B). A B

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66 Figure 3 11 AZT exposure suppresses neurosphere forming cells. Single cell suspensions from postnatal SVZ plated in the serum free media containing EGF, FGF and heparin give rise to neurospheres within 10 days. One day following the primary cells were pl ated, a single pulse of 0.3 and 30M AZT was exposed on the cell culture for 1, 3 and 10 days. At the end of exp osure time, cell culture media wa s refreshed, and the neurosphere size and number is quantified on day 10. AZT exposure d oes not inhibit neuros phere formation but perturbed the formation of neurospheres. While 1 day long AZT exposure d oes not affect neurosphere number compared to their control levels (A), 3 and 10 day long AZT exposure on neurosphere forming cells cause s a significant reduction i n neurosphere size both in concentration and exposure time dependent manner ( B, C respectively). In addition, the t otal number of neurospheres of control and AZT treated groups i s not significantly different (p>0.05) (D) One way ANOVA, Dunnett's Multiple Comparison Test of significance; N=3 for all groups ; *p<0.05, **p<0.001 Error bar s represent standard deviation A D C B

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67 Figure 3 12 AZT exposure increases the proportion of senescent neurosphere cells. One day foll owing plating, primary cells were exposed to a single pulse of 30M AZT. On the 7 th day neurospheres were dissociated and cytospinned onto glass slides and stained to detect SA B Gal activity. Representative images of dissociated neurospheres with DAPI (ps eudocolored magenta) and SA B Gal staining showing an increase in SA B Gal (+) cells in 30M AZT treated neurospheres (B) compared to control group (A). Quantification of SA B Gal labeled cells showed that 30M AZT exposure increase s the number of SA B Gal stained cells compared to the control group (C). Unpaired t test; N=3 for all groups ; *p<0.05 Error bars represent standard deviation A B C

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68 Figure 3 13 AZT severely perturbs formation of neural colonies der ived from both neural stem and progenitor cells. Dissociated primary neurosphere cell suspension wa s plated at a low density in semi solid, serum free collagen media containing growth supplements. AZT was added to the cell culture at 0.3 and 30 M concentr ations. After 21 days in culture, colonies we re classified into one of four categories based on diameter. AZT treatment of SVZ stem and progenitor cells cause s a concentration dependent decrease in neural colony formation. Representative images of neural c olonies showing AZT exposure at 0.3 (B) and 30M (C) concentrations yield s smaller neural colonies compared to the control group (A). AZT pre treatment of primary neurospheres d oes not affect formation of neural colonies (D) as severe as of colonies which were exposed to AZT directly (E). 30M AZT pre treatment disturbe s formation of colonies smaller than 2mm in diameter, colonies derived from neural progenitor cells, but not colonies larger than 2mm which are colonies derived from stem cells (D). One way ANOVA, Dunnett's Multiple Comparison Test of significance; N=3 for all groups; *p<0.05, **p<0.001. Error bars represent standard deviation. NCFC frequency (%) = Number of colonies/total cells plated*100 C B A D E

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69 CHAPTER 4 IN VIVO AZIDOTHYMIDINE ADMINISTRATI ON DISTURBS NEUROGENESIS Background The Effect of AZT on Adult Neurogenesis At the end of 2008, 33.4 million people worldwide were estimated to be living with human immunodeficiency virus 1 (HIV 1) infection. Besides, about 2.7 million of adults and child ren are newly infected and 2 million adults and children died due to AIDS. Azidothymidine (AZT) also known as Zidovudine or Retrovir is a thymidine analog and is an integral part of HAART as a nucleoside reverse transcriptase inhibitor (NRTI). It has been used in HIV treatment and prophylaxis of infection inhibits HIV 1 reverse transcriptase by acting as a competitive inhibitor of thymidine and causes proviral DNA termination. AZT is one the antiviral drugs classified into high CNS penetration (rank 1) ran king (Letendre et al. 2008) Indeed, it is shown that AZT can enter the CNS by passive diffusion across the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCSFB) Thomas & Segal, 1997; Kearney & Aw eeka, 1999; Cysique et al. 2004; Evers et al. 2004; Letendre et al. 2004) while it is removed from the brain via an active probenecid sensitive transport efflux (Dykstra et al. 1993; Takasawa et al. 1997a; Takas awa et al. 1997b) More importantly, AZT is shown to incorporate into the periventricular region of the brain (Busidan et al. 2001) Surprisingly, little attention has been focused on investigating the effect of AZT exposure on the CNS. While subventricular zone (SVZ) stem and progenitor cells are located immediately subjacent to the ependymal lin ing of the anterolateral wall of lateral ventricle, the mouse hippocampus is located within the posteriomedial aspect of the lateral ventricle forming a part of the posteriosuperior border of the third ventricle. In the

