VIRVE KÄRKKÄINEN

Multipotent neural stem/progenitor cells as a model of genetic neuronal diseases

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Snellmania L22, Kuopio, on Friday, September 7th 2012, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences Number 131 Molecular Brain Research Group Department of Neurobiology A.I.Virtanen Institute for Molecular Sciences Faculty of Health Sciences, University of Eastern Finland Kuopio 2012

Kopijyvä Kuopio, 2012 Series Editors: Professor Veli-Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN: 978-952-61-0890-2 (print) ISBN: 978-952-61-0891-9 (pdf) ISSN: 1798-5706 (print) ISSN: 1798-5714 (pdf) ISSN-L: 1798-5706

III

Author’s address:

Department of Neurobiology, A.I.Virtanen Institute for Molecular Sciences University of Eastern Finland 70211 KUOPIO FINLAND

Supervisors:

Professor Jari Koistinaho, M.D., Ph.D. Department of Neurobiology A.I. Virtanen Institute for Molecular Sciences KUOPIO FINLAND Johanna Magga, Ph.D. Department of Pharmacology and Toxicology University of Oulu OULU FINLAND Professor Karl Åkerman, M.D., Ph.D. Institute of Biomedicine, Physiology University of Helsinki HELSINKI FINLAND Docent Maija Castren, M.D., Ph.D. Institute of Biomedicine, Physiology University of Helsinki HELSINKI FINLAND

Reviewers:

Professor Irma Holopainen, M.D., Ph.D Department of Pharmacology University of Turku TURKU FINLAND Professor Petri Lehenkari, M.D., Ph.D Institute of Biomedicine University of Oulu OULU FINLAND

Opponent:

Adjunct Professor Jouni Sirviö, Ph.D. Oy Sauloner Ltd KUOPIO FINLAND

IV

V Kärkkäinen, Virve Multipotent neural stem/progenitor cells as a model of genetic neuronal diseases, 85p. University of Eastern Finland, Faculty of Health Sciences, 2012 Publications of the University of Eastern Finland. Dissertations in Health Sciences nro. 2012. 85p. ISBN: 978-952-61-0890-2 (print) ISBN: 978-952-61-0891-9 (pdf) ISSN: 1798-5706 (print) ISSN: 1798-5714 (pdf) ISSN-L: 1798-5706

ABSTRACT Neural stem/progenitor cells (NPC) can be used for therapeutic purposes in several ways. Cultured NPCs offer a unique cell source to study the neural mechanisms in normal situations as well as in the pathogenesis of genetic diseases. In neurodegenerative disorders, potential therapies to recover the damaged neurons are NPC transplantation or activation of a patient’s own NPCs. However, the mechanisms of NPC differentiation are still poorly known and before any clinical use, it is of utmost importantance to know how the differentiation of NPCs is regulated. These four studies provide new information about the differentiation of NPCs. Fragile X syndrome (FXS) is caused by the defiency of fragile X mental retardation protein (FMRP). We investigated how FMRP deficiency affects the differentiation of NPCs. We show that FMRP deficiency directs the NPC differentiation into neuronal phenotypes, but developing neurons have fewer and shorter neurites and smaller body volume. Furthermore, FMRP deficient NPCs have increased occurrence of intense oscillatory Ca2+ responses to neurotransmitters (NT) compared to controls. In the second study, we used Ca2+ imaging techniques to monitor the neurotransmitter responsiveness in an early state of NPC differentiation. We found that during the early stage of differentiation, cells responded to various NTs and could be distinguished based on their NT responses. During development, cells progressively lose their metabotropic responses and gain ionotropic responses while they simultaneously develop into neuronal cells. Next, we studied the effect of Alzheimer’s disease (AD) -linked mutation and environment on NPC differentiation. We treated NPCs with synthetic amyloid- (A ) in vitro and also transplanted NPCs into AD-linked mutant mouse brain. We show that both AD-linked mutation in NPCs and AD-brain environment have effects on NPC differentiation. Transplanted NPCs survived and migrated better when transplanted into AD mouse brain. In addition, transplanted NPCs stimulated brain neurogenesis even in highly A -burdened brain. Oxidative stress (OS) is one of main characteristics in AD brain. Activation of a transcription factor nuclear factor erythroid 2-related factor (Nrf2) during the OS leads to activation of cellular defense mechanisms. We also show that the activation of Nrf2 protects NPCs against A -induced toxicity by enhancing their survival, proliferation and neuronal differentiation. Furthermore, we discovered another important function for Nrf2: the ability to regulate injury-induced neurogenesis. National Library of Medical Classification: WL 102, QV 126 Medical Subject Headings (MeSH): Neural Stem Cells; Neurogenesis; Cell Differentiation; Neurotransmitters Receptors; Neurotransmitter; Fragile X Syndrome; Fragile X Mental Retardation Protein; Alzheimer Disease; Amyloid beta-Peptides; NF-E2-Related Factor 2; Oxidative Stress; Neurobiology

VI

VII

Kärkkäinen, Virve Monikykyiset hermoston kantasolut mallina perinnöllisille neuronaalisille sairauksille, 85p. Itä-Suomen yliopisto, terveystieteiden tiedekunta, 2012 Publications of the University of Eastern Finland. Dissertations in Health Sciences Nro 131. 2012. 85p. ISBN: 978-952-61-0890-2 (print) ISBN: 978-952-61-0891-9 (pdf) ISSN: 1798-5706 (print) ISSN: 1798-5714 (pdf) ISSN-L: 1798-5706

TIIVISTELMÄ Hermoston kantasoluja voidaan kasvattaa laboratoriossa. Neurosfeereinä eli eräänlaisina hermokantasolupallosina kasvavat hermoston kantasolut tarjoavat työkalun tutkia hermosolun erilaistumista. Geneettisesti muunnelluilla kantasoluilla voidaan tutkia kuinka geenivirhe vaikuttaa kantasolujen erilaistumiseen. Lisäksi hermoston kantasolut voivat tarjota mahdollisuuden hoitaa vaikeita hermorappeumasairauksia viljeltyjä hermoston kantasoluja solusiirteenä käyttämällä tai aktivoimalla potilaan omia jo olemassa olevia kantasoluja korvaamaan tuhoutuneita hermosoluja. Jotta hermoston kantasoluja voitaisiin käyttää terapeuttisiin tarkoituksiin, täytyy ensin ymmärtää kuinka erilaistuvaa hermosolua säädellään sekä miten geenivirheet ja sairauden patologia vaikuttavat kehittyviin hermosoluihin. Tämän väitöstutkimuksen tarkoituksena oli tuottaa tietoa hermoston kantasolujen erilaistumisesta. Tutkimme säätelevätkö klassiset hermovälittäjäaineet hermoston kantasoluja ja miten kehitysvammaisuutta aiheuttavaan Fragile X oireyhtymään (FXS) liittyvän fmr1-geenin mutaatio, joka johtaa FMR-proteiinin (FMRP) puutteeseen vaikuttaa hermoston kantasolujen erilaistumiseen. Lisäksi tutkimme miten Alzheimerin taudin (AT) patologia ja geenivirhe vaikuttavat hermoston kantasolujen erilaistumiseen ja suojaako transkriptiotekijä Nrf2:n aktivaatio erilaistuvia hermoston kantasoluja amyloidi(A ) -peptidin toksisilta vaikutuksilta. Tutkimuksemme osoitti, että solut, joilta puuttui FMRP, erilaistuvat useammin neuroneiksi, joiden sooma oli pienempi ja joilla oli vähemmän ja lyhyempiä neuriitteja kuin kontrollisoluilla. Lisäksi suurempi joukko erilaistuvia soluja reagoi hermovälittäjäainestimulaatioon tuottamalla solunsisäisen kalsiumpitoisuuden oskillaatioita. Kalsiumkuvannuksen avulla osoitimme, että erilaistuvat hermosolut reagoivat monien hermovälittäjäaineiden stimulaatioon, joka tarkoittaa sitä, että välittäjäaineet osallistuvat hermosolujen erilaistumisen säätelyyn. Kolmannessa tutkimuksessa havaitsimme että sekä AT:n patologia mm. runsas A -peptidin keräytymä aivoissa että perinnöllistä AT:a aiheuttava mutaatio hermoston kantasoluissa vaikuttavat niiden elinkykyyn, liikkumiseen ja erilaistumiseen. Tärkeä havainto oli myös se, että siirretyt kantasolut pystyivät stimuloimaan hiiren omaa hippokampuksen neurogeneesiä jopa vanhoilla AT-hiirillä. Osoitimme myös, että Nrf2 suojaa erilaistuvaa hermoston kantasolua A -peptidin toksisilta vaikutuksilta ja Nrf2:lla on myös toinen tärkeä fysiologinen rooli, neurogeneesin säätely. Luokitus: WL 102, QV 126 Yleinen suomalainen asiasanasto (YSA): hermosto; kantasolut; erilaistuminen; hermosolut; välittäjäaineet; fragiili X oireyhtymä; Alzheimerin tauti; neurobiologia

VIII

IX

Acknowledgements This study was carried out in the Department of Neurobiology A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, during the years 2003 – 2012 and financially supported by Ministry of Education, Finland, Sigrid Juselius foundation, Finland, Tekes, Finland, Olvi foundation, Finland and University of Eastern Finland, Finland. I want to express my deepest gratitude to my principal supervisor Professor Jari Koistinaho, M.D., Ph.D., for giving me the opportunity to continue my Ph.D. project in the Molecular Brain Research (MBR) Group, his scientific guidance, personal contribution and support for my project. I am also very grateful to my other supervisors Professor Karl Åkerman, M.D., Ph.D. and Docent Maija Castren, M.D., Ph.D. for giving me the opportunity to start this project and work with neural stem cells. I am also very thankful to Ph.D. Johanna Magga for her support, ideas and encouraging words during the years in MBR-group. I express my gratitude to Professor Irma Holopainen M.D. Ph.D. and Professor Petri Lehenkari M.D. Ph.D., the official reviewers, for their constructive comments and criticisms to improve this thesis. I also wish to thank Professor Garry Wong, who revised the language of this manuscript. I am thankful to my co-authors who contributed to this work; Topi Tervonen, Ph.D., Seppo Heinonen, M.D., Eero Castren, M.D., Kim Larsson, Ph.D., Cathy Bakker, Ph.D., Ben Oostra, Ph.D., Verna Louhivuori, M.Sc., Tarja Malm, Ph.D., Antti Kurronen, M.Sc., Katja Kanninen, Ph.D., Ekaterina Savchenko, M.D., Yuriy Pomeschick, M.D., and Anna-Liisa Levonen, Ph.D. I wish to thank all my colleagues from Cell Biology and MBR groups: Veera Pevgonen, Jonny Näsman, Kim Larsson, Genevie Bart, Lauri Louhivuori, Hanna Peltonen, Miia Antikainen, Laila Kaskela, Mirka Tikkanen, Piia Valonen, Katja Puttonen, Aino Kinnunen, Piia Vehviläinen, Marika Ruponen, Minna Oksanen, Riitta Kauppinen, Sarka Lehtonen, Susanna Boman, Anu Muona, Rea Pihlaja, Sisko Juutinen, Paula Korhonen, Anni Lehikoinen, Taisia Rolova, Gundars Goldsteins, Velta Keksa-Goldsteine, Eveliina Pollari, Merja Jaronen, Riikka Heikkinen, Hiramani Dunghana, Iurii Kidin, Sighild Lemarchant, Yajuvinder Singh and Sara Wojciechowski. You have created a pleasant and cheerful working atmosphere in the lab. I also wish to thank Jari Nissinen, Jouko Mäkäräinen and Pekka Alakuijala for technical support, Sari Koskelo, Kaija Pekkarinen, Riitta Laitinen, Amanuens Arja Afflect, Eija Susitaival, Hanne Tanskanen, Docent Riitta Keinänen, Ph.D. and Riikka Pellinen are thanked for secretarial and admistrative help. I warmly thank my parents Kaisu and Martti Kärkkäinen, parents-in-law Eino and Aino Savolainen, other family members Jaakko, Maija, Kerttu, Alisa, Ville, Tarja, Olli, Kari, Tiina, Teija, Henri, Carita, Anna, Emmi and friends Leila, Jarmo, Tiina, Anna, Pirjo, Paavo, Birgi, Timo who all have helped me in numerous ways and share my life outside the lab. I owe my deepest thanks to Kimmo for his endless love and patience during these years and for our wonderful sons Tatu and Saku. Virve Kärkkäinen

X

XI

List of original publications

This dissertation is based on the following original publications:

I

II

Castren M, Tervonen T, Kärkkäinen V, Heinonen S, Castren E, Larsson K, Bakker C E, Oostra B A and Åkerman K. Altered differentiation of neural stem cells in fragile X syndrome. PNAS 102:17834-17839, 2005. *Kärkkäinen V, *Louhivuori V, Castren M and Åkerman K. Neurotransmitter responsiveness during early maturation of neural progenitor cells. Differentiation 77:188-198, 2009.

III

Kärkkäinen V, Magga J, Koistinaho J and Malm T M. Brain environment and mutations linked to familiar Alzheimer´s disease affect the survival, migration and differentiation of neuronal progenitor cells. Submitted.

IV

Kärkkäinen V, Savchenko E, Pomeshchik Y, Kurronen A, Levonen A-L, Magga J, Kanninen K and Koistinaho J. Nrf2 protects neural progenitor cells against A toxicity and promotes endogenous neurogenesis. Manuscript. *Authors with equal contribution In addition, this thesis contains previously unpublished data.

The publications were adapted with the permission of the copyright owners.

XII

XIII

Contents 1 INTRODUCTION ..................................................................................................................... 1 2 REVIEW OF THE LITERATURE ............................................................................................ 3 2.1 Adult neural stem/progenitor cells (NPCs)....................................................................... 3 2.1.1 Adult neurogenesis ........................................................................................................ 3 2.1.2 Neurogenic regions ....................................................................................................... 4 2.1.3 Regulation of adult neurogenesis ................................................................................. 5 2.1.4 Cultivation of NPCs in vitro and therapeutic possibilities ......................................... 6 2.1.5 Neurotransmitters in the regulation of NPCs ............................................................. 8 2.2 Fragile X syndrome (FXS) ................................................................................................. 12 2.2.1 Clinical features and neuropathology of FXS............................................................ 12 2.2.2 Fragile X mental retardation protein (FMRP) ........................................................... 13 2.2.3 FMRP deficiency and NPCs ........................................................................................ 15 2.3 Alzheimer disease (AD) .................................................................................................... 16 2.3.1 General features of AD ................................................................................................. 16 2.3.2 Proteins involved in AD pathology ............................................................................ 18 2.3.3 Neuroinflammation ...................................................................................................... 22 2.3.4 Oxidative stress (OS) .................................................................................................... 23 3 AIMS OF THE STUDY ............................................................................................................ 28 4 MATERIALS AND METHODS.............................................................................................. 29 4.1 Brain tissue (I-IV) ............................................................................................................... 29 4.2. Isolation and culture of NPCs (I-IV) ............................................................................... 29 4.3 Lentiviral transduction (III-IV) ......................................................................................... 30 4.3.1 Quantitative real-time PCR (IV) ................................................................................. 30 4.4 In vivo experiments (I, III-IV) ............................................................................................ 30 4.4.1 Animals ......................................................................................................................... 30 4.4.2 Intracerebral NPC transplantation (III)...................................................................... 31 4.4.3 BrdU injections (I, IV) .................................................................................................. 31 4.4.4 Kainic acid injections (IV) ........................................................................................... 31 4.4.5 Brain processing (III – IV) ........................................................................................... 31 4.4.6. Immunocytochemistry for brain slices (III – IV) ...................................................... 32 4.5 In vitro experiments (I-IV) ................................................................................................ 32 4.5.1 Ca2+ -imaging (I-II) ....................................................................................................... 32 4.5.2 Cell proliferation, neurite length and cell-body volume analysis (I) ...................... 33 4.5.3 A treatments (III-IV) .................................................................................................. 33 4.5.4 Immunocytochemistry for NPCs................................................................................ 34 4.6 Analysis of immunorectivities.......................................................................................... 35 5 RESULTS ................................................................................................................................. 36 5.1 FMRP deficiency has impact on differentiation of NPCs (I).......................................... 36 5.1.1 FMRP deficiency alters neuronal differentiation of NPCs ....................................... 36 5.1.2 Neurotransmitter responses of differentiating FMRP deficient NPCs ................... 37 5.1.3 Production of new cells in FMRP deficient mice ...................................................... 37 5.2 Differentiating NPCs responded to neurotransmitters (II)............................................ 38 5.2.1 Neurotransmitter responses after 1 – 4 day differentiation ..................................... 38

XIV

5.2.2 Neurotransmitter responses after 5 - 6 day differentiation ...................................... 39 5.2.3 Neurotransmitter responses after 8 day differentiation ........................................... 39 5.3 AD mimicking genotype and environment affects the survival, migration and differentiation of neuronal progenitor cells (III) ..................................................................... 40 5.3.1 Survival and proliferation of engrafted NPCs .......................................................... 40 5.3.2 Survival and proliferation of A treated NPCs in vitro ............................................ 40 5.3.3 Migration of transplanted NPCs ................................................................................ 40 5.3.4 Migration of cultured neurosphere derived NPCs ................................................... 41 5.3.5 Phenotype of transplanted NPCs. .............................................................................. 41 5.3.6 Phenotypes of differentiated NPCs in vitro ............................................................... 41 5.3.7 Endogenous neurogenesis .......................................................................................... 42 5.4 Nrf2 mediates protection of NPCs against A toxicity and promotes endogenous neurogenesis (IV) ....................................................................................................................... 42 5.4.1 Nrf2 promotes NPC proliferation and differentiation in vivo .................................. 42 5.4.2 Nrf2 promotes NPC proliferation and differentiation but not migration in vitro .. 43 5.4.3 Nrf2 counteracts the negative effects of A 1-42 on NPC viability and neuronal differentiation ............................................................................................................................. 43 6 DISCUSSION .......................................................................................................................... 44 6.1 FMRP deficiency has effect on differentiation of NPCs (I) ............................................ 45 6.2 The role of neurotransmitters in differentiation of NPCs (II) ........................................ 47 6.3 The effects of AD genotype and AD-like neural environment on differentiation of cultured or transplanted NPCs (III).......................................................................................... 49 6.4 Transplanted NPCs stimulated endogenous neurogenesis (III).................................... 52 6.5 Nrf2 mediates the protection of NPCs against A toxicity and promotes endogenous neurogenesis (IV) ....................................................................................................................... 53 7 CONCLUSIONS AND SUMMARY ....................................................................................... 55 8 REFERENCES.......................................................................................................................... 57

XV

Abbreviations amyloid beta ACh acetylcholine AD Alzheimer´s disease AMPA -amino-3-hydroxy-5-methyl -4-isoxazolepropionic acid APdE9 transgenic AD mice carrying Swedish mutation in APP-gene and PS1-gene with deletion in exon 9 ApoE apolipoprotein E APP amyloid precursor protein ARE antioxidant response element sAPP soluble amyloid precursor protein ATP adenosine triphosphate BDNF brain-derived neurotrophic factor BrdU 5-bromo-2´ -deoxyuridine BSA bovine serum albumin 2+ Ca calcium ion 2+ [Ca ]i intracellular calcium concentration 2+ [Ca ]o extracellular calcium concentration CDK4 cyclin dependent kinase 4 cDNA complement deoxyribonucleic acid CNS central nervous system DAG diacylglycerol DCX doublecortin DG dentate gyrus DNA deoxyribo nucleic acid E embryonic day EGF epidermal growth factor ER endoplasmic reticulum ES embryonic stem cells FAD familiar Alzheimer´s disease FGF fibroblastic growth factor FMR1 fragile X mental retardation gene Fmr1 KO fragile X knockout FMRP fragile X mental retardation protein Fura-2AM fura-2-acetoxymethyl ester FXS fragile X syndrome GABA -aminobutyric acid GFAP glial fibrillary acidic protein GFP green fluorescent protein GLAST glutamate aspartate transporter Glu glutamate

G protein

guanine nucleotide binding protein GPCR G protein coupled receptor GSK3 glycogen synthase kinase 3 GWAS genome wide association study h hippocampal hnRNP heterogenous nuclear ribonucleoproteins IDE insulin degragating enzyme IP3 inositol 1,4,5-trisphosphate iPS induced pluripotent stem cells + K potassium ion Ki67 nuclear protein associated with cell proliferation KA kainic acid Keap1 Kelch ECH associated protein 1 KH domain K homology domain KO knockout LTD long-term depressio LTP long-term potentiation lv lateral ventricle LV lenti viral LY367385 mGluR1 antagonist MAP1B microtubule glutamate receptor 2+ Mg magnesium ion mGluR metabotropic glutamate receptor MPEP 2-methyl-6-(phenylethynyl) pyridine hydrochloride 2+ Na sodium ion NE norepinephrine NFT neurofibrillary tangles NGS normal goat serum NeuN neuronal specific nuclear protein NMDA N-methyl-D-aspartate NPC neural stem/progenitor cells Nrf2 nuclear factor erythroid 2related factor Nrf2-/Nrf2 deficient

XVI

NT P PB PBS PD PEST PI PIP2 PKC PLC PSEN OB Ox-A OxoM OS RGG box RGS RNA mRNA ROC ROS SAD SOC Sox2 SGZ SP SVZ SYTO 13 TRP Tuj-1 TUNEL VEGF VOC wt

neurotransmitter post natal day phosphate buffer phosphate buffered saline Parkinson’s disease penicillin-streptomycin propidiumiodide phosphatidyl-inositolbis-phosphate protein kinase C phospholipase C presenilin olfactory bulb orexin-A oxotremorine oxidative stress arginine-glycine-glycine box which binds RNA regulators of G-protein signaling ribonucleic acid messenger ribonucleic acid receptor-operated calcium channel reactive oxygen species sporadic Alzheimer´s disease store-operated calcium channel transcription factor, important for self-renewal subgranular zone substance P subventricular zone green fluorescence nucleic acid stain transient receptor potential channels III -tubulin terminal deoxynucleotidyl transferasemediated dUTP nick end labeling vascular endothelial growth factor voltage-operated calcium channel wild type

1 Introduction Currently there are no effective cures for neurodegenerative diseases. The hope to develop a completely new therapeutic approach for these severe diseases was realized in 1992 when Reynolds and Weiss isolated a cell population in the adult brain with stem cell characteristics. These cells were defined as multipotent neural stem/progenitor cells (NPCs), which have the capacity to differentiate into all neuronal cell types and glia, both in vitro and in vivo. In the adult brain, NPCs locate in two restricted regions the dentate gyrus (DG) in hippocampus (subgranular zone, SGZ), and in the wall of the lateral ventricle (subventricular zone, SVZ) where they continuously produce new neurons throughout life (Gage 2002). Adult neurogenesis is speculated to be important in learning and memory, however, the exact role is still unresolved as well as how it is regulated. Various growth factors, hormones and neurotransmitters are thought to be the main regulators in adult neurogenesis (Hagg 2005; Pathania et al 2010). In addition, pathological stages and aging have an impact on neurogenesis. For example, in Alzheimer´s disease (AD) and in Fragile X syndrome (FXS), endogenous neurogenesis and neuroplasticity are impaired and poorly functioning endogenous neurogenesis may contribute to learning and memory impairment (Taniuchi et al., 2007; Lazarov et al., 2011). Cultured human and mouse NPCs provide a unique cell source to study the neural mechanisms of pathogenesis of various human neurological disorders such as FXS. The loss of neurons is characteristic to disorders such as AD and Parkinson´s disease (PD). NPCs can be potentially used for therapeutic purposes in neurodegenerative diseases in several ways. A brain´s own neurogenesis has limited recovery capacity and is unable to compensate for neuronal damages completely. The recovering capacity can be potentially enhanced in two alternative ways: by transplantation of exogenous NPCs directly into the damaged brain area, or by stimulation of the patient’s own NPCs pharmacologically to proliferate, differentiate, and replace or recover damaged neurons. The source of NPCs for cell transplantation is still under discussion. NPCs can be isolated directly from fetal tissue, differentiated from embryonic stem cells (ES), or reprogrammed from somatic tissue as pluripotent stem cells (iPS). Taking into account the advantages and disadvantages, the most promising alternative is iPSderived NPCs because their availability is unrestricted, and when derived from the patient´s own cells, NPCs do not cause rejection. However, iPS-derived NPCs obtained from patients carrying a mutation are genetically similar to all of the patient’s cells. In some cases, the mutation may have an effect on the function of transplanted cells and thus reduce the effectiveness of stem cell therapy. Another possibility is to stimulate a patient´s own neurogenesis. In several diseases, such as AD, the levels of growth factors may be altered which may also alter endogenous neurogenesis and their recovering capacity. Restoring the levels of

2

growth factors by using environmental enrichment (e.g. de novo synthesis or delivery of growth factors) at the site of disease may have neuroprotective effects. As an example, delivery of brain derived growth factor (BDNF) into the brain has been shown to be beneficial in animal models of AD (Blurton-Jones et al., 2009). Before the cell replacement therapy of the neurodegenerative disease can be successfully applied in clinics, the mechanisms of NPC survival, migration, and differentiation need to be well understood. It is also equally important to clarify how the pathology of the disease being treated may affect the transplanted cell survival, migration, and differentiation. The main characteristics of AD brain are accumulation of neurotoxic A plaques and tau aggregations, neuroinflammation and reactive oxygen species (ROS). These may have impact on delivered as well as endogenous NPCs. NPCs and their differentiation form the link between all of the four studies presented in this thesis. We chose several approaches to study NPC differentiation in vitro and in vivo. In addition, we utilized genetic mouse models to study NPC differentiation and neurogenesis in neurodegenerative diseases. Here, we show that several neurotransmitters are involved in the early stages of neuronal maturation. We also used FXS and AD mouse models with typical genetic mutations causing these disorders. Moreover, we investigated how fragile X mental retardation protein (FMRP) deficiency in FXS affects the differentiation of both human and mouse NPCs. Next, we provide information about the effects of both AD-linked genotype as well as AD-mimicking environment on survival, proliferation, migration, and differentiation of transplanted NPCs. Finally, we investigated the ways to alleviate AD pathology by studying NPCs and the transcription factor Nrf2. We show that together with the neuroprotective effects, Nrf2 also has an important function to promote endogenous neurogenesis.

3

2 Review of the literature 2.1 ADULT NEURAL STEM/PROGENITOR CELLS (NPC) 2.1.1 Adult neurogenesis Adult neurogenesis is a process which includes proliferation, differentiation, migration, and synaptic integration of newly produced neurons. It was traditionally thought that neurogenesis occurs only during embyonic and perinatal stages of mammalian development. In the 1960s, a new method of 3H-thymidine autoradiography to detect cells undergoing DNA synthesis in preparation for mitosis was generated. This marker for proliferating cells was immediately utilized by Altman (1962, 1963, 1966, 1969) and Altman and Das (1965, 1966, 1967) who were the first to report new neurons in the adult rat and cat brain. Fifteen years later, these findings were confirmed by Kaplan´s ultrastructural studies (Kaplan and Hinds, 1977; Kaplan, 1981; Kaplan and Bell, 1983, 1984). Even thought further evidence for proliferating neurons, and thus for the existence of NPCs in the dentatus gyrus of adult mammalian brain, came from studies by Stanfield and Trice (1988) and Gue´neau et al., (1982), the pioneering work of these investigators encountered considerable skepticism and was not taken seriously in the field. It was not earlier than in the 1990s when neurogenesis in the adult rodent was finally established. In 1992, Reynolds and Weiss isolated proliferating cells with stem cell characteristics in the adult brain and the old dogma of “no new nerve cells formed after birth” could be forgotten. The finding of NPCs and the idea that they theoretically are able to produce new nerve cells and perhaps aid in recovery of brain damage, provided an opportunity to explore an entirely new research field. In addition, culture and differentiation of these cells opened a new field for in vitro studies to study the mechanisms of neurogenesis and their unique potential for future therapy in neurological disorders. In the adult mammalian brain, there are two spatially restricted neurogenic regions: subventricular zone (SVZ) in the wall of lateral ventricle; and subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus. At these two sites of the brain, NPCs proliferate and produce new neurons throughout life (Alvarez-Buylla and Lim, 2004; Lie et al., 2004). Restricted populations of NPCs are also found in other brain areas such as in the striatum, cortex, optic nerve, septum, corpus callosum, spinal cord, retina, hypothalamus, and caudal portions of SVZ, (Palmer et al., 1995, 1999; Weiss et al., 1996; Shihabuddin et al., 1997; Lie et al., 2002). NPCs are classified as being multipotent stem cells meaning that they are highly undifferentiated cells. They have self-renewal capacity and they may generate all neural cell types: neurons, astrocytes, and oligodendrocytes in their own environment (Gage 2002; Galli et al., 2003; Bull and Bartlett 2005; Kornblum 2007). Compared to pluripotent stem cells, which are able to differentiate into all cell types of the body, the differentiation potential of multipotent cells is more restricted