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70 adult brain SVZ neural stem cells (t ype B) have an apical ending which directly contacts with the ventricle and a basal process ending on blood vessels (Mirzadeh et al. 2008) Moreover, it is shown that the NSCs contacting blood vessels lack astrocyte endfeed and precyte coverage (Tavazoie et al. 2008) This modified blood brain barrier structure exposes SVZ stem cells to various signals including AZT from the vascular s ystem. In addition, the relatively superficial location of neural stem cell (NSC) niches of both SVZ and dentate gyrus with respect to the ventricular spaces makes it likely that passive diffusion of AZT from the CSF is the primary mechanism to access to n eural tissue may be sufficient to expose these persistent germinal matrices to significant levels of AZT. Altogether, the present data support the hypothesis that AZT has adverse effects on both adult and perinatal neurogenesis in vivo In the first part of this chapter, we focused on investigating the possible toxic effects of therapeutically relevant doses of AZT exposure on cellular proliferation in the neurogenic niches SVZ and dentate gyrus of hippocampus of the adult brain. Here, we have performed tw o different experiments in which short term exposure of low and moderate AZT doses have been examined. In this study, young adult mice were administered 20 and 100 mg/kg/day AZT for 14 days. Neurogenesis was assessed by 5 bromo 2 deoxyuridine (BrdU) incor poration within dentate gyrus and SVZ. The quantification of BrdU (+) cells revealed that a two week course of both low and moderate dose AZT administration does not change the number of BrdU (+) cell number in both dentate gyrus and SVZ. While the survi val of dentate gyrus cells is not affected, BrdU (+) SVZ cells show a very significant decrease in mice administered with moderate dose of AZT administration.

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71 The Effect of in utero AZT Exposure on Neurogenesis The AIDS Clinical Trials Group Protocol 076 (ACTG 076) study established that AZT administration to pregnant women with HIV infection prenatally and during labor and to newborn infants, reduces the rate of perinatal HIV infection by about two thirds (Connor et al. 1994) In addition, when the AZT monotherapy regimen is combined with elective Cesarean Section (CS) delivery, transmission rates of about 2% were reported (1999; Mofenson, 2000) Currently, in order to preve nt mother to child transmission of HIV 1, AZT monotherapy is administered at 14 34 weeks of pregnancy at 600mg/day, during labor at 2mg/kg loading dose followed by 1mg/kg/hour and at 8mg/kg/day dose for the neonate for 6 weeks (Witt et al. 2004) It is shown that AZT crosses the placenta rapidly wi th similar concentrations in maternal plasma amniotic fluid and cord blood plasma .The AZT monotherapy regimen is shown to result in an average plasma concentration of 0.82 (Capparelli et al. 2005) AZT monotherapy is shown to decrease the mother to infant viral transmission rates however there are several adverse effects of perinatal AZT monotherapy have been reported. In 1999, Blanche et al. suggested that perina tal exposure of AZT may occasionally lead to mitochondrial toxicity shown by abnormality in respiratory chain complex activity, some alterations in brain morphology, neurological anomalies, cognitive and impairment, and episodes of seizures in children exp osed to AZT in utero and after birth (Blanche et al. 1999; Blanche et al. 2006) In addition, it is shown that the proportion of birth defects was greater in the central nervous system (CNS), heart and chromosomes after prenatal AZT exposure in a medicaid population (Newschaffer et al. 2000) Moreover, by analyzing data from the National Institute of Child Health and

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72 Human Development (NICHD) International Site Development Initiative Perinatal Study, Joao et al. reported CNS anomalies such as anencephaly, microcephaly, agenesis of the corpus callosum, ventricular cysts following anomalies in cardiovascular and musculoskeletal system even though the prevalence of these congen ital anomalies in CNS was found relatively low (Joao et al. ) In addition to the human studies which are mostly limited with cohort studies, a number of animal studies were conducted to evaluate possible adverse effects of prenatal exposure of AZT. AZT was detected in DNA of fetal liver, lung, heart, skeletal muscle, brain, testis, and placenta in Macaca mulatta (Poirier et al. 1999; Slikker et al. 2000) Moreover, transplacental exposure was found t o cause mitochondrial dysfunction. Alterations of oxidative phosphorylation complexes were shown in mitochondria of Erythrocebus patas brain, heart, and muscle (Ewings et al. 2000; Gerschenson et al. 2000; Gerschen son & Poirier, 2000) Moreover, DNA attrition was shown in monkeys and mice exposed to the drug in utero (Olivero et al. 1997) Furthermore, telomeric shortening was observed in tissues like brain, lung and liver of transplacentally treated mice (Olivero, 2007) Finally, the offspring of AZT treated rodents were shown to have neurobehavioral abnormalities such as deficits in motor responses, investigative/exploratory and social behavior, learning and spatial tasks suggesting that AZT interferes with CNS development (P etyko et al. 1997; Busidan & Dow Edwards, 1999; Calamandrei et al. 1999a; Calamandrei et al. 1999b; Rondinini et al. 1999; Venerosi et al. 2000; Calamandrei et al. 2002a; Calamandrei et al. 2002b; Venerosi et al. 2003; Melnick et al. 2005; Veneros i et al. 2005)

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73 In the previous chapter we reported reduced proliferation and differentiation potential of neural stem and progenitor cells following AZT treatment in vitro In addition to findings listed above, given the fact that after a single dose o f 150 mg/kg AZT administration to E20 pregnant rats, the distribution of AZT in fetus brain is heterogeneous with relatively greater amounts of AZT in the periventricular area (Busidan et al. 2001) we hypothesize that in utero administration exposes the n eurogenic regions to AZT, and may disrupt prenatal and early postnatal neurogenesis. In order to examine whether perinatal exposure of AZT perturbs prenatal and early postnatal neurogenesis, 250 mg/kg/day AZT was administered to C57BL/6 pregnant mice subc utaneously from day 12 of gestation to Postnatal Day 3. The AZT concentration and exposure period was chosen on the basis of literature reports. We show that overall pregnancy was not affected by AZT exposure so that the litter size and pup we ight did not change. We showed a significant decrease in the potential of inducible neurogenesis from astrocyte monolayer cells isolated from pups treated with AZT in utero On the other hand, the expansion potential of monolayers was not affected by AZT a dministration. In addition, we show altered proliferation of neurosphere forming cells giving rise to smaller neurospheres. Finally, the cellular proliferation in the neurogenic regions of the pup brain is examined. We show only a slight decrease in Ki67(