4

(Kazanis 2011). The population of NPCs includes not only multipotent stem cells but also bipotent cells, which are called progenitor cells. Progenitor cells show a more restricted proliferation and self-renewal capacity compared to multipotent cells. Because it is impossible to distinguish multipotent and bipotent cells, they are together known as NPCs (Bull and Bartlett 2005). 2.1.2 Neurogenic regions The subventricular zone (SVZ) located along the lateral wall of the lateral ventricles shows the highest neurogenic rate in the adult brain. In the rat brain, as many as 30 000 new neuroblasts can be produced each day (Alvarez-Byulla et al., 2000). New SVZ neuroblasts migrate along the rostral migratory stream at their final destination into the olfactory bulb (OB) and differentiate into local periglomerular inter neurons (Lois and Alvarez-Buylla 1994; Belluzzi et al., 2003). The subgranular zone (SGZ) in the hippocampal dentate gyrus (DG) is the second most active neurogenic area and generates about 9 000 neuroblasts per day. In contrast to olfactory bulb, hippocampal neuroblasts in SGZ migrate only a short distance into the inner granule cell layer and differentiate to neurons (Cameron and McKay, 2001). A more detailed analysis of SVZ neurogenesis showed that one SVZ NPC population consists of relatively quiescent NPCs. These quiescent cells give rise to actively proliferating cells, which are also called transit amplifying progenitors (Doetsch et al., 1996), which then give rise to immature neuroblasts. Neuroblasts migrate along the rostral migratory stream into the OB and differentiate into interneurons, especially granule and periglomerular cells (Lois and Alvarez-Buylla 1994; Belluzzi et al., 2003). SVZ progenitors express Nestin and Sox2 and interestingly, despite their stem cell characteristics, also express glial fibrillary acidic protein (GFAP) and glial glutamate transporter (GLAST). Both of these markers are typically associated with differentiated astrocytes (Doetsch et al., 1997; Colak et al., 2008; Kriegstein and Alvarez-Buylla 2009). In vitro, proliferating NPCs have neurosphere-forming ability. Neurospheres are freely floating cell clusters (Doetsch et al., 2002). Analysis of SGZ NPCs has demonstrated that there are early precursor cells which have morphological and antigenic features similar to radial glia. Similar to the SVZ, there is an abundant population of slowly proliferating progenitors also in the SGZ which express GFAP, Sox2, and Nestin. Slowly proliferating progenitors then give rise to fast proliferating precursor nonradial cells, which still express Sox2 and Nestin, but not GFAP. These cells give rise to neuroblasts, which express doublecortin (DCX) and differentiate into glutamatergic dentate granule cells (Seki and Arai 1993; Fukuda et al., 2003; Seri et al., 2004; Mu and Gage 2012). Later, more mature neurons express neuronal specific nuclear protein (NeuN) (Figure 1).

Cerebellum RMS

SVZ

CA LV

DG

SGZ

SGZ

Time Ma rkers

Hippocampus

Nich e cells existing granule neurons

Endothelial cells

Glia cell

S GZ

Olfactory bulb

Developm ental process

Cerebral cortex

Granular layer M ole cular lay er

5

Stem cell maintenance self-renewal Nestin GFAP

Neuronal fate specification

DCX PSA-NCAM

Newborn neuron maturation integration

NeuN

Tbr2 ~25 h

~4 days

4-10 days (maturation) 2 - 4 weeks (integration)

Figure 1. (A) Adult NPCs are found in the SVZ and SGZ of the mammalian brain. (B) Shematic representation of the dentate gyrus SGZ which is the other main region in the adult mammalian brain where new neurons are generated continuously throughout life (modified from Ma et al., 2009a and Ming and Song, 2005).

Taken together, in both adult neurogenic regions, SVZ and SGZ NPCs show first astroglial morphology, then generate precursors of neuronal commitment (neuroblasts), which are able to migrate to their targets, differentiate to mature neurons, and finally integrate into the neural network. Despite the fact that thousands of new neurons are generated per day, only a small proportion of these survive and finally integrate into neural circuits (Kemperman et al., 2004; Ma et al., 2009a,b, Cameron 2001). Dead and dying neurons are removed by phagocytosis glial cells which play the major phagocytic role in the central nervous system. In addition, DCX positive NPCs have been shown to play an unexpected phagocytic role, but this may occur in only specific neurogenic areas (Lu et al., 2011). 2.1.3 Regulation of adult neurogenesis Postnatal neurogenesis is a dynamic process modulated by various genetic, environmental and pharmacological factors. The specific neurogenic area, where NPCs are laying is also called a “niche”, which plays a dominant role in the regulation of NPCs. The niche comprises various cell types including endothelial cells, astrocytes, ependymal cells, microglia, mature neurons, and vascular cells which all have their own roles in the regulation of proliferation, migration, fate specification, maturation, or synapse formation of NPCs (Palmer et al., 2000; Shen et al., 2008; Tavazoie et al., 2008; Kojima et al., 2010; Morrens et al., 2012; Barkho et al., 2006; Lim et al., 2006; Ekdahl et al., 2003; Sierra et al., 2010). Many factors are known to clearly influence the rate of neurogenesis. However, the exact mechanism of how adult neurogenesis is regulated is not completely

6

elucidated. Regulators could be categorized into intracellular and extracellular players. Intracellular players are for example cell-cycle regulators, transcription factors, genetic, and epigenetic factors (Zhao et al., 2008). The main extracellular players are cytokines, hormones and growth factors including neurotrophins (Pathania et al., 2010; Zhao et al., 2008). Several classical neurotransmitters have also been reported to directly or undirectly regulate NPCs (Sahay and Hen, 2007; Warner-Schmidt and Duman, 2006). Other important factors which are known to affect the rate of neurogenesis are for example environmental enrichment and exercise, which increases neurogenesis (Kemperman et al., 1997; van Praag et al., 1999), whereas aging (Bizon and Gallagher 2005), stress (Lemaire et al., 2000), oxidative stress (Yoneyama et al., 2011), and a number of toxins and drugs (Eisch and Harburg 2006; Nixon and Crews 2002) have shown the opposite effects. Many neurological diseases associate with alterations in neurogenesis (Grote and Hannan 2007). Neuronal injury and ischemia have been shown to stimulate NPCs to become active and migrate towards the lesioned area (Yoneyama et al., 2011; Arvidsson et al., 2002; Nakatomi et al., 2002) and neurological diseases including AD and FXS have been associated with either impaired or increased neurogenesis (Choi et al., 2008; Luo et al., 2010; Lazarov et al., 2011; Taniuchi et al., 2007; Jin et al., 2004). There is a body of evidence that new neurons are generated in the adult brain and the rate of neurogenesis is highly sensitive to various physiological and environmental stimuli. However, the exact role of adult neurogenesis has not been fully elucidated and is still under intensive investigation. Several studies suggest that newly produced neurons in the hippocampus may play an important role in learning and memory (Imayoshi et al. 2008; Kemperman and Gage 2002; Lledo et al., 2006; Deng et al., 2009; Gould et al., 1999; Tronel et al., 2010). It has been shown that boosting adult neurogenesis facilitates pattern separation and memory when investigated using Morris water maze analyses (Sahay et al., 2011; Stone et al., 2011). In addition, decreased neurogenesis results in cognitive impairment which is typical in aging and particularly in AD patients (Clelland et al., 2009; Lazarov et al., 2010). The role of the SVZ neurogenesis is less clear compared to hippocampal neurogenesis. Previous studies suggest that SVZ neurogenesis is involved in synaptic plasticity and function in olfaction (Nissant et al., 2009; Doetsch and Hen 2005). Recently, it has been also shown that both SGZ and SVZ neurogenesis are involved in offspring recognition (Mak and Weiss 2010). 2.1.4 Cultivation of NPCs in vitro and therapeutic possibilities After Reynolds and Weiss (1992) demonstrated the methods to isolate cultured NPCs, NPCs have been routinely used in in vitro experiments. NPCs can be kept in an undifferentiated stage by adding growth factors: epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF). In the presence of growth factors in serum-free media, NPCs proliferate and form freely floating cell aggregates called ´neurospheres´. Expanded neurospheres are dissociated to single cells which form

7

secondary neurospheres in culture. This procedure can be repeated several times to obtain an exponentially growing cell population (Galli et al., 2003). Actually, single neurospheres are not homogenous populations of neural stem cells. A neurosphere contains only 10-50% of cells with real stem cell characteristics; the rest of the cells are differentiating progenitor cells and even more mature cells (Galli et al., 2008). Removal of mitogens from the culture media causes spontaneous differentiation of NPCs and the cells differentiate into neurons, astrocytes, and oligodendrocytes. Cultured human or mouse NPCs offers excellent tools to study and understand the mechanisms of normal neurogenesis as well as its disturbances in various neurological diseases. Especially, NPCs offer valuable tools for studying the mechanisms underlying neurogenesis in disorders, which are caused by single-gene defects such as FMRP mutation in FXS. By using genetically modified mouse models or human NPCs generated from affected aborted fetuses, it is possible to study how genetic as well as environmental factors affect the behavior of NPCs (Bhattacharyya et al., 2008a). NPCs can also be useful for drug screening, when testing the drugs which have effects on developing neurons (Ma et al., 2009b). In addition, NPC technologies and stem cell therapy have become a potential option to treat several neurodegenerative disorders. Because a typical feature of several neurodegenerative disorders such as PD, AD, spinal cord injury, and stroke is the irreversible loss of neurons in the CNS (Lindvall and Kokaia 2006), a possible treatment could be stem cell transplantation. Transplanted in vitro-expanded NPCs are either expected to directly replace damaged or lost neurons or they may have trophic function which support the neurons at risk to die or even stimulate endogenous NPCs (Park et al., 2010). Several experiments carried out in animal models have given promising results, which encourage the tests of stem cell therapy also in humans (Lindvall and Kokaia 2006; Shetty and Hattiangady 2007; Einstein and Ben-Hur 2008; Blurton-Jones et al., 2009; Yamasaki et al., 2007). The best NPC source for stem cell therapy is still under discussion. Human NPCs can be isolated from aborted fetuses, derived from pluripotent embryonic stem cells (ES), or induced pluripotent stem cells (iPS). ES cells are isolated from the inner cell mass of a developing blastocyst. ES cells are pluripotent and may have the capacity to give rise to cells from all three germ layers. Pluripotent stem cells are also able to form teratomas. After differentiation into more restricted NPCs they lose the ability to form teratomas. Multipotent NPCs could also be isolated from aborted fetuses directly. In both alternatives, ES-derived NPCs or NPCs isolated from aborted fetuses, are associated with ethical problems and cause host-versus-donor rejections, when the transplanted cells from outside sources cause immunereactions and cells will die (Bithell and Williams 2005). In addition, because the availability of aborted features and developing human blastocysts is limited, the amount of ES- and fetusderived NPCs is restricted. Thus, a feasible cell source for cell therapies is NPCs derived from the patient´s own tissue. Therefore, the best option for a stem cell source probably are the iPS-derived NPCs. iPS cells are first generated from a patient’s own somatic cells by reprogramming them back to the pluripotent stage

8

(Takahashi and Yamanaka 2006; Takahashi et al., 2007). These iPS cells showed similar ability to form teratomas as ES. When the iPS cells are differentiated to NPCs and thereafter to neural cells they lose the ability to form teratomas (Wernig et al., 2008). Compared to ES cells, because the starting material is the patient’s own mature cells, it is possible to avoid the rejection problems and the usage of immunosuppressive drugs. The use of the patient’s own somatic cells also excludes ethical concerns. However, problems can occur in patients with genetic disease if the mutations which cause the disease also have negative effects on functions of NPCs (Choi et al., 2008). It is known that many pathological conditions such as ischemia and stroke increase endogenous neurogenesis which is, however, not sufficient to fully compensate for all neuronal damage (Kokaia and Lindvall 2003). This observation raises the notion that it could be possible to activate a patient’s own neurogenesis and cell mobilization during or after the pathological stages (Ma et al., 2009b; Gage 2004). Even though the number of newly produced neurons probably is relatively small, compared to dead and damaged neuronal cells, the stimulation may still enhance the regenerative and recovery processes during the disease (Mu and Gage 2011). One important group of molecules, which regulates neurogenesis are growth factors. The levels of growth factors have been shown to be altered in several pathological states. For example in AD brain, the decreased levels of BDNF may contribute to the progression of the disease (Blurton-Jones et al., 2009). In previous studies, enhanced levels of bFGF, BDNF, and vascular endothelial growth factor (VEGF) have been shown to stimulate NPCs and thereby they may alleviate the symptoms of diseases in animal models (Nakatomi et al., 2002; Kim et al., 2009; Blurton-Jones et al., 2009). Other factors that could be useful for stimulation of patient own neurogenesis are cytokines, certain drugs, and even enhanced physical activity (Mu and Gage 2011). The stimulation of a patient’s own NPCs offers many advantages when compared to cell transplantation. By stimulating endogenous neurogenesis it is possible to avoid rejection complications, location-specific recruitment is better achieved and generated progenitors possibly maturate and integrate better than exogenously transplanted NPCs (Ma et al., 2009b). However, the mechanisms which regulate NPCs and modulate endogenous neurogenesis should be fully characterized before any clinical application. Despite intensive studies, the mechanisms which regulate NPCs are still poorly understood. Furthermore, it is also equally important to understand how the diseased brain environment and genetic mutations affect the behavior of NPCs. 2.1.5 Neurotransmitters in the regulation of NPCs The classical role of neurotransmitters is to mediate chemical communication between neurons. In addition, neurotransmitters play an important role in cell development during the embryonic stage (Nquyen et al., 2001; Emerit et al., 1992) and may also have a role in the regulation of adult neurogenesis (Gould et al., 1994; Cameron et al., 1995; Hagg et al., 2009). Neurotransmitters could even be master

9

regulators, that control various stages of neurogenesis (Platel et al., 2010b). However, the importance of neurotransmitters in the regulation of NPCs is largely unknown and further investigation is needed. Neurotransmitters are speculated to affect differently various NPC subpopulations and stages of neurogenesis (Merkle et al., 2007). They are also speculated to have an indirect role on NPCs. For example, the usage of a serotonin reuptake inhibitor increases BDNF, which further increases neurogenesis (Duan et al., 2008). Activation of several neurotransmitter receptors stimulates the elevation of intracellular calcium (Ca2+) levels, which then triggers plastic changes in neurons (Spitzer et al., 2004; Deisseroth et al., 2004). Ca2+ ion is a universal intracellular messenger that controls a wide range of cellular functions such as gene transcription, neurotransmitter release, vesicular secretion, activation of enzymes, apoptosis, fertilization, and many more. Even though Ca2+ is a very common intracellular messenger, it also mediates very specific functions. Specificity is possible by using different entry, exit, and distribution routes inside cells. Intracellular Ca2+ elevation is mediated either by transporting extracellular Ca2+ via ionotropic receptors inside the cell or via G protein-coupled receptors (GPCR) which then activate intracellular Ca2+ release from intracellular stores (Clapham 1995; 2007; Berridge et al., 2003). Ionotropic receptors Ca2+ enters cells by passing through specific Ca2+ channels. The movement between inside and outside the cell is dependent on the electrochemical gradient. In excitable cells, voltage-operated Ca2+ -channels (VOCs) generate the fast Ca2+ fluxes which then control fast cellular processes. This group of channels includes also channels which respond to external stimuli. They are called receptor-operated Ca2+ -channels (ROCs). For example the N-methyl-D-aspartate (NMDA) receptor responds to external glutamate, opening the Ca2+ channel, allowing Ca2+ to flow inside the cell, and then link to other signaling components. Other channels are second-messenger-operated channels (SMOCs) that are controlled by internal messengers and store-operated channels (SOCs) which open in response to depletion of internal Ca2+ stores. Many of these channels are classified into the large transient receptor protein (TRP) ion-channel family, which are known to be involved in important slow cellular processes such as cell proliferation (Berridge et al., 2003). G protein coupled receptors (GPCR) GPCRs are a large membrane protein superfamily, which mediate a wide variety of cellular responses, including the responses of various neurotransmitters. Most neurotransmitters have been shown to activate G protein coupled metabotropic receptors and, in contrast to ionotropic receptors, mediate slow responses (Wettschureck and Offermanns 2005). GPCRs form a seven transmembrane -helical bundle which has three intra- and extracellular loop regions. GPCRs are divided into six protein families based on their structure and sequence similarities:

10

rhodopsin receptors (A), secretin receptors (B), glutamate receptors (C), fungus pheromone receptors (D), cAMP receptors (E), and frizzled/smoothened receptors (F) (Rosenbaum et al., 2009; Tuteja 2009). Biological effects occur when neurotransmitters bind to the GPCR, recruit heterotrimeric G proteins (GTP-binding protein), and activate GTP-GDP exchange. This exchange results in the dissociation of the GTP-bound -subunit and -subunit from the GPCR (Karnik et al., 2003). Both G -subunit and G -subunit can independently mediate several signaling pathways such as activation of phospholipases, modulation of adenylate cyclases, and gating of ion channels (Gether 2000). G proteins are also divided into four families: Gq/G11, Gi/Go, Gs, and G12/G13 proteins. In the nervous system, Gq/G11 are widely expressed and modulate neuronal fuctions (Wettschureck and Offermanns 2005). Activation of the specific neurotransmitter receptor activates usually Gq/G11 protein, which then couples to phosholipase C (PLC) and leads to hydrolysis of phosphatidyl-inositol-bis-phosphate (PIP2). After hydrolytic cleavage, PIP2 generates inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which then activates protein kinase C (PKC). IP3 mediates Ca2+ mobilization from intracellular stores, which finally promotes several Ca2+-dependent processes such as activation of enzymes, secretion, or gene transcription. Inside the neuronal cells, the main intracellular Ca2+ stores are in endoplasmic reticulum (ER). After Ca2+ release it is pumped back into the ER via sarcoplasmic-reticulum-associated Ca2+ ATPase (Berridge et al. 2000; Berridge et al., 2004). Neurotransmitters Neurotransmitters are classified as classical neurotransmitters such as -amino butyric acid (GABA) and glutamate (Glu), and as neuromodulators such as dopamine, serotonin and acetylcholine (ACh) (Young et al., 2011). In previous studies, at least dopamine, serotonin, GABA, ACh, noradrenaline (NE), and glutamate have been shown to have effects on different stages of NPC differentiation. However, the regulation of SVZ neurogenesis by neurotransmitters is better understood than the role of neurotransmitters in SGZ neurogenesis. Both dopamine and serotonin are known to regulate mood, motivation, and movement. In addition, both have effects on SVZ neurogenesis. Dopaminergic afferents have been found to be in direct contact with transit amplifying cells in SVZ (Hoglinger et al., 2004) and cultured SVZ neurospheres have been demonstrated to express dopamine receptors (Coronas et al., 2004; Hoglinger et al., 2004; Winner et al., 2009). In mouse models of PD, when dopaminergic inputs are prevented, also proliferation and differentiation of SVZ NPCs have been shown to be decreased (Borta and Hoglinger 2007; O´Keeffe et al., 2009a,b; Cova et al., 2010; Freudlieb et al., 2006; Baker et al., 2004). Furthermore, increased SVZ neurogenesis has been observed by administration of a dopaminergic agonist (Yang et al., 2008). Serotonin regulates positively both SGZ and SVZ NPC proliferation and neurogenesis throughout several serotonergic receptor subtypes (Banasr et al., 2004; Brezun and

11

Daszuta 1999). In addition, direct in vivo infusion of serotonin increases proliferation and neurosphere formation of cultured SVZ NPCs (Hitoshi et al., 2007). GABA is the main inhibitory neurotransmitter in adult brain. Functional GABA receptors are identified also on astrocytes and neuroblasts from SVZ (Stewart et al., 2002; Nguyen et al., 2003; Gascon et al., 2006). Striatum, which is located adjacent to the SVZ, is mainly composed of GABAergic neurons, therefore it is not surprising that GABA has a negative effect on proliferation and differentiation of SVZ NPCs (Nguyen et al., 2003; Liu et al., 2005). One important neurotransmitter, glutamate, acts either via ionotropic receptors AMPA, kainate, and NMDA (Hollman and Heinemann 1994), or via group I-III metabotropic receptors 1-8 (mGluR1-8) (Goutinho and Knopfel 2002). Both ionotropic and metabotropic receptor types were identified in SVZ NPCs (Brazel et al., 2005; Young et al., 2011). Neuroblasts express AMPA, kainate, NMDA and mGluR5 receptors (Platel et al., 2007; Platel et al., 2008b; Platel et al., 2010a) and interestingly, during the migration along rostral migratory stream, the number of NMDA receptors in neuroblasts is increased (Platel et al., 2010a). The expression of various types of glutamate receptors in NPCs indicates that these receptors mediate also various cellular functions. Glutamate has already been shown to affect proliferation, migration, and cell survival. Inhibition of kainate receptors stimulated the migration of neuroblasts (Platel et al., 2008a, b). Mice without functioning mGluR5 or mice with mGluR5 antagonist showed a reduced number of proliferating cells in SVZ (Di Giorgi-Gerevini et al., 2004, 2005). Moreover, mice without functional NMDA receptors resulted in increased apoptosis of migrating neuroblasts which eventually led to reduced neurogenesis in olfactory bulb (Platel et al., 2010a; Lin et al., 2010). Finally, important neurotransmitters which have been shown to affect NPCs are ACh and NE. ACh signals are mediated via ionotropic nicotinic or metabotropic muscarinic ACh receptors. Even though the number of cholinergic afferents is relatively small, they are widespread in the brain (Young et al., 2011). There are several studies which show that the cholinergic system and ACh are involved in the regulation of adult hippocampal neurogenesis, especially proliferation of NPCs. Cholinergic stimulation increased proliferation of hippocampal NPCs (Itou et al., 2011). Lesions in medial septum systems have been shown to decrease the proliferation of NPCs (Cooper-Kuhn et al., 2004; Mohapel et al., 2005; Van der Borght et al., 2005). Increased ACh obtained via use of a acetylcholine esterase inhibitor also increased proliferation of NPCs (Mohapel et al., 2005; Narimatsu et al., 2009). Also, in vitro stimulation of muscarinic ACh receptor was found to be involved in proliferation and differentiation of NPCs (Zhou et al., 2004). Recently, NE has been shown to affect the proliferation of SGZ NPCs (Jhaveri et al., 2010; Masuda et al., 2011).

12

2.2 FRAGILE X SYNDROME (FXS) 2.2.1 Clinical features and neuropathology of FXS Fragile X syndrome (FXS) is a common X-linked inherited form of human mental disability; one in every 4000 males and one in every 8000 females suffer from this syndrome (Huber et al., 2002). FXS was first documented in 1943 as a familiar form of X chromosome-linked cognitive impairment. The disease was called Martin-Bell syndrome until 1991 when the Fragile X Mental Retardation 1 (FMR1) gene was found and the disease was renamed FXS (Fu et al., 1991; Verkerk et al., 1991). The severity and symptoms of the disease vary widely between the FXS individuals and also between the genders. Even though both males and females can be affected, the symptoms and phenotypes are typically more severe in males. The severity of dysfunctions in females is dependent to the degree of X-inactivation on the abnormal chromosome (Willemsen et al., 2011). Typical characteristics of FXS males are recognizable long face, large ears, hyperextensible joints, and enlarged testicles. Delayed speech and language development often leads to the diagnosis and the phenotype includes mild to severe intellectual disability, autistic behavior, hyperactivity, attention deficits, social anxiety, sensory-processing problems, and epilepsy. Postmortem analysis of FXS brains has revealed morphological changes in dendritic spines (Bear et al., 2004; Wilson and Cox 2007). Dendritic spines are small membranous protrusions from dendrites of neurons, which receive input from the synapse of an axon. They act as storage sites, support the electrical signal transmission, increase the surface area of dendrites, and thereby increase the number of possible contacts with other nerve cells (Yuste 2011). Dedritic spines also have been shown to express GluRs (e.g. AMPA and NMDA receptors) which mediate a wide variety of signals. Cognitive function, motivation, learning, memory, and long-term potentiation (LTP) are especially dependent on spine plasticity. During normal neurogenesis dendritic spines mature or they are pruned (eliminated). If the maturation or pruning of spines is disturbed, spines show immature morphological features (e.g. long “necks” or lack of “head”), which then may also alter signal transduction (Chonchaiya et al., 2009). Correct regulation of the morphology of dedritic spines is important and the morphological abnormalities are widely associated with mental retardation (von Bohlen and Halbach, 2010). Dendrities in many brain regions of FXS individuals have morphologically longer, thinner, and otherwise immature spines. These morphological changes were long thought to indicate defective spine pruning (Bear et al., 2004; Wilson and Cox 2007). Recently, it was shown that both formation and elimination of spines (so called spine turnover) is increased in a mouse model of FXS (Fmr1 KO mice) compared to wt mice. In addition, Fmr1 KO mice have a higher amount of instable, so called transient spines, with smaller head diameter and longer neck compared to stable spines. A high number of transient spines of Fmr1 KO mice may contribute to the immature spine phenotype in Fmr1 KO mice (Pan et al., 2010).

13

Numerous studies have shown that FXS is a monogenic disorder and the loss of function of the FMR1 gene underlies the pathogenesis of FXS. Over 95 % of FXS cases are caused by the CGG trinucleotide repeat mutation (O´Donnell and Warren, 2002). CGG repeats are observed in the 5´untranslated region of the FMR1 gene. The length of the CGG trinucleotide repeat varies even within a normal population. During maternal transmission, CGG repeats may become unstable resulting in the expansion of the repeat in the next generation. In unaffected individuals, the CGG region contains 5 – 50 repeats, whereas premutation carriers have 50 – 200 CGG repeats. In a full mutation of FXS individuals, CGG repeats are massively increased and show over 200 repeats. The full mutation leads to methylation of CGG repeats and FMR1 promoter which then silences the FMR1 gene and causes the loss of gene expression and finally loss of FMRP results in the phenotype of FXS (Ashley et al., 1993; Verkerk et al., 1991; Pfeiffer and Huber 2009; Santoro et al. 2011; Willemsen et al., 2011). In premutation carriers, the level of FMR1 mRNA has been shown to be increased; but surprisingly, the levels of FMRP have been reported to be decreased (Tassone et al., 2007). Having intermediate numbers of CGG repeats does not result in FXS, but may cause two other disorders. Premutation carrying females have a risk for fragile X-related primary ovarian insufficiency which leads to ovarian failure during the teenage years and approximately 5 years earlier menopause. Ovarian dysfunction depends on repeat size, but the molecular mechanisms behind this disease are still largely unknown (Allingham-Hawkins et al., 1999; Murray 2000; Willemsen et al., 2011). Still another disease reported in premutation carriers is fragile X-associated tremor/ataxia syndrome, which is a late-onset neurodegenerative disorder. The symptoms of this disease vary greatly between premutation carriers. Characteristics of this disease together with tremor and ataxia are progressive cognitive decline (Jacquemont et al., 2004). To study the molecular mechanisms causing FXS, a Fmr1 knockout (KO) mouse model is widely used. These mice completely lack the expression of FMRP and they show similar morphological abnormalities in synapse structures and altered synapse function as FXS individuals (Comery et al., 1997; Braun and Segal 2000; Irwin et al., 2000; Nimichinsky et al., 2001; Galvez et al., 2003). In addition, Fmr1 KO mice show deficits in spatial learning, hyperactive behavior, and audiogenic epilepsy (Bakker et al. 1994; Kooy et al. 1996; Paradee et al. 1999; Dobkin et al. 2000; Chen and Toth 2001; Nielsen et al. 2002; Frankland et al. 2004; Qin et al. 2005; Spencer et al. 2011). 2.2.2 Fragile X mental retardation protein (FMRP) FMRP belongs to a family of RNA binding proteins, nuclear ribonucleoproteins, which have strong affinity to RNA (Van de Bor and Davis 2004). FMRP contains three RNA-binding motifs: two K homology domains and an arginine-glycineglycine box which binds to RNA in a sequence-specific manner via these domains (Blackwell et al., 2010; Ashley et al., 1993). However, other types of interactions act

14

through adaptive brain cytoplasmic 1 protein, which first binds to the FMRP and then interacts with the FMRP targets (Zalfa et al., 2003; Zalfa et al., 2005). FMRP is ubiquitously expressed both in fetal and adult tissues, most abundantly in the brain and testes (Devys et al., 1993). In the brain, the highest FMRP expression is detected in neurons, where it is found in the neuronal cell body as well as in dendrites and spines. FMRP is associated with ribosomes both in cytoplasmic and endoplasmic reticulum (ER) (Antar et al., 2004; Feng et al., 1997; Wilson and Cox 2007). FMRP is also found in the nucleus, indicating that this protein shuttles between the nucleus and cytoplasm (Devys et al., 1993; Van de Bor and Davis 2004). In nucleus, FMRP has been suggested to bind to its own mRNA and thereby facilitate (chaperone) transportation of mRNA out of the nucleus (Eberhart et al., 1996). It has been estimated that FMRP interacts with 4 % of brain mRNAs (Brown et al., 2001). These mRNAs include many mRNAs which are involved in neuronal and synaptic transmission and they also include several candidate genes for autism (Darnell et al., 2011). In the testes, FMRP has a role in testes development and maintenance of male fecundity. Male FXS patients have been shown to have enlarged testes and defects of spermatogenesis leading to reduced fecundity (Turner et al., 1980; Nistal et al., 1992). Several studies have shown that FMRP acts as a translational repressor for those mRNAs, which associate with FMRP (Laggerbauer et al., 2001; Li et al., 2001; Mazroui et al., 2002). One role of FMRP is to repress the translation of microtubuleassociated protein 1B (MAP1B) (Lu et al., 2004). MAP has an important role in stabilization of microtubules during the elongation of dendrites, neurites and their morphological structures (Gonzalez-Billault et al., 2004). Thus, the absence of FMRP may lead to altered microtubule dynamics which promotes the alterations of spine morphology (Lu et al., 2004; von Bohlen and Halbach 2010). By regulating protein translation at the synaptic site FMRP modulates synaptic plasticity that is important for learning and memory (Antar and Bassell 2003; Bagni and Greenough 2005; Bassell and Warren 2008). Synaptic plasticity, defined as the ability to change synaptic structure, can be divided into two categories: long-term potentiation (LTP) and long-term depression (LTD). Both of these plasticity forms include several cellular and molecular mechanisms, which either enhance or weaken the function of specific synapses. The mGluRs are involved in LTD. In normal brain, activation of group 1 mGluRs increases the synthesis of FMRP, which then negatively regulates the protein translation involved in the internalization of AMPAR (Bagni and Greenough 2005). In the spines of FXS patients or similarly in FMR1 deficient mice, the absence of FMRP results in increased translation of LTD proteins. LTD proteins are involved in AMPA and NMDA internalization leading to lower number of receptors on the postsynaptic membrane and plasticity changes (Bagni and Greenough 2005).