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74 Results In Vivo AZT Administration Does Not Affect Brdu (+) Cell Number in Adult Neurogenic Area Given the information that AZT can pass through the BBB and BCSFB, and classified as rank 1 drug with high CNS penetr ation, we aimed to determine if in vivo neurogenic niches SVZ and dentate gyrus of hippocampus are located relatively superficially with respect to ventricles also in dicates that passive diffusion from the CSF may be sufficient to expose these persistent germinal matrices to significant levels of AZT. In addition, the subventricular zone (SVZ) stem cells (type B) have contact with both lateral ventricle and the blood v essels we claim that AZT may show its toxicity on SVZ stem cells more significantly. In the present day, for human adults the total recommended daily amount of AZT is 600 mg/kg which is about 10mg/kg. In addition, the FDA recommended pediatric dosage is 2 4 600mg/kg/day. In order to equate mouse and human doses, we used mg/ m 2 which is 600mg AZT/day, administered to human with 1.1710m 2 body surface area would be equal to 133mg/kg d ose administered to mouse with 0.007m 2 body surface area. First, we conducted an in vivo experiment where only low dose AZT doses via intraperitoneal (i.p.) injections for t wo weeks. On the day following the last AZT injection, all animals received 4 BrdU injections (100mg/kg) separated by two hours. In order to asses immediate cell proliferation and survival of BrdU (+) cells, in the following 1 week and 4 weeks, the animals were sacrificed and their brains were

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75 processed for BrdU immunolabeling in SVZ and dentate gyrus ( Figure 4 1 ). Our results show that 2 week long, low dose administration of AZT does not change the number of BrdU (+) cell number b oth in dentate gyrus and SVZ ( Figure 4 2 ). In the second set of our experiments, we examine the effect of moderate dose of AZT administration on adult neurogenesis in vivo In our first set of experiments the dose regimen of AZT we have tested was 67 mg/kg/day AZT for human patients, which was lower than any therapeutic dose. Here, we injected AZT at 100mg/kg/day dose, of which human equivalent dose is 335mg/kg via i.p. injections for two weeks. On the day following last AZT injec tion, all animals received 8 BrdU injections (50mg/kg) separated by 2 hours. Two survival times of 1 week and 4 weeks of BrdU (+) was cells examined. We show that only highly proliferative SVZ region show a significant decrease in survival of BrdU (+) cell number ( Figure 4 3 ). In Utero AZT Administration Does Not Affect the Litter Size or Pup Weight HIV infection from mother to c hild, also called perinatal or vertical transmission, occurs during pregnancy, labor, delivery or breas tfeeding. In 1994, the AIDS Clinical Trials Group Protocol 076 (ACTG 076) study established that AZT monotherapy plays a beneficial role in reducing mother to child transmission of HIV (Connor et al., 1994) Since then, AZT given to pregnant women infected with HIV and their newborns reduced the risk of HIV transmission. However, little is known about the impact s of this therapy on developing brain more specifically on prenatal neurogenesis In order to investigate whether in utero exposure of AZT perturbs prenatal and early postnatal neurogenesis, AZT at 250 mg/kg/day (~5mg/day) dosage which was chosen on the basis of literature reports, was administered to C57BL/6 pregnant mice subcutaneously from day 12 of gestation to Postnatal Day 3. There was no significant

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76 difference in the weight and number of mouse offspring between control and AZT treated groups ( Figure 4 4 A B). In add ition, at the end of AZT administration, the weight of pregnant mice was also not altered compared to the control group Figure 4 4 C). In Utero AZT Administration Does Not Affect t he Expansion Potential o f Astrocyte Monolayer Cell Population Derived f SVZ Cells In order to investigate the effect of in utero treatment of AZT on neurogenic stem/progenitor cell expansion, primary MASC were generated from P3 offspring. Following decapitation, the SVZ tissue surrounding the lateral ventricles was dissected from offspring brains. The dissociated SVZ tissue was re suspended in neural growth medium consisting of N2 media containing 5% FBS, recombinant human epidermal growth factor and basic fibroblast growth factor. The sin gle cell suspension was plated onto tissue culture T 25 flasks and incubated at 37C in 5% CO2. After two days of incubation, the neural growth medium was refreshed, and cells were supplemented every other day with EGF and FGF until the primary passage of monolayer cell culture reached confluence. Confluent primary astrocyte monolayer cells were passaged at a density of 17,500 cells/ cm 2 and supplemented every other day with EGF and bFGF for 7 10 days until confluence was established. Six passages of MASC d erived from in utero AZT exposed offspring were analyzed and quantified with a Z2 Coulter Counter. Our results show that in utero AZT administration causes a decrease in the expansion assage, further passages show recovery ( Figure 4 5 A).