15

A

B

FMRP

INT.

NMDA receptor mGluR receptor AMPA receptor

Ribosomes regulator of AMPA internalization INT. AMPA internalization cytoskeleton regulator

Figure 2. A schematic model of FMRP at wt synapse and synapse of a FXS patient or Fmr1 KO mouse. (A) In the normal spine, stimulation of mGluR enhances the synthesis of FMRP. FMRP negatively regulates the translation of proteins which are involved in ionotropic receptor internalization (AMPA and NMDA) during LTD and the proteins that regulate the cytoskeleton (such as MAP1B). (B) In the FXS spine, the lack of FMRPmediated repression leads to an increase or decrease in the translation of proteins involved in the regulation of cytoskeletion and in ionotropic receptor internalization during LTD leading to reduced receptor number on the postsynaptic site and thinner spines (modified from Bagni and Greenough, 2005).

2.2.3 FMRP deficiency and NPCs FMRP is widely expressed throughout the brain during embryonic brain development (Devys et al. 1993; Hinds et al., 1993; Abitbol et al., 1993), especially in the areas which contain proliferating progenitor cells and newly born neurons (Rife et al., 2004). Peak levels of FMRP expression have been detected at the end of the first postnatal week, declining gradually thereafter (Lu et al., 2004; Wang et al., 2004). These results indicate that FMRP may have an important role during development. NPCs offer great possibilities to study the mechanisms of early neuronal development in FXS. Cultured human NPCs propagated from FXS fetuses or Fmr1 KO mice completely lack the synthesis of FMRP and were used to study effects of FMRP deficiency on the neurobiology of NPCs (Jakel et al., 2004).

16

However, the way FMRP regulates embryonic development via proliferation, migration and differentiation of neuronal cells has not been well characterized. Another important question is whether FMRP has a role also in adult neurogenesis and how FMRP deficiency affects learning and memory. It has been suggested in several studies that adult neurogenesis is critical for hippocampus-dependent learning (Ming and Song, 2005) and the disturbed regulation of neurogenesis may lead to learning and memory deficits. Recent studies have suggested that FMRP plays an important role in adult hippocampus-dependent learning and neurogenesis and the deficiency of FMRP leads to impaired learning and reduced hippocampal neurogenesis in Fmr1 KO mice (Guo et al., 2011; Brennan et al., 2006; Zhao et al., 2005). It has been shown that FMRP regulates several factors such as CDK4 and cyclin D1, which are cell-cycle regulators, (Miyashiro et al., 2003) and glycogen synthase kinase 3 (GSK3 ). These signaling molecules are involved in the regulation of proliferation and differentiation of NPCs (Bhattacharyya et al., 2008a; Callan et al., 2010; Luo et al., 2010; Lazarov et al., 2011). Increased expression of both CDK4 and cyclin D1 has been shown to increase proliferation of NPCs (Jablonska et al., 2007; Kenney et al., 2000). Therefore, it is not suprising that due to lack of FMRP negative regulation, the proliferation of NPCs is increased in the mouse model of FXS (Luo et al., 2010). GSK3 is known to be important for cellular signal transduction; and it acts as a negative regulator of both canonical Wnt signaling pathways and -catenin (Hur and Zhou, 2010). Levels of GSK3 have been shown to be increased both in Fmr1-KO mice and FXS patients (Luo et al., 2010; Min et al., 2009). FMRP is known to negatively regulate the level of GSK3 and due to lack of FMRP, the levels of GSK3 are elevated. Elevated GSK3 then inhibits the Wnt signaling pathway, which again alters neurogenin1 expression. Neurogenin1 is a transcription factor that promotes neuronal differentiation and inhibits astrocyte differentiation at the early stage of differentiation (Guo et el., 2011; Luo et al., 2010; Hur and Zhou 2010; Sun et al., 2001). Luo and coworkers (2010) recently demonstrated that hippocampal Fmr1-deficient adult NPCs have a higher proliferation rate, and differentiate more into glial cells and less into neurons. Recent studies have also shown that inhibition of GSK3 rescues hippocampal neurogenesis and enhances hippocampus-dependent learning. Althogether, these results indicate that GSK3 inhibition could be a potential therapeutic target for FXS (Guo et al., 2012; Min et al., 2009).

2.3 ALZHEIMER´S DISEASE (AD) 2.3.1 General features of AD AD is the most common neurodegenerative disorder in the elderly. It is multifactorial, heterogenous, and progressive disease, which finally leads to loss of neurons in specific brain areas. Neuronal loss occurs typically in areas which are associated with cognitive functions. Clinical characteristics of AD include the

17

progressive memory loss, deficits in other cognitive skills, and psychiatric problems. Pathologically, sporadic (SAD) and familiar (FAD) forms of AD have similar pathological features including dramatic neuronal and synaptic loss in the hippocampus and cerebral cortex, appearance of neuritic plaques (amyloid plaques), neurofibrillary tangles (NFT), and reduced brain size compared to healthy brain. Inflammation and OS are also characteristic to AD brain (Marques et al., 2010) as well as microvascular dysfunction and/or degeneration, accompanied with defects in the function of blood-brain barrier and vascular factors (Zlokovic 2011). The most typical pathological hallmarks of AD, extracellular amyloid plaques and NFT, were first described in 1907 by Dr. Alois Alzheimer in the brain of a patient who suffered from dementia. Despite these findings, intensive AD research started only in the mid 1960s, when the first reports on the deficits of specific neurotransmitter systems were announced. The first hypothesis was that reduced production of ACh caused AD. Therefore, the earliest AD therapies were developed to preserve ACh (Arce et al., 2009). These therapies only relieve the symptoms, but do not cure the disease, which indicates that AD is a more complex disease. In the 1980s, amyloid plaques were reported to be composed of extracellular deposits of amyloid and NFT composed of hyperphosphorylated tau protein filaments. Finally, in the 1990s, the mutations involved in amyloid precursor protein (APP) proteolytic processing and the fate of A came into focus and then the modern era of AD research began (Selkoe 2001; Glenner et al., 1984; De Strooper 2010). Presently, it is estimated that dementia affects approximately 24 million people around the world. The majority of these people have typical AD pathology and dementia, and are thus classified as AD. In addition, the number of people suffering from AD will be expected to rise, because the life expectancy of people is increased. In 2020, approximately 42 million people are estimated to suffer from dementia (Ballard et al., 2011). Even though the pathology of AD is under extensive investigation, the mechanisms behind this disease are still poorly known. The complex nature of this disease makes it challenging for drug development. Therefore, effective treatment for this disease is still lacking. Current treatments for AD only relieve the symptoms, but do not stop the progression of the disease or cure neuronal damage. The multifactorial nature of this disease will probably require a combination of different therapeutic approaches in order to be effective (De Strooper 2010). However, in the near future the development of new therapeutic approaches would be of utmost importance to avoid the enormous economical and social impacts of this devastating disease (Marques et al., 2010). Sporadic (SAD) and familiar form (FAD) of AD AD patients can be categorized as having sporadic and familiar forms of AD. Most cases (95 %) of AD are classified as the sporadic form of AD (SAD), which usually occurs only in the elderly, after the age of 60 (late-onset). A minor proportion of cases (5 %) are classified as the familiar form of AD (FAD), which usually occurs

18

before the age of 60 (early-onset) (Marques et al. 2010). Table 1 summarizes the major genes that are linked to AD. These genes are involved in the production, clearance, or uptake of A peptide. The dominant mutations (missense) appear in genes which encode APP (chromosome 21), presenilin 1 (PSEN 1) (chromosome 14), and presenilin 2 (PSEN 2) (chromosome 1). Patients who carry this type of mutation acquire AD symptoms under middle age (early-onset AD). Currently there are no specific gene mutations linked to late-onset AD. The only evident risk so far to acquire late-onset AD is aging. The expression of apolipoprotein E (ApoE) 4 allele instead of ApoE 3 allele increases the risk of AD more than seven fold (Corder et al., 1993; Selkoe et al., 2001; Ashford 2004). The role of allele e4 is not fully elucidated, but it has been suggested to be important in A clearance (Reiman et al., 2009). Using a relatively new approach called genome-wide association study (GWAS), a number of genes, in addition to ApoE e4, have been identified that may increase a person´s risk for late-onset Alzheimer´s, including BIN1, CLU, PICALM and CR1 (Shi et al. 2012). Some non-genetic risk factors linked to sporadic AD are hypertension, diabetes, and obesity (Beydoun et al., 2008; Holscher 2011; Wysocki et al., 2011). Interestingly, no matter which type of AD (sporadic or familiar), the typical pathological features are remarkably similar. Table 1. Confirmed genetic factors linked to AD (modified from Selkoe, 2001). Chromosome 21 14 1 19

Gene defect -APP mutations Presenilin 1 mutations Presenilin 2 mutations ApoE4 polymorphism

Phenotype A A 1-42 A 1-42 A

2.3.2 Proteins involved in AD pathology Amyloid precursor protein (APP) and the formation of -amyloid (A ) Currently, the most dominant hypothesis concerning the pathogenesis of AD is “the amyloid cascade hypothesis” which suggests that the overproduction of -amyloid (A ) and/or total amyloid load causes toxic effects and leads to neuronal dysfunction and finally neuronal death (Walsh and Selkoe 2007). A is the natural product of metabolism and it is produced by proteolytic cleavage of APP. APP is a membrane spanning glycoprotein which consists of 695 to 770 amino acids (Selkoe 2001). APP is generated in the endoplasmic reticulum (ER), transported through the Golgi apparatus and then transported to the cell membrane (Xu et al., 1997; Hartmann et al., 1997). APP gene is located on choromosome 21 (Goate et al., 1991). On the cell surface, APP is either cleaved by specific secretases or degraded via the endosomal/lysosomal degradation pathway (Nordstedt et al., 1993; Caporaso et al., 1994). Specific secretases, which are involved in the cleavage of APP precursors are -, - and - secretases. APP cleavage begins with -secretase or -secretase which then leads to nonamyloidogenic or amyloidogenic pathways, respectively (Figure 3). The first step in the generation of A (amyloidogenic pathway) is the cleavage of APP with -secretase when sAPP is released into extracellular fluid. The remaining

19

fragment in the plasma membrane is then cleaved by -secretase resulting in A peptides. The size of A peptides varies from 39 to 43 amino acids. The most important and common forms of A s are A 1-40 and A 1-42, the latter being also the most amyloidogenic form of A . Compared to A 1-40 form, A 1-42 is more hydrophilic, fibrillogenic, and thus a more easily aggregating form (Zhao et al., 2004; Zhao et al., 2007; Chow et al., 2010; Wang et al., 1996). The nonamyloidogenic pathway does not produce A . Cleavages of APP by - and -secretases result in a p3 peptide, secreted ectodomain sAPP , and APP intracellular domain (Ring et al., 2007; Chow et al., 2010). When A is released in monomeric form, it undergoes spontaneous conformational changes starting from soluble fragments, which aggregate to oligomers and finally form large A fibrils or so called A plaques (Querfurth and LaFerla 2010).

AD pathology Aß Aß Aß Aß Aß Aß Aß Aß

Aß Aß Aß Aß

oligomerization

Neuron death

ß Amyloid plaque

fibrillization

Inflammation Oxidative stress

ß

P3

sAPP

sAPP

Aß

Full-length APP

extracellular space cytoplasm

Amyloidogenic pathway

Non-amyloidogenic pathway

Figure 3. APP cleavage products in non-amyloidogenic (blue arrow) and amyloidogenic (yellow arrow) pathways. The non-amyloidogenic pathway involves cleavages of APP by - and -secretases and results in long secreted APP (sAPP ) and C-terminal fragments (purple). The amyloidogenic pathway involves cleavage by - and -secretases and results in long secreted APP (sAPP ), C-terminal fragments (purple protein domain) and s (yellow protein domain). A oligomerizes and then fibrillizes leading to formation of plaques and AD pathology (modified from Chow et al., 2010).