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77 In Utero AZT Administration Causes a Very Significant Decrease i n Inducible Neurogenesis Potential o f Astrocyte Monolayer Cells Derived f SVZ Cells To induce di fferentiation of Passage 1 astrocyte monolayer culture derived from primary MASC of SVZ of in utero AZT administered offspring, cells at a density of 17,500 cells/ cm 2 were plated onto poly L ornithine coated glass coverslips in 12 well plastic plates in n eural growth medium, and were supplemented every other day with EGF and bFGF. Four days later, the growth medium was withdrawn and replaced with an equivalent volume of N2 medium (without serum and growth factors) to induce formation of neuroblast cells. 4 8 hours after withdrawal of neural growth medium, cells were fixed with 4% paraformaldehyde for B III tubulin immunocytochemical analysis. Our results show that in utero exposure of AZT causes a very significant decrease in inducible neurogenesis potential SVZ cells ( Figure 4 5 B). In Utero AZT Administration Yields Smaller Neurospheres than Control Group In order to determine if in utero exposure of AZT alters the growth potent ial of the neurosphere forming cell progeny, we isolated SVZ cells of P3 offspring. Single cell dissociates were plated at clonal density in non adherent conditions including EGF, FGF and heparin. After 7 10 days, the number and size of neurospheres were a ssessed and classified based on diameter (40m, 40 80m, >80m). Neurosphere forming cells obtained from in utero AZT exposed pups yield smaller primary neurospheres with no significant difference in total neurosphere number when compared to those obtained from control offspring ( Figure 4 6 A B).

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78 In Utero AZT Administration Causes a Slight Decrease in Ki67 (+) Cell Number in Given the information that in utero exposure causes incorporation of AZT into DNA, alter ations in oxidative phosphorylation complexes in mitochondria, and telomeric shortening in the brains of offspring, we examined whether in utero AZT administration affects the neurogenic SVZ stem and progenitor cells causing a decrease in cell proliferatio n in vivo We injected 0 and 250 mg/kg/day AZT to C57bL/6 pregnant mice subcutaneously from day 12 of gestation to Postnatal Day 3. On the day following the last AZT injection, all pups were sacrificed and their brains were processed for Ki67 immunolabeling. The quantification of Ki67(+) cells within the brains revealed that in utero AZT administration causes only a slight decrease compared to the control groups ( Figure 4 7 ). Conclusion In previous chapter we show th at AZT causes severe disruption on neurogenic cell expansion and differentiation leading to a senescence profile in vitro Here we investigate if AZT administration disturbs normal levels of neurogenesis in mouse brain when applied at clinically relevant c oncentrations. First, we designed an experimental paradigm to model AZT administration to the adult patients. We administered AZT to young adult male mice at 0, 1, 10, and 20mg/kg/day doses via intraperitoneal (i.p.) injections for 2 weeks. In order to lab el dividing cells within the neurogenic regions of mice brain, all animals were given 100mg/kg BrdU injections on the day following last AZT injection. To assess the immediate/ short term cell proliferation one group of animals was sacrificed 1 week follow ing the last BrdU injection. In addition, another group was sacrificed 4 weeks following the last BrdU injection to examine the survival of

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79 BrdU (+) cells. The quantification of the BrdU immunolabeling in SVZ and dentate gyrus of hippocampus show that the immediate/short term neurogenesis is not disturbed by AZT administration. Similarly, compared to the control groups 2 week long AZT treatment at 20mg/kg/day dosage does not cause a difference in the survival of BrdU (+) cells in SVZ and dentate gyrus. Sinc e the human data support the hypothesis that AZT exposure affects periventricular area of the brain where the neurogenic niches reside, we further analyzed our experimental paradigm. In order to equate mouse and human doses, this time we used mg/m 2 convers recommendation. Accordingly, 10mg/kg dose, which is 600mg AZT/day, administered to human with 1.1710m 2 body surface area would be equal to 133mg/kg dose administered to mouse with 0.007m 2 body surface area. In our second set of in vivo experiment, we examined the effect of clinically the most relevant dosage of AZT, 100mg/kg/day, administered for 2 weeks. As we show with low dose (20mg/kg/day) administration, AZT at 100mg/kg dosage does not cause a significant decrease in the imm ediate/short term neurogenesis. On the other hand, we show statistically a very significant decrease in survival of B III tubulin (+) cells in SVZ but not in dentate gyrus of hippocampus. Given the fact that the animals we used in our experimental model of AZT administration were healthy animals with intact blood brain barrier it is expected to have limited penetration of AZT through the blood brain barrier to CSF to expose SVZ and dentate gyrus to harmful levels of AZT. On the other hand, HIV infection is shown to disrupt the blood brain barrier with an increase in the blood vessel diameter due to inflammation of the vessel walls, alterations in basal lamina, disrupted endothelial cell and tight junction structure. Blood brain barrier disruption leads the e ntry of drugs

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80 including AZT into the central nervous system. Since our in vivo AZT administration paradigm does not reflect the HIV pathogenesis within the brain, we suggest that the decrease in the survival of BrdU (+) cells within SVZ should be considere d as a very significant disruption of adult neurogenesis. Given the fact that, the enhanced entry of AZT into the central nervous system by modulating transporters, developing blood brain barrier permeable derivatives and efflux inhibitors of AZT is one the major goals in the field, we claim that direct exposure of AZT to the neurogenic regions would cause a dramatic disruption on the neurogenesis. It remains for future studies to design experiments modeling HIV infection within the brain and modified pas sage of AZT through the blood brain barrier to investigate the effects of direct exposure of excessive AZT concentrations on neurogenic niches. In addition to the adult brain, we examined whether perinatal exposure of AZT perturbs prenatal and early postna tal neurogenesis. Currently, in order to prevent mother to child transmission of HIV 1, AZT monotherapy is administered at 14 34 weeks of pregnancy at 600mg/day, during labor at 2mg/kg loading dose followed by 1mg/kg/hour and at 8mg/kg/day dose for the neo nate for 6 weeks (Witt et al. 2004) To cre ate an experimental model mimicking this regimen, we injected clinically relevant doses of AZT to pregnant mice from day 12 of gestation to Postnatal Day 3. According 2 conversion calculations, an average women patient with 1.6 m 2 body surface area is given to 600mg/kg/day AZT for the last 6 months of pregnancy. Accordingly, the clinically relevant experimental dosing would be at 131.25mg/kg/day. Since a pregnant mouse give birth to 5 to 10 pups, the distribution of AZT per pup would be less amount that a human newborn. Hence we tested AZT

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81 dosages at 250mg/kg/day and 125mg/kg/day. Since AZT can pass through the breastmilk, we continued giving AZT injections to the adult mice till postnatal 3 rd day. The gestation, pup yie ld, mortality, and weight were analyzed. Since the group administered with 125mg/kg/day did not give birth to enough number of pups due to the lack of pregnancy, we had to eliminate the data from this group. We show that overall pregnancy was not affected by high dose AZT exposure so that the litter size and pup weight did not change. In order to investigate the effect of in utero treatment of AZT on neurogenic stem/progenitor cell expansion, primary monolayers of MASC were generated from P3 offspring SVZ a nd passaged six times. Our data show a significant decrease in the expansion potential of primary MASCs derived from in utero AZT administered pup brain. Consistent with our in vitro data, we show a recovery in disturbed expansion potential of primary cell s in further passages. Similarly, we show a very significant decrease in differentiation potential of monolayers of MASC derived from in utero AZT administered pup SVZ. Moreover, the neurosphere forming potential of SVZ cells was also disturbed. Consistent with our in vitro data, neurosphere forming cells derived from in utero AZT exposed pup SVZ give rise to smaller neurospheres while the yield is not changed. Finally, we analyzed dividing Ki 67 (+) cells within the neurogenic regions of pup brains. Even t hough there is a slight decrease in the number of Ki 67 expressing cells in AZT administered pup brains, the effect was not statistically significant. Due to experimental limitations we were not able to analyze the brain section with double immunolabeling. Because of that, we cannot claim that the Ki 67(+) cell number reflects the level of dividing neurogenic cells. Altogether, we show that in utero

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82 potential significantly as s een in our in vitro cell culture paradigms. Future studies are needed to examine the role of these effects on neurodevelopment and functioning. It is important to determine clinically safe and effective dosages to lead to a better health quality for HIV (+ ) adults, children and infants.

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83 Figure 4 1 Representative images of Dentate Gyrus and SVZ with BrdU and NeuN immunolabeling. (A) A coronal hemisection through the adult mouse hippocampus with BrdU (green) and Ne uN (red) double staining shows the BrdU (+) dividing cells in the granule cell layer of hippocampal dentate gyrus. (B) A coronal hemisection through the mouse SVZ subjacent to the ependymal lining of the lateral ventricle (LV). A B LV

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84 Figure 4 2 2 week long, low dose administration of AZT does not change the number of BrdU (+) cell number both in dentate gyrus and SVZ. Young adult male mice were administered AZT at 20mg/kg/day dosage for 2 weeks. Quantification of BrdU labe ling on on a 1 in 6 series of sagittal sections in a single focal plane was performed. (A) The short term cell proliferation 1 week after the last AZT injection, both in dentate gyrus and SVZ i s not changed by 20mg/kg/day AZT administration for 2 weeks. (B ) The survival of cells 2 weeks after the last AZT injection i s not affected by AZT. There i s only a slight decrease in BrdU (+) cell number in SVZ. Unpaired t test of significance; N=4 for all groups ; p>0.05. NS: non significant. Error bars represent stan dard deviation. A B

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85 Figure 4 3 2 week long moderate treatment regimen caused a significant decrease in BrdU (+) cell number only in SVZ area. Young adult male mice were administered AZT at 100mg/kg/day dosage for 2 weeks Quantification of BrdU labeling on on a 1 in 6 series of sagittal sections in a single focal plane was performed. (A) The short term cell proliferation 1 week after the last AZT injection, both in dentate gyrus and SVZ i s not changed by 100mg/kg/day AZT administration for 2 weeks. (B) The survival of dentate gyrus cells 2 weeks after the last AZT injection i s not affected by AZT. There i s a significant decrease in BrdU (+) cell number in SVZ. Unpaired t test of significance; N=4 for all groups ; **p<0.01. NS: non significant. Error bars represent standard deviation. A B

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86 Figure 4 4 In utero exposure of AZT does not affect the litter size or pup weight. AZT at 250 mg/kg/day dosage was administered to C57BL/6 pregnant mi ce subcutaneously from day 12 of gestation to Postnatal Day 3. (A, B) There i s no significant difference in the weight and number of mouse offspring between control and AZT treated groups. (C) At the end of AZT administration, the weight of pr egnant mice i s also not altered compared to the control group. Unpaired t test of significance; N=4 for all groups ; p>0.05. Error bars represent standard deviation. A B C

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87 Figure 4 5 In utero exposure of AZT on MASC derived at 250 mg/kg/day dosage was administered to C57BL/6 pregnant mice subcutaneously from day 12 of gestation to Postnatal Day 3. Primary MASC was generated from P3 offspring. Six passages of MASC derived from in ut ero AZT exposed offspring were quantified. Passage 1 MASC s of SVZ derived from in utero AZT administered offspring w ere induced to differentiate into neuroblasts by withdrawal of serum and mitogens, EGF and FGF. 48 hours following the withdrawal, number of B III tubulin (+) neuroblasts was quantified. (A) In utero AZT administration causes a decrease in the expansion potential of MASC only in the first passage, further passages show recovery. All other comparisons are not significantly different. (B) In ute ro exposure of AZT causes a very significant decrease in inducible SVZ cells. Unpaired t test; N=3 for all groups ; *p<0.05, **p<0.01. Error bars represent standard deviation. A B