However, the mutation located just before the -secretase cleavage site and after secretase or -secretase cleavage sites of APP are quite rare (familiar AD) and appear

20

only in about two dozen families wordwide. Another way to develop AD pathology is the overproduction of structurally normal APP. As an example, Down syndrome (trisomy 21) patients have elevated gene dosage of APP and frequently develop AD pathology in old age (Selkoe, 2011). While accumulation of oligomeric A is clearly associated with dementia, the function of its monomeric form is not well understood. Moreover, the physiological functions of the secreted APPs (sAPP and sAPP ) are also poorly understood. The secreted sAPP has been suggested to be subsequently processed by unknown proteases. The resulting ligand may then bind to death receptor DR6, which in turn activates caspase-6. This sAPP driven caspase-6 activation is thought to be involved in axonal pruning during embryogenesis (Nikolaev et al., 2009). Because sAPP seems to be involved in cell death, it is speculated that this pathway could also contribute to AD pathogenesis. Contrary to sAPP , sAPP seems to have a neuroprotective role and synapse promoting activities (Bandyopathyay et al., 2007). Some studies have shown that sAPP has a role in neuronal plasticity and survival (Mattson et al., 1993; Furukawa et al., 1996). In in vitro models sAPP has been shown to act as a growth factor by increasing cell proliferation, cell mobility and differentiation of many types of cells (Pietrzik et al., 1998; Sabo et al., 2001; Saitoh et al., 1989). Recently, it was demonstrated that sAPP affects the proliferation of NPCs as well (Demars et al., 2011), possibly directing their differentation towards astrocyte lineage (Kwak et al., 2006; Kwak et al., 2010). Similar effects were also seen in NPCs when transplanted into APP and A -rich environments in vivo (Marutle et al., 2007), thus suggesting that application of stem cell therapy to replace lost or dysfunctional neurons in neurodegenerative diseases, such as AD may not be straightforward. toxicity Although the formation of A is quite well understood, the mechanisms which make toxic to neurons remains elusive. In AD, A plaques, as well as NFT, are usually detected in the cortex, hippocampus, basal forebrain, and amygdala (Mattson et al., 2004). Analysis of post-mortem AD brain has shown that the loss of synapses and neurite damage occur typically near the A plaques, indicating that accumulation of fibrillar A has a direct neurotoxic role (Pratico and Trojanowski 2000). This is supported also by in vitro studies showing that fibrillar A contributes to early dendritic and synaptic injuries leading to neuronal dysfunction (Walsh et al., 2002). Fibrillar A has also been shown to activate microglia to produce inflammatory mediators such as nitric oxide and reactive oxygen species (ROS), which then facilitate neuronal death (Akiyama et al., 2000; Kitazawa et al., 2004). However, the magnitude of A plaques correlates poorly with cognitive disturbances. More recent analyses suggest that A -mediated neurotoxicity might be highly dependent on the conformation stage of A (Aizenstein et al., 2008; Pike et al., 2007) and, as already mentioned, the peptide length affects A toxicity substantially. The most neurotoxic form of A has been shown to be soluble oligomers and intermediate amyloids (Walsh and Selkoe 2007). Cognitive defects in AD brain

21

correlate better with the levels of A oligomers compared to the number of A plaques (Lue et al., 1999). Dimeric and trimeric forms of A have been shown to be toxic for synapses (Walsh et al., 2005; Klyubin et al., 2008). In addition, soluble oligomeric A has been shown to be more toxic than the fibrillar form (Tomic et al., 2009). In addition to the obvious A toxicicity to mature neurons, A appears to have effects also on proliferation, migration, and differentiation of endogenous NPCs. Several studies have been carried out in vitro by using synthetic A to evaluate the impact of A 1-42 and A 1-40 on NPCs. These results are quite variable which is possibly due to different forms or preparations of A or its concentrations applied to the cells. Both A 1-40 and A 1-42 have been shown to stimulate proliferation of NPCs. 1-40 has also been found to promote neuronal differentiation, whereas A 1-42 has been reported to promote the differentiation towards the astrocyte phenotype (Chen and Dong 2009). Lopez-Toledano and Shelanski (2004) have reported that A treatments do not disturb proliferation and differentiation of NPCs and in fact, A 1-42 but not A 1-40 treatment may even have neurogenic effects. The opposite effects have also been reported; A 1-42 impairs the proliferation, migration, and neuronal differentiation of cultured NPCs (Haughney et al., 2002a; Haughney et al., 2002b). Importantly, Mazur-Kolecka and coworkers (2006) showed that A 1-40 impaired migration of human NPCs. Also, endogenous neurogenesis has been shown to be altered both in transgenic AD mouse models and also in human AD brain when analyzed from postmortem tissues. These studies reported controversial results of increased and decreased neurogenesis in AD (Jin et al., 2004; Boekhoorn et al., 2006; Taniuchi et al. 2007; Wen et al., 2004; Ermini et al., 2008; Verret et al., 2007; Zhang et al., 2007). Cells have protective systems against A toxicity. For example, many proteases, including neprilysin and insulin-degrading enzymes (IDE), are known to regulate the levels of A . Neprilysin is a membrane-anchored zinc endopeptidase which degrades both monomeric and oligomeric forms of A (Kanemitsu et al., 2003) and the reduction of this enzyme directly affects the accumulation of A (Iwata et al., 2001). IDE is a thiol metalloendopeptidase, which is known to degrade various small peptides such as A (Qiu et al., 1998). In mice, the deletion of IDE has been shown to lead to an over 50 % reduction of A degradation (Farris et al., 2003). On the contrary, overexpression of both degrading enzymes leads to increased A degradation and reduced brain A levels (Leissring et al., 2003). Neurofibrillary tangles (NFTs) Another histopathological hallmark observed in AD brain is the accumulation of intracellular NFTs (Kosik et al., 1986). NFTs consists of tau protein, which is the major neuronal microtubule associated protein. In cells, tau binds to the microtubules, regulates their growth and shrinkage, and thus stabilizes the neuronal cytoskeleton (Burbank and Mitchison 2006). In the normal brain, tau protein is phosphorylated with numerous kinases, which usually exist in soluble forms in

22

axons. In various neurodegenerative disorders, tau protein becomes hyperphosphorylated and subsequently becomes insoluble, then begins to aggregate, and finally forms NFTs (Hanger et al., 2009). Phosphorylated forms of tau protein reduce its affinity to microtubules, and prior to NFT formation, it may interfere with protein function, which again is important in axonal transport and contributes to synaptic dysfunction and degeneration of neurons (Mandelkow et al., 2003; Roy et al., 2005). The mechanism which leads to hyperphosphorylation of tau protein is not completely understood. It is speculated that hyperphoshorylation is caused by upregulation or abnormal activation of tau kinases, mutation of tau gene or covalent modification of tau protein. Furthermore, indirect events such as A -mediated toxicity, OS and inflammation may have negative effects also on tau protein (Ballatore et al., 2007; McNaull et al., 2011). It is known that various kinases are involved in tau phoshorylation. Such kinases include: glycogen synthase kinase 3 (GSK-3 ), cyclin-dependent protein kinase-5, protein kinase A, calcium/calmodulindependent protein kinase II, casein kinase-1, mitogen-activated protein kinase ERK1/2, and stress-activated protein kinases (Iqbal et al., 2005, 2009; Sengupta et al., 2006). In addition, increased NFT burden may have an effect on immune responses. Although the relationship between A deposition and tau aggregation is not completely known, it is certain that hyperphoshorylation of tau is linked to neuroinflammation and is thus involved in the progression of AD (Lee et al. 2010). 2.3.3 Neuroinflammation One of the main pathological features of AD is chronic inflammation. In the AD brain inflammatory response in neurons, so called neuroinflammation, it is thought that accumulation of A deposits and NFT leads to neuroinflammation. Both A deposition and NFT activate inflammatory cells to produce several pro- and antiinflammatory mediators, some of which may also have neurotoxic properties. Important inflammatory cells in the brain are microglia and astrocytes, in certain cases also the macrophages and lymphocytes, and the main released immunomediators are cytokines, chemokines, and ROS (Tansey et al., 2007; Akiyama et al., 2000). Immunomediators are known to recruit monocytes and lymphocytes as well as activate microglia and astrocytes to proliferate and produce more inflammatory factors (Lossinsly and Shivers, 2004; Das and Basu, 2008). Microglia are the first activating inflammatory cells, which then orchestrate the other endogenous immune responses. They express major histocompatibility complex type II and produce pro-inflammatory cytokines, chemokines, ROS, complement protein and extracellular proteases (Moore and O´Banion, 2002). One of the main functions of microglia is their phagocytic property (Streit et al., 1999). In the AD brain A plaques attract microglia. There are scavenger receptors at the surface of microglia which then interact with A by forming the adhesion between microglia and A plaque. After interaction with opsonized A microglia phagocytize A . However, this process may result in the upregulation of pro-inflammatory mediators

23

and ROS production which then further contributes to OS and neuroinflammation (El Khoury et al., 1996). Another method microglia internalizes extracellular A is through fluid phase macrocytosis. The mechanisms of macrocytosis are different than phagocytosis or receptor-mediated endocytosis. The macropinocytose vesicle is formed by the enclosure of membrane ruffles and this vesicle formation is dependent on cytoskeletal structures consisting of both actin and tubulin. After internalization, A goes to endolysosomal compartments and is degradated (Mandrekar et al., 2009). Microglia also release IDE, which acts as a protease capable of cleaving small proteins, such as insulin and glucagon. As already mentioned, IDE also degrades A (Qiu et al., 1998; Qiu et al., 1997). IDE missense mutations in type 2 diabetes mellitus diminish IDE secretion and lead to disturbed A degradation (Farris et al., 2004). Another important inflammatory cell type is astrocytes, which are also the most common cells in the brain. Astrocyte functions in the CNS are not completely elucidated but they are known to support the endothelial cells of the blood brain barrier, supply nutrients and growth factors to nervous tissue, and maintain extracellular ion balance. In the injured brain and spinal cord, astrocytes heal the brain by forming a ´glia scar´ and secrete proinflammatory and anti-inflammatory molecules (Tuppo and Arias 2005). In AD brain, A plaques have been shown to induce to development reactive astrocytes (Dickson 1997), which similar to microglia, secrete chemokines, cytokines, and neurotoxic ROS (Johnstone et al., 1999; Smits et al., 2002) as well as surround A plaques and play a role in A clearance and degradation by phagocytosing A deposits (Wyss-Coray et al., 2003; Koistinaho et al., 2004). Recently, it was shown that when transplanted into the brain of AD mice, the astrocytes migrate near the A deposits and phagocytize A . These results indicate astrocytes play an important role in A clearance and may thus provide one alternative therapeutic approached for AD (Pihlaja et al., 2008; 2011). 2.3.4 Oxidative stress (OS) During normal oxygen metabolism, several by-products are generated in all aerobic organisms. These molecules such as superoxide, hydrogen peroxide, and hydroxyl radicals are collectively called ROS; and they have an important role in cell signaling. Cells have mechanisms to keep the amount of ROS in equilibrium, but in situations when ROS production exceeds the cellular recovery capacity, intracellular OS increases. Accumulation of ROS causes cellular damage, which finally trigger both necrotic and apoptotic cell death (De Vries et al., 2008). In more detail, accumulation of ROS affects directly membrane lipids, proteins, DNA, RNA, and leads to damage in membrane structures, proteins and nucleic acids (Zhu et al., 2006). Furhermore, oxidation of cytoplasmic protein decreases the antioxidative defense machinery (Rodrigues Siqueira et al., 2005) and causes damage in the mitochondrial DNA respiratory chain (Wang et al., 2005). Accumulation of ROS inside cells leads to activation of cellular defensive mechanisms. Various endogenous antioxidant enzymes are responsible for

24

maintaining the redox balance inside cells. ROS production is known to induce the transcription of various cytoprotective enzymes, which all share a common promoter element called antioxidant response element (ARE). ARE is an enhancer sequence which controls the expression of both basal and induced genes. All these induced genes are responsible for detoxification of oxidative stress, xenobiotics, heavy metals, or ultraviolet light (Kaspar et al., 2009; Nquyen et al., 2009; Rushmore et al., 1991) and belong to phase II enzymes and antioxidant proteins. One important phase II enzyme is glutathione S-transferase (Lee et al., 2005). Keap-Nrf2-ARE pathway The activation of the transcription of detoxification and antioxidant genes during OS is dependent on the activation of nuclear factor E2-related factor 2 (Nrf2). Nrf2 proteins belong to the cap ´n´ collar subfamily of basic-region leucine zipper transcription factors (Itoh et al., 1997) and contain cis-acting element(s) ARE. Under normal conditions, Nrf2 is expressed in a constitutive manner, degraded within minutes and mainly kept in cytoplasm rather than nucleus (Figure 4). A cytoplasmic Kelch-like ECH protein 1 (Keap1) has an important role for the activation and stabilization of Nrf2. Under normal conditions, Keap1 acts as a Nrf2 repressor by binding to it and promotes its proteasomal degradation. When the ROS production is increased, Keap1 loses its activity and allows Nrf2 to dissociate from Keap1. Released Nrf2 passes into the nucleus, binds to the ARE element and then activates gene transcription of important antioxidant enzymes. After the cellular ROS production is returned back to normal, Nrf2 dissociates from the nucleus and gets degraded (Baird and Dinkova-Kostova 2011; Dinkova-Kostova et al., 2002; Itoh et al., 2003). Previous in vivo and in vitro studies have shown that Nrf2 plays a central role in the regulation of gene expression of various detoxification enzymes and antioxidant genes. In vivo, Nrf2-deficient mice have shown lower basal levels of gene expression of those detoxification enzymes and antioxidant genes and also lack the ability to induce those genes. These studies also showed that Nrf2 deficiency led to higher vulnerability to OS-induced damages and the effects of carcinogens (Chan and Kan, 1999; Chan et al., 2001; Khor et al., 2006). In vitro studies showed that in cultured Nrf2 deficient fibroblasts, the glutathione levels were decreased which was associated with higher sensitivity to OS (Kwong et al., 1999; Chan and Kwong, 2000).

25

Basal state

Cytoplasm

Cytoplasm

Keap1 Keap1

Induced state

Keap1

Nrf2

Nucleus

Nucleus

ARE

Nrf2

Keap1

Nrf2

ARE

transcription

Figure 4. A schematic model of regulation of the Keap1-Nrf2 pathway in basal state and after induction (modified from Baird and Dinkova-Kostova 2011).

Oxidative stress and Alzheimer´s disease High level of polyunsaturated fatty acids, high oxygen consumption, and low levels of antioxidants make the CNS extremely vulnerable to OS (Floyd and Hensley, 2002; de Vries et al. 2008). OS is known to be increased in normal aging. Aging not only increases the amount of ROS, but also impairs the cellular defense mechanisms against them (Katzman 1986). ROS are mainly produced in mitochondria and because of close proximity to the ROS production site, mitochondrial DNA is especially vulnerable to ROS-induced injuries. Malfunctioning mitochondria and defects in mitochondrial DNA, may cause declines in mitochondrial functions and simultaneously further increase ROS production (Lin and Beal, 2006). OS is associated with pathogenesis of AD and many other neurodegenerative diseases such as PD and Huntington disease (Halliwell 2006). In AD, brain OS is thought to play an important role in progression of disease. Production of A has been shown to be induced in OS both in vitro and in vivo (Hensley et al., 1994; Mark et al., 1997; Murakami et al., 2005; Tabner et al., 2005), and vice versa, increased OS has been shown to induce the production of A which again contributes to the progression of disease (Paola et al., 2000). OS is believed to exist before A formation and thus could be the earliest sign of developing AD (Nunomura et al., 2001). Importantly, the effect of OS has been shown to be increased in AD brain, not only due to increased production of OS, but also because the antioxidant defense mechanisms are weakened (Katzman, 1986). The levels and activation of cytoprotective enzymes superoxide dismutase and glutathione peroxidase have been shown to be decreased in the AD mice (Olcese et al., 2009). In human AD, the brain levels of hippocampal Nrf2 is reduced (Ramsey et al., 2007) and in hippocampal neurons, Nrf2 is localized preferentially in cytoplasm rather than in nucleus. These results suggest that induction of Nrf2-mediated transcription may be disturbed during OS in AD brain (De Vries et al., 2008).