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88 Figure 4 6 In utero AZT administration yields smaller neurospheres than control group. AZT at 250 mg/kg/day dosage was administered to C57BL/6 pregnant mice subcutaneously from day 12 of gestation to Postnatal Day 3. Single cell dissociates isolated from SVZ of P3 offspring were plated in nonadhesive serum free medium supplemented with EGF, FGF and heparin. After 7 10 days of initial culture, the number and size of neurospheres were quantified based on diamete r. (A) Neurosphere forming cells obtained from in utero AZT exposed offspring yield smaller primary neurospheres with no significant difference in total neurosphere number (B) when compared to those obtained from control offspring. Unpaired t test; N=3 for all groups ; *p<0.05 Error bars represent standard deviation. A B

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89 Figure 4 7 In utero exposure of AZT does not affect proliferating Ki 67 (+) cell number C57BL/6 pregnant mice subcutaneously from day 12 of gestation to Postnatal Day 3. On the day following the last AZT injection, all pups were sacrificed and their brains were processed for Ki 67 immunolabeling. The quantification of Ki 67(+) c ells within the brains revealed that in utero AZT administration causes only a slight decrease compared to the control group. Unpaired t test; N= 4 for all groups ; p>0.05. Error bars represent standard deviation.

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90 CHAPTER 5 DISCUSSION AND CONCL USIONS Sinc e 1987 AZT has been used in the treatment and prevention HIV 1 infection, either alone or in combination with other antiviral agents. In addition, AZT monotherapy has been recommended for use in pregnancy to reduce vertical transmission of HIV 1 from mothe r to fetus during pregnancy, labor and delivery or breastfeeding. On the other hand, it is still classified in Pregnancy Category C of Food and Drug Administration due to the potential risks of AZT usage to the fetus in human pregnancy (Walker et al. 2007; Durand Gasselin et al. 2008; Read et al. 2008; Foster et al. 2009) It is shown that in a nonbreastfeeding population, AZT monotherapy administered at 14 34 weeks of gestation (600mg/day), continuous intravenou s infusion during labor (2mg/kg loading dose followed by 1/mg/kg/h) and 6 weeks of oral dosing to the newborn reduced the mother to child viral transmission rate from 25.5% to 8.3%. tients and in reducing vertical viral transmission, several studies showed adverse effects of AZT such as bone marrow suppression, pancytopenia, anemia, macrocytosis, cardiomyopathy, hepatic steatosis, fatal lactic acidosis, myopathy, peripheral neuropathy distal symmetrical neuropathy, carcinogenicity (Ayers et al. 1996; Chow et al. 1997; Zhang et al. 1998; Diwan et al. 1999; Anderson et al. 2003; Lee et al. 2003; Lewis et al. 2003; Lai et al. 2004; Lewis et al. 2004; Torres et al. 2007) Some of these pathologies, e.g. myopathies, cardiomyopathy, hepatotoxicity, are shown to resolve were due to AZT and were not symptoms of A IDS (Lynx & McKee, 2006)

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91 In addition to the adverse effects seen in adults, it is shown that in utero exposure of AZT is genotoxic and mutagenic in fetal cells of humans, causing increased somatic mutations in infants and their mothers (Diwan et al. 1999; Olivero et al. 1999; Walker et al. 2007; Witt et al. 2007) Indeed, abnormality in mitochondrial respiratory chain complex activity, alterations in brain morphology, neurological anomalies, cognitive and impairment, and episo des of seizures in children exposed to AZT in utero and after birth is reported (Blanche et al. 1999; Blanche et al. 2006) In addition, the proportion of birth defects was shown to be greater in the central nervo us system, heart and chromosomes after prenatal AZT exposure (Newschaffer et al. 2000) Moreover, even though with a low prevalence, congenital central nervous system anomalies such as anencephaly, microcephaly, ag enesis of the corpus callosum, ventricular cysts following anomalies in cardiovascular and musculoskeletal system of were reported (Joao et al. ) Experimental animal models of the in utero AZT treatment support these findings. AZT was detect ed in DNA of fetal liver, lung, heart, skeletal muscle, brain, testis, and placenta in Macaca mulatta (Poirier et al. 1999; Slikker et al. 2000) Alterations of oxidative phosphorylation complexes were shown in mi tochondria of Erythrocebus patas brain, heart, and muscle (Ewings et al. 2000; Gerschenson et al. 2000; Gerschenson & Poirier, 2000) DNA attrition was shown in monkeys and mice exposed to the drug in utero (Olivero et al. 1997) Furthermore, telomeric shortening was observed in tissues like brain, lung and liver of transplacentally treated mice (Olivero, 2007) Besides, the offspring of AZT treated rodents were shown to have neurobehavioral abnormalities such as deficits in motor responses, investigative/exploratory and social