26

Nrf2 and therapeutic potential for AD It has been reliably demonstrated that Nrf2 response is an inducible and highly protective mechanism against OS and various toxic compounds. These findings, together with the hypothesis that the Nrf2-ARE pathway is impaired in AD brain, as well as the notion that OS is important in the pathogenesis of AD, provide the basis for the idea that the Nrf2-ARE pathway may be a potential target for the development of new therapeutic approaches against OS to alleviate AD. For therapeutic purposes, a cells’ own Nrf2-mediated defense system could be induced by using small doses of Nrf2 inducers, enhance the amount of Nrf2 by using Nrf2 overexpressing cells, or inject Nrf2 directly into the brain. Several in vitro and in vivo studies support the hypothesis that Nrf2 could be a useful target for future treatment. First, cultured astrocytes and neurons lacking Nrf2 have increased vulnerability against OS (Lee et al., 2003), and when the Nrf2 is induced, cultured neurons have higher susceptibility to OS or mitochondrial toxins (Calkins et al., 2005; Jakel et al., 2005; Lee et al., 2003; Shih et al., 2003). In vivo results are in line with in vitro studies; Nrf2-deficient mice were more sensitive to OS and toxins (Calkins et al., 2005) and induction of their own Nrf2 defense mechanisms protected against neurotoxicity (Burton et al., 2006; Chen et al., 2009; Jakel et al., 2007; Kraft et al., 2006; Satoh et al., 2006, Satoh et al., 2008; Shih et al., 2005a and b). Kanninen and coworkers (2008) have demonstrated that overexpression of Nrf2 protected cultured neurons against A toxicity and Frautschy and coworkers (2001) demonstrated that the antioxidant treatment resulting in various responses, including induction of Nrf2, also relieved cognitive defects in an AD mouse model. Furthermore, lentiviral Nrf2 injections into the hippocampus of AD mouse brain caused significant alleviation in memory deficits. However, the mechanism behind this is unknown, but it is suggested that the direct delivery of a Nrf2 gene probably saturates Keap1 and allows Nrf2 to translocate into the nucleus and further induce the expression of cytoprotective genes (Kanninen et al., 2009). The amount of Nrf2 could be also increased by transplanting Nrf2-overexpressing cells into the brain. Transplanted Nrf2-overexpressing astrocytes and NPCs, which then differentiated into astrocytes, were shown to be beneficial for the protection of neurons against malonate-induced lesions (Calkins et al., 2005; Calkins et al., 2010). As previously discussed, one therapeutic approach for AD could be stem cell therapy. High OS level in the AD brain environment have clear effects on survival, proliferation, migration, and differentiation of transplanted NPCs and therefore could reduce therapeutic effectiveness. One strategy to prevent OS-related injuries could be to transplant Nrf2-overexpressing NPCs which then probably are more sustainable against OS-related injuries. Several studies clearly have shown that Nrf2 is the master regulator of the endogenous antioxidant response. However, further protective roles of Nrf2 were also reported earlier. Nrf2 was shown to inhibit Fas-mediated apoptosis (Kotlo et al., 2003; Su et al., 2003) and induce the expression of a class of proteosomal proteins, which have an important role in removal of aberrantly modified protein and thus

27

protein aggregation (Kwak et al., 2003; Park et al., 2009). In addition, Nrf2 was shown to attenuate inflammatory response (Li et al., 2008) as well as overproduction of proinflammatory mediators (Koh et al., 2009). In 2009, Zhao and coworkers published interesting results, in which they showed that the overexpression of Nrf2 promotes neuronal differentiation whereas the inhibition of endogenous Nrf2 results in opposite effects. These results indicate that if Nrf2 has a role in neuronal differentiation in cell replacement therapy, the up-regulation of Nrf2 not only protects against OS, but also may stimulate NPCs towards the neuronal lineage and offer extra benefits for the treatment of neurodegenerative disorders.

28

3 Aims of the study Multipotent NPCs provide excellent tools to study the mechanisms of developing neuronal cells in vitro both in normal situations as well as in genetic disease. NPCs are also a source for cell transplantation, which could be a future therapeutic approach for severe neurodegenerative disorders such as AD. NPCs could be used for therapeutic purposes also by stimulating a patient’s own endogenous NPCs directly in order to replace damaged neurons. The differentiation of NPCs is regulated by various factors, which are not well characterized and are of utmost importance to be identified before any therapeutic use of NPCs. One intriguing regulator could be classical neurotransmitters, that others have suggested to play an important role in maturation of NPCs. In addition, it is certain that the genetic background has effects on behavior of NPCs. Mutations that cause FXS and AD may also alter the function of NPCs. Genetic background also matters when using iPS derived NPCs created from a patient´s own cells, assuming that the mutation alters the behavior of transplanted NPCs. Moreover, the altered brain environment in disease could cause unwanted effects on transplanted NPCs. In AD brain, both A and OS-rich environment may affect the behavior of transplanted NPCs. The alleviation of OS related damages could also be beneficial in stem cell therapy. The aims of this thesis were: 1. To assess whether FMRP deficiency affects the differentiation of mouse and human NPCs. 2. To study the role of neurotransmitters involved in the regulation of an early stage of NPC differentiation. 3. To assess whether AD-linked geno- and phenotypes affect the survival, differentiation and migration of NPCs in vitro or after transplantation to the brain. 4. To assess whether the transcription factor Nrf2 has protective effects against toxicity in cultured NPCs and whether Nrf2 plays a role in neurogenesis.

29

4 Materials and methods 4.1 BRAIN TISSUE (I-IV) Neural stem/progenitor cells (NPCs) were generated from mouse hippocampus, mouse lateral ventricle, or human fetuses (lateral ventricle). Animal experiments were conducted according to the national regulation on the usage and welfare of laboratory animals and approved by the Animal Experiment Committee in the State Provincial Office of Southern Finland. Postmortem human fetal tissue was obtained in accordance with the guidelines of the National Institute of Health, the government of Finland, and the local ethics committee of the Kuopio University Hospital. All investigations were conducted according to the principles of the Declaration of Helsinki. NPCs were isolated from wt mice or transgenic Fmr1 KO (Bakker et al., 1994), APdE9 (Jankowsky et al., 2004), and Nrf2 KO mice (Itoh et al., 1997). Human NPCs were isolated from aborted 18-week-old Fragile X fetus with a methylated repeat expansion of 276 – 300 trinucleotide repeats in the 5` untranslated region of the FMR1 gene, and due to social reasons, aborted control fetuses at 7, 12 and 18 weeks of gestation which was determined by intrauterine ultrasound examinations. The developmental stage and genotype of the used mouse and human NPCs in the different studies are listed in table 2. Table 2. NPCs genotype, age and source of the NPCs used in vitro studies referred as number I-IV. NPC Cell genotype Age mouse lv NPC Fmr1 KO and wt E13 mouse lv NPC Fmr1 KO and wt P6 human lv NPC FXS 18-week-old human lv NPC wt 7, 12 and 18-week-old mouse lv NPC wt P6 / P7 mouse h NPC APdE9 and wt E18 mouse h NPC Nrf2 -/- and wt E18 lv = lateral ventricle, h = hippocampal, E = embryonic day, P = postnatal day, APdE9 = AD transgenic mice, Fmr1 KO = fragile X knockout mouse model, Nrf2 -/- = Nrf2 mice lacking Nrf2 gene

Stydy I I I I II III IV

4.2. ISOLATION AND CULTURE OF NPCS (I-IV) Tissues (hippocampus or tissue from the wall of the lateral ventricle) was dissected out from the brain and dissociated with 1.33 mg/ml trypsin (Sigma), dissolved in Hank´s balanced salt solution (HBSS, Gibco, Life Technology) with 2 mM glucose, 0.7 mg/ml hyaluronidase (Sigma), and 0.2 mg/ml kynurenic acid (Sigma) at 37oC for 30 min. Cells were centrifuged at 1500 rpm for 5 min, resuspended in 0.9 M sucrose in 0.5 X Hank’s balanced salt solution, and centrifuged at 2000 rpm for 10 min. The cells were then resuspended in 2 ml Earl’s balanced salt solution (EBSS, Gibco, Life Technology) and centrifuged through a gradient of the same salt solution with 4%

30

bovine serum albumin at 1500 rpm for 7 min. Dissociated cells were then plated in culture medium (DMEM/F12, Gibco, Life Technology) containing 2 mM Lglutamine, 15 mM HEPES, 100 U/ml penicillin, 100 mg/ml Streptomycin, B27 supplement, 20 ng/ml epidermal growth factor (EGF, PeproTech, London, UK), and 10 ng/ml basic fibroblast growth factor (FGF-2, PeproTech, London, UK), and cultivated at 37°C in a humidified atmosphere at 5% CO2. EGF and FGF-2 mitogens were added every third day and the culture medium refreshed every 3 - 4 days. Neurospheres were passaged every 5 - 8 days with papain (0.5 mg/ml, Sigma) and carefully triturated mechanically by using a Pasteur pipette. NPCs were proliferated up to 10 passages. 4.3 LENTIVIRAL TRANSDUCTION (III-IV) For transplantation experiments, enhanced green fluorescence protein (GFP) expressing NPCs were used. For transduction experiments, wt and APdE9 neurospheres were first dissociated. Single NPCs were infected with a multiplicity of infection of 3 at a density of 500 000 cells / ml with a lentiviral vector carrying an enhanced GFP (LV GFP) (Kanninen et al., 2009) for 18 hours. During the infection, culture media was supplemented with growth factors to keep cells in an undifferentiated stage. On the following day, the virus-infected media was removed by centrifugation at 1000 rpm for 1 min. Cells were replated in fresh culture media supplemented with growth factors and expanded in culture. The transduction efficiency was about 70 – 80% as analyzed with flow cytometry. To manufacture Nrf2 overexpressing NPCs, wt NPCs were infected with lentiviral vector carrying a human Nrf2 (LV Nrf2). The transduction efficiency was analyzed with quantitative real-time PCR. 4.3.1 Quantitative real-time PCR (IV) For quantitative real-time PCR, 100 000 LV-Nrf2 transduced NPCs were homogenized and RNA extracted by using RiboPure Kit (Ambion, Life Technology). cDNA synthesis was performed by using Random hexamer primer (Fermentas, Thermo Scietific) and Maxima reverse transcriptase (Fermentas, Thermo Scientific). The relative Nrf2 expression levels were detected by using qRT-PCR protocol from ABI PRISM 7700 Sequence directors (Applied Biosystems, Life Technology). The expression levels were normalized to ribosomal RNA and were in Nrf2 transduced cells about 1000 x higher compared to control cells. 4.4 IN VIVO EXPERIMENTS (I, III-IV) 4.4.1 Animals Animal experiments were conducted according to the national regulation of the usage and welfare of laboratory animals and approved by the Animal Experiment Committee in State Provincial Office of Southern Finland. For all in vivo experiments,

31

age- and weight –matched adult male mice were housed under a 12 h light-dark cycle and given food and water ad libitum throughout the study. For cell proliferation analysis (study I), pregnant (E13) Fmr1 KO and their wt littermate mice were used. For the transplantation experiment (III), 4, 9 and 18 month-old transgenic APdE9 mice expressing familiar AD-linked APP Swe and PS1dE9 mutant genes (APP/PS1) (Jankowsky et al. 2004) and their wt littermates were used. For kainic acid (KA) injection (study IV), Nrf2 -/- (Nrf2 KO) and wt mice were used. All mice were in a C57BL/6J genetic background. 4.4.2 Intracerebral NPC transplantation (III) For intracerebral NPCs, transplantation GFP positive APdE9 and wt NPCs were used. Single NPCs were resuspended in PBS to obtain a final cell density (100 000 cells/µl) and injected immediately into the mouse hippocampus. Mice were anesthetized with isoflurane (5% for induction and 1.8% for maintenance of surgical anesthesia) and immobilized in a stereotaxic apparatus (Kopf Instruments, USA). NPCs were injected uni- or bilaterally in the volume of 2 µL at the speed of 0.25 µL / min. The following coordinates were used: +0.32 cm ML, -0.27 cm AP, and -0.27 cm DV. The injection needle was held still for 10 min before lifting it up. The wound was closed with sutures and the animals were monitored until they regained full consciousness. 4.4.3 BrdU injections (I, IV) For detection of proliferation of transplanted NPCs, BrdU were dissolved in saline and administered intraperitoneally (i.p) at a dose of 50 mg / kg every 24 h for the first seven days. To investigate the production of new cells in the embryonic fmr-1 KO and wt, brain, BrdU was administered to pregnant mice at a dose of 100 mg / kg every 12 h for four days. BrdU-GFP-NPCs and BrdU expressing cells were detected in brain sections by immunostaining. 4.4.4 Kainic acid injections (IV) Kainic acid (KA) is known to induce endogenous neurogenesis in mice (Jessberger et al., 2005; Choi et al., 2007; Jaako et al. 2009). In this study, i.p injection of KA (Sigma, dissolved in 0.9 % NaCl) with a dose of 15 mg / kg was given to Nrf2 KO (n = 17) and wt (n = 14) to induce neurogenesis. Animals in the control groups were injected with an equal volume of 0.9 % NaCl as a vehicle into Nrf2 KO (n = 8) and wt mice (n = 9). After injection, mice were monitored for seizure-associated behavior and 4 Nrf2 KO and 7 wt mice without seizure associated behavior were excluded from further observations. Additionally 4 Nrf2 KO mice died within 2 h after KA injection. 4.4.5 Brain processing (III – IV) Mice were terminally anesthetized with Avertin 4 - 8 weeks after NPC transplantation and 9 d after KA injection and transcardially perfused with heparined saline (2,500 IU/L) for 2 min before removing the brain. Thereafter, the left