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92 behavior, learning and spatial tasks suggesting that AZT interferes with CNS development (Petyko et al. 1997; Busidan & Dow Edwards, 1999; Calamandrei et al. 1999a; Calamandrei et al. 1999b; Rondinini et al. 1999; Venerosi et al. 2000; Calamandrei et al. 2002a; Calamandrei et al. 2002b; Venerosi et al. 2003; Melnick et al. 2005; Venerosi et al. 2005) Indeed, Busidan et al. have shown that after a single dose of 150 mg/kg AZT administration to E20 pregnant rats, the distribution of AZT in fetus brain is heterogeneous with relatively greater amounts of AZT in the periventricular area. It is known that AZT can pass through the BBB and BCSFB with passive diffusion (Thomas & Segal, 1997; Kearney & Aweeka, 1999; Cysique et al. 2004; Evers et al. 2004; Letendre et al. 2004) Even though the level of AZT in the CNS is low compared to the other systems, it is classified into high rank of CNS penetration eff ectiveness (CPE) (rank 1) based on its chemical features, measured CSF concentrations and effectiveness of AZT in the CNS (Wu et al. 1998; Letendre et al. 2008; Im et al. 2009) On the other hand, the potential e ffects of direct exposure of excessive AZT concentrations and immune response to the toxicity on the CNS are not known. Even there is extensive literature on AZT toxicity on different systems and cell types, surprisingly little attention has been focused on investigating the neurotoxic effects of AZT administration. In this study, we focused on investigating the possible toxic effects of AZT on neural stem and progenitor cell functioning. We claim that the superficial location of neurogenic niches with res pect to the ventricular spaces and recently revealed cellular architecture of SVZ type B cells, which contact with both

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93 ventricle and blood vessels forming a modified BBB, exposes neural stem cells to various signals including AZT from CSF and vascular sys tem. First, we investigated whether AZT exposure perturbs neural stem and progenitor cells in vitro In order to examine the population expansion potential of neural stem and progenitor cells isolated from naive mouse SVZ, we exposed multipotent astrocyte monolayer cells (MASC) to clinically relevant AZT concentrations. We show that AZT has a strong antiproliferative effect on MASCs. Interestingly, while treated primary cells were more resistant showing a recovery, the late passages of cells were more vul nerable to AZT. In addition to the impaired expansion potential, we examined whether AZT leads to a disruption on inducible neurogenesis from MASC. We exposed monolayer with AZT at the time of induction stimulus, which is withdrawal of serum and mitogen wi thdrawal. We show that even the lowest concentration of AZT we examined, 0.03M, exposed to cells even for only 2 hours at the time of serum and mitogen withdrawal causes a significant decrease in both expansion of population and differentiation to neurobl asts however that effect was more severe on B III tubulin (+) neuroblasts. On the other hand, MASC treated with the same concentration range of AZT for three days before supplement withdrawal did not show such a dramatic response. In order to investigate the possible mechanism causing AZT to impair the expansion potential and to abolish inducible neurogenesis, we assessed the activation of cleaved caspase 3 as an early marker in cellular apoptosis and the Terminal Uridine Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay, which detects DNA fragmentation of late stage apoptotic cells, on MASC exposed to AZT for 48 hours applied at the time of withdrawal. We show that there is already a baseline level of cells

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94 expressing caspase 3 in withdrawn control group while 30M AZT causes a significant increase in the percentage of cells expressing caspase 3. In addtion AZT exposure causes significant increase in the percentage of TUNEL (+) cells. Stress induced senescence, which is permanent arrest o f cell division, can be induced by exposure to a variety of factors, such as UV and gamma radiation, pharmacological agents, and oxidative stress; and is characterized by DNA damage. In order to examine if AZT, as a genotoxic agent, is causing any increase in senescent associated B galactosidase activity, we exposed MASC with single pulse of 0.3M and 30M AZT for 48 hours starting at the time of serum and mitogen withdrawal. Our data show that in addition to the baseline level of senescent cells in control group due to the cell culture conditions, AZT exposure causes an increase the number of SA B Gal (+) cells in a concentration and exposure time dependent manner. Since DNA damage at any location including the mitochondria might lead to cellular senescenc e, we performed JC 1 assay to understand whether AZT induced SA B Gal activity is due to mitochondrial damage. However, our results show that AZT exposure does not perturb mitochondrial membrane polarization of serum and mitogen withdrawn MASC. We also co nducted experiments examining the effect of AZT on neurosphere cell culture system. Consistent with previous findings with astrocyte monolayers, AZT exposure reduces the size of neurospheres, alters their morphology, and increases the proportion of senesce nt neurosphere cells. Moreover, to examine the effect of AZT on stem and progenitor cells, neural colony forming cell assay (NCFCA) was used. We show AZT severely perturbs formation of neural colonies formed by both neural stem and progenitor cells isolate d from SVZ.

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95 Our results show that reduced expansion of stem progenitor cells is concurrent with the onset of a senescent phenotype in AZT treated cells that alters cell differentiation potential and susceptibility to apoptosis suggesting that AZT inhibits differentiation at the point of mitotic expansion, most likely through inhibition of early S phase of and subsequent differentiation processes. Here we show AZT exposure results in a striking inhibition of stem/progenitor cell population expansion and wit hdrawal induced differentiation in a dose dependent manner. The rapid blockage of differentiation by AZT suggests a direct toxic effect rather than a telomerase inhibition which is thought to be one of the main mechanisms of AZT toxicity. However the redu ction in AZT exposed neurosphere diameter and MASC proliferation could be explained by perturbations in nucleoside phosphorylation kinetics, telomerase inhibition, or mitochondrial dysfunction. Here, we show that there was no disruption of mitochondrial fu nction by AZT exposure. Overall, the adverse effect of AZT leading to a cellular senescence may be related with synergistic interaction of several toxicity mechanisms. To examine whether in vivo exposure of AZT would cause any disruption on adult neurogene sis, we injected adult animals with AZT at clinically relevant low and moderate concentrations for a short term period. Neurogenesis was assessed by 5 bromo 2 deoxyuridine (BrdU) incorporation within dentate gyrus and SVZ. The quantification of BrdU (+) ce lls revealed that a two week course of both low and moderate dose AZT administration does not change the number of BrdU (+) cell number in both dentate gyrus and SVZ. While the survival of dentate gyrus cells is not affected, BrdU (+) SVZ cells show a ve ry significant decrease in mice administered with

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96 moderate dose of AZT administration. Here we did not see a strong antiproliferative effect of AZT as seen in cell culture systems in vitro If AZT exerts a direct toxic effect, we expect to see significant differences in short term neurogenesis. However, we did not observe such a difference between BrdU (+) cell of control and AZT treated groups. Similarly, long term survival was not affected. The intact structure of the brain and blood brain barriers of adu lt animals might prevent the passage of AZT through the neurogenic niches. On the other hand, HIV infection is associated with the disruption of BBB with an increase in the diameter of blood vessels due to inflammation of the vessel walls, alterations in the basal lamina, loss of glycoproteins in endothelial cells, endothelial cell apoptosis, and tight junction disruption (Toborek et al. 2005; Guillevin, 2008) As BBB disruption leads the HIV infected cells to ente r the brain, it also facilitates the entry of drugs into the CNS (Varatharajan & Thomas, 2009) HIV infection in the CNS leads to the development of asymptomatic neurocognitive impairment, HIV associated mild neurocognitive disorder (MND), and A IDS dementia complex (ADC) or HIV associated dementia (HAD) with impairment in cognitive activity, memory, attention, and motor and behavioral functioning (Antinori et al. 2007) .Therefore prevention of HIV infectio n in the CNS is one of the major goals in the field. In order to enhance levels of antiretroviral drugs including AZT in CNS and to make them more efficient, researchers focus on developing new strategies such as developing BBB permeable derivatives of ant iretroviral drugs and efflux inhibitors, and modulating transporters (Li et al. ; Saiyed et al. ; Zhivkova & Stankova, 2000; Eilers et al. 2008; Miller et al. 2008; Quevedo et al. 2008; Im et al. 2009) We suggest that in case of increased delivery of AZT into the

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97 brain, a direct exposure to the CNS would cause more dramatic changes as we have shown with MASC, NS, NCFCA cell culture systems. Finally, we examined whether perinatal exposure of AZT perturbs prenatal and early postnatal neurogenesis, the C57BL/6 pregnant mice were treated with 250 mg/kg/day AZT subcutaneously from day 12 of gestation to Postnatal Day 3. The AZT concentration and exposure period was chosen on the basis of literature reports We show that the concentration of AZT was not so toxic that overall pregnancy was affected; the litter size and pup weight did not change. We showed a significant decrease in the potential of inducible neurogenesis from astrocyte monolayer cells isolated from pups treated with AZT in utero Except the first passage of MASC, the expansion potential of monolayers was not affected. The decrease in the first passage of MASC was recovered in further passages. In addition, we show altered proliferation of neuro sphere forming cells giving rise to smaller neurospheres as seen in our in vitro findings. Since the neurosphere forming cells were passaged for only once, we cannot claim that this decrease would be recovered as in MASCs. Finally, the cellular proliferati on in the neurogenic regions of the pup brain is examined. We show only a neurogenesis was not affected directly by AZT toxicity. Summary Together, these data reveal uncharacterized negative consequences of AZT treatment on neural stem and progenitor cells. Most of the toxic effects of AZT occur after long time scales, beyond the capability of our in in vivo experiments with short term treatment models. The long term u se of AZT as a part of anti HIV therapy might affect the stem and progenitor cells within the adult brain. Given the fact that HIV infection

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98 leads to development of neurological deficits and that human HIV (+) patients are treated with AZT over years, it i s important to determine what extent AZT regimens might perturb normal levels of neurogenesis to exacerbate or contribute to these neurological problems. We expect our results will reveal new insights regarding the effect of AZT on stem/progenitor cell fun ctioning, and the development of new treatment approaches to prevent the HIV infection in CNS.

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116 Zhou, F.X., Liao, Z.K., Dai, J., Xiong, J., Xie, C.H., Luo, Z.G., Liu, S.Q. & Zhou, Y.F. (2007) Radiosensitization effect of zidovudine on human malignant glioma cells. Biochemical and biophysical research communications 354 351 356.

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117 BIOGRAPHICAL SKETCH Meryem Demir was born in 1978 in Trabzon, Turkey. She graduated from Bahcelievler Anadolu High School in 1996 in Istanbul. Following graduation, she attended Istanbul University, Cerrahpasa M edical School in Istanbul, Turkey and obtained a B.S. degree in Biomedical Sciences in May 2000. She enrolled in the Institute of Biomedical Engineering at Bogazici University Istanbul in September 2000 and graduated with a MSc. d egree in Septemb er 2003. In August 2004 she entered the Interdisciplinary Program in Biomedical Sciences (IDP) at the University of Florida College of Medicine leading to the degree for D octor of P hilosophy In May 2005 Meryem joined the laboratory of E ric D. Laywell. During her graduate study she worked on characterizing the effects of Azidothymidine on adult and perinatal neurogenesis. Upon completion of her Ph.D. in December 2010, Meryem plans to pursue a career dedicated to the study of stem cell bi ology.