32

hemisphere was dissected, postfixed by immersion in 4 % paraformaldehyde (PFA) solution for 21 h at 4 °C and cryoprotected in 30 % sucrose for 48 h. Then, both left and right hemispheres were frozen in liquid nitrogen and stored at – 70 °C until later use. Coronal 20-µm-thick sections were cut with a cryostat throughout the hippocampal formation (Leica Microsystems GmbH, Germany). 4.4.6. Immunocytochemistry for brain slices (III – IV) Free floating brain sections were washed with 0.1 M PB pH 7.4 for 6 x 30 min, transferred to glass slides and air-dried for 20 min. After rehydration sections were permeabilized with 0.4 % Triton X-100 in PBS for 30 min and washed for 3 x 5 min in PBS with 0.5 % Tween (PBST) pH 7.4 before blocking unspecific sites using 10 % normal goat serum (NGS, Chemicon International, Millipore) or a mouse-on-mouse blocking solution (MOM, Vector Laboratories, UK). The sections were incubated with primary antibodies prepared in 5 % NGS PBST at RT with slow agitation overnight. The sections were then washed again in PBST for 3 x 5 min before adding secondary antibodies in 5 % NGS PBST at RT for 2 h and protected from light. For double staining, additional primary and secondary antibodies were added subsequently and stained similarly as the first ones. For detection of the proliferated NPCs, BrdU/GFP double staining was performed with the following modification in the staining protocol. After rehydration the brain sections were permeabilized in precooled ethanol-acetic acid mixture (absolute ethanol and glacial acetic acid 2:1) at – 20 C for 5 min and washed with PBST for 3 x 5 min. Then sections were treated with 2 M HCl at 37 C for 30 min, washed for 3 x 5 min in PBST, and incubated in 0.1 M borate buffer pH 8.5 at RT for 10 min and washed again for 3 x 5 min. Before adding the primary antibody, sections were treated with 0.5 % MOM-blocking reagent in PBST for 1 h, and washed for 3 x 5 min. Because HCl treatment abolishes GFP fluorescence, the sections were eventually stained with anti-GFP antibody. Primary and secondary antibodies are listed in Table 3. 4.5 IN VITRO EXPERIMENTS (I-IV) 4.5.1 Ca2+ -imaging (I-II) For the Ca2+ -experiment, several (15 – 25) neurospheres with the diameter on average of 200 µm were transferred on poly-DL-ornithine (0.5 mg/ml in PBS, Sigma) or poly-D-lysine (10 µg/ml, Sigma) / laminin (5 µg/ml laminin, GIBCO, Life Technology) coated cover glasses and differentiated without EGF and FGF for 1 – 8 d. After attachment, single neurosphere cells started to migrate out of neurospheres and differentiate. After differentiation of the defined time period, the neurospheres were incubated with 4 µM Fura-2 acetoxymethyl-ester in a Na+ -Ringers solution (pH 7.4) consisting of (in mM) 137 NaCl, 5 KCl, 1 CaCl2, 1.2 MgCl2, 0.44 KH2PO4, 4.2 NaHCO3, 10 glucose, 1 probenicid and 20 HEPES. Thereafter the coverglasses were attached to the bottom of a thermostated perfusion chamber and perfused at a rate 2 ml/min (37 C). Individual NPCs (10 – 50) from the edge of neurospheres were

33

simultaneously recorded using 340 and 380 nm light excitation achieved with a filter changer under control of an InCytIM-2 system (Intracellular Imaging corp., Cincinnati, OH) and dichroic mirror (DM430, Nikon). The emission was measured through a 510 nm barrier filter with a COHU integrating CCD camera. A rationed (340/380) image was achieved every second. NPCs were challenged with various substances which elevated intracellular Ca 2+. The following neurotransmitters were used: substance P (SP), neurotensin (NT), oxotremorine (OxoM), ACh, ATP, NE, and glutamate in the presence and absence of extracellular Ca2+. For depolarization, NPCs were challenged with high potassium chloride K+ (70 mM) (Na+ -Ringer solution was mixed with a Ringer solution where the NaCl is replaced with KCl). For detection of the type of metabolic glutamate receptors (mGluR), group I mGluR antagonists MPEP and LY367385 were used (both from Tocris Cookson Ltd., Bristol, UK). 4.5.2 Cell proliferation, neurite length, and cell body volume analysis (I) The incorporation of 0.5 µCi/ml (1 Ci = 37 GBq) [3H ]thymidine (GE Healthcare Ltd, UK) was investigated in cell cultures seeded at a density of 105 cells per ml in culture media supplemented with growth factors. After 24 h incubation, cells were precipitated with 50 µl/ml trichloroacetic acid for 20 min at 4 C, solubilized in 80 µl of 1 M NaOH for 20 min at room temperature, and neutralized with 0.25 ml of 1 M HCl. The radioactivity of the lysates was counted with scintillation fluid (OptiPhase HiSafe 3) in a liquid scintillation counter (1450 MicroBeta, Wallac). To measure the length of neurites and cell body area, NPCs were first differentiated 1 or 5 days on the cover glasses as a single-cell suspension or as neurospheres. After Tuj-1 staining the neurons at the outer radius of the differentiated neurospheres were imaged with 40 and 60 objectives and the length of neurites and cell body area was measured by using Neurolucida Software (NeuroBright Field, VT) or Image Pro Plus (Media Cybergenics, Silver Spring, MD) software. 4.5.3 A treatments (III-IV) To investigate the effects of A on proliferation, migration, differentiation, and survival of developing NPCs, A -treatment assays were performed. For all experiments, A 40 or A 42 peptides (American Peptide, Sunnyale, USA) were reconstituted in sterile water at a concentration of 155 µM and used immediately after reconstitution except prefibrillized A 42 which was incubated for seven days at 37 C before use. Proliferation assay In the proliferation assay, 15 000 single NPCs (after neurosphere dissociation) were plated into a 96-well plate in the medium supplemented with growth factors (EGF and bFGF) and different A peptide preparations. Cells were allowed to proliferate and form new freely floating neurospheres for 48 hours. Newly formed

34

neurospheres were imaged (4xobjective) and the average sizes of neurospheres (diameter >40µm) were counted. Six to eight replicate images per each concentration were used. Migration assay In migration and differentiation assays, 10 – 15 single neurospheres (average diameter 200 – 400µm) were transferred to the poly-DL-ornithine (0.5 mg/ml in PBS, Sigma) coated well plate and allowed to differentiate without additional growth factors, but in the presence of different A peptide preparations for 2 – 4 days. For the evaluation of cell migration neurospheres were imaged as 6 – 8 replicate neurospheres per each A concentration (10xobjective). The average migration distance from the edge of a neurosphere was measured. The 30 farthest migrated cells per neurosphere were included in migration analysis. Differentiation assays For differentiation and viability assays, single NPCs were resuspended at the density of 200 000 cells / 0.5 ml in growth media and allowed to differentiate in the poly-DLornithine coated well plate without growth factors and with different A preparations for 7 days. Differentiated single NPCs and neurospheres were then fixed by using 4% formaldehyde and immunostained. For analysis, ten replicate images per each concentration were analyzed. When neurospheres were used, images (10 or 20xobjective) were taken at the periphery of neurosphere colony area, where the cells migrated separately. Viability assays For detection of dead and live cells, propidium iodide (PI) (5 µM in DMSO, Molecular Probes) and SYTO 13 (1 µM in DMSO, Molecular Probes, Life Technology) were added in culture wells (single NPCs or neurospheres) and incubated for 10 min at 37 C. Immediately after incubation, cells were imaged (6-8 replicates) and the number of live and dead cells counted. All images were taken by using Olympus IX71 microscope with MT10 illumination system attached with the DP70 digital camera, running DP software (all from Olympus). 4.5.4 Immunocytochemistry for NPCs For characterization of phenotype of differentiated NPCs, fixed cells were first permeabilized with ice-cold methanol for 20 min and then washed 3 x 15 min with 0.1 M PBS pH 7.4. After each step of this protocol, cells were washed similarly. Next, non-specific sites were blocked with 20% NGS, in PBS for 1 h. Thereafter, primary antibody with 5% NGS was added and incubated over night at RT. On the following day, secondary antibodies were incubated for 2 h at RT and protected from light. For double staining, another primary and secondary antibody were used. For nuclear stainings Hoechst 33342 (2.5µg/ml Sigma) or DAPI (0.1 µg/ml) were used. The

35

primary and secondary antibodies used in this study are listed in table 3. After staining, the wells were filled with PBS for future use and cover glasses were mounted with SlowFade antifading reagent (Molecular Probes, Life Technology). Table 3. Primary and secondary antibodies used in immunohistochemistry of cells and/or brain sections. Antibody Primary antibodies mouse anti-FMRP mouse anti-Tuj-1 mouse anti-NeuN rabbit anti-GFAP rabbit anti-Tbr1 rabbit anti-Tbr2 rabbit anti-BLBP rabbit anti-mGluR5 quinea big anti-GLAST rabbit anti-Nestin rabbit anti-DCX rabbit anti-PanA mouse anti-BrdU rabbit anti-GFP mouse anti-CD45 TUNEL Secondary antibodies Cy3 AMCA Alexa 488 Alexa 568 Dylight 405

Dilution

Manufacturer

Use (study)

1:1000 1:500 1:500 1:500 1:500 1:500 1:500 1:500/1:250 1:400 1:500 1:500 1:300 1:50

Euromedex Biosite Chemicon Sigma/DAKO Chemicon Chemicon Chemicon Chemicon Chemicon Abcam Cell Signaling Biosource Roche Applied Sciences /Amersham Bioscience BD Bioscience Serotec Chemicon

cells (I) cells (I-IV) cells (I-II) cells/slices (I-IV) cells (II) cells (II) cells (II) cells (I-II) cells (II) cells/slices (III) slices (III-IV) slices (IV) slices (III) cells (I) slices (III) slices (III) slices (III)

1:200 1:100 detection kit 1:500 1:250

Chemicon Jackson Immuno Research 1:500/1:200 Molecular Probes 1:200 Molecular Probes 1:200 Jackson Immuno Research

cells (I-II) cells (I-II) cells/slices (III-IV) cells/slices (III-IV) slices (IV)

4.6 ANALYSIS OF IMMUNOREACTIVITIES For quantitative analysis, the brain sections and cells from in vitro experiments were imaged by using fluorescence/light microscope Olympus IX71 equipped with highresolution PD70 digital camera running DP software (all from Olympus), AX70 microscope equipped with a digital camera (Color View 12 or F-view, SoftImaging Systems), laser scanning microscope Zeiss LSM5 Pascal and Leica TCS SP2 AOBS with subsequent analysis program, Zeiss LSM 700 confocal microscope and ZEN 2009 light edition analysis program. Results are shown as mean ± standard error of mean (SEM). Statistical analysis was performed by using SPSS software (SPSS Inc Chicago, IL, USA) or GraphPad Prism software (GraphPad Software, San Diego, CA). For two group comparison Student ttest was used and for more groups one way and two way ANOVA followed by LSD or Tukey’s post-hoc test. In addition Mann-Whitney test was used. The statistical significance is indicated as *p40µm) of wt and Nrf2-/- neurospheres were approximately 30 % smaller compared to LVNrf2 neurospheres. Next, for migration and differentiation assays wt, LV-Nrf2, and Nrf2-/- neurospheres were differentiated for 7 days. In the migration assay, the average migration length was measured after 7 days of differentiation. We found that LV-Nrf2 NPCs migrated slightly, but not significantly longer compared to wt and Nrf2-/- NPCs. The neuronal phenotype of differentiated NPCs (neurosphere origin) was determined by Tuj-1 staining. Tuj-1 positive NPCs were found around the neurospheres and the number of Tuj-1 positive NPCs was significantly higher in LV-Nrf2 NPCs compared to wt and Nrf2-/- NPCs. These in vitro results suggest that overexpression of Nrf2 promotes both proliferation and differentiation but not migration of NPCs. 5.4.3 Nrf2 counteracts the negative effects of A 1-42 on NPC viability and neuronal differentiation Next, we examined whether Nrf2 regulates viability, proliferation, migration, and differentiation of NPCs when exposed to A 1-42 and A 1-40. A 1-42 exposure reduced significantly the cell viability of differentiated wt and Nrf2-/- NPCs, but did not affect LV-Nrf2 transduced NPCs. In the proliferation experiment, A 1-42 reduced the proliferation of both wt and Nrf2-/- NPCs, but the size of LV-Nrf2 transduced neurospeheres was not changed. A 1-40 treatment reduced proliferation only in Nrf2/- NPCs, while in wt NPCs, proliferation remained unchanged; and in LV-Nrf2 transduced NPCs, proliferation increased almost by 35%. In the migration assay, 1-42 treatment reduced migration of all cell types. However, Nrf2-/- NPCs migrated slightly shorter distances compared to wt or LV-Nrf2 transduced NPCs. In the differentiation study, A 1-42 did not change the number of Tuj-1 positive cells in either wt or LV-Nrf2 NPCs, whereas the number of Tuj-1 positive neurons were reduced 33 % in Nrf2-/-.

44 Table 4. Main findings of this thesis. Study Main findings I

FMRP deficient neurons showed smaller cell body volume, lower number and shorter primary neurites. Differentiated FMRP deficient NPCs showed an increased amount of apoptotic cells in the cell population which was GFAPpositive. FMRP deficient NPCs showed a higher number of cells which showed intracellular calcium oscillation during the ACh stimulation.

II

At the early stage of differentiation (1-4 days), neurosphere derived NPCs responded to ACh, NE, ATP, and they had small responses to elevated K+. Several cells had also glutamate (mGluR5) and SP responses. After 5-6 days of differentiation, the number of mGluR5 responding cells progressively decreased and the number of glutamate (iGluR) and elevated K+ responding cells increased. After 8 days of differentiation, cells showed only robust iGluR, NMDA and elevated K+ responses and they were identified as Tuj-1 positive neurons.

III

When studied in vivo, transplanted NPCs survived better and migrated longer distances when transplanted into the hippocampus of APdE9 mice compared to NPCs transplanted into wt mice. In addition, transplanted NPCs stimulated endogenous neurogenesis even in APdE9 mice. When studied in vitro, APdE9 NPCs differentiated more into Tuj-1 positive cells compared to wt NPCs. Synthetic A 1-42 treatment significantly reduced migration of NPCs.

IV

When studied in vivo, KA-induced proliferation and endogenous neurogenesis were significantly lower in the hippocampi of Nrf2-/- mice. When studied in vitro, Nrf2 overexpressing NPCs differentiated more and Nrf2-/- NPCs less into Tuj-1 positive neurons compared to wt controls. During the A 1-42 treatment Nrf2 overexpressing NPCs had a higher survival, proliferation, and neuronal differentiation rate than wt controls or Nrf2-/NPCs.

45

6 Discussion Human and mouse multipotent NPCs provide a unique cell source for in vitro and in vivo studies of neural mechanisms in the pathogenesis of various human neurological disorders. Furthermore, considering that stem cell-based therapy may be an alternative therapy for many neurodegenerative diseases in the future, NPCs could represent a potential cell source for such purposes. The potentially therapeutic effect of NPCs could be obtained either by transplanting them into the affected brain area or by stimulating a patient’s own NPCs to proliferate and differentiate towards the type of neuron that has become dysfunctional or is lost during the course of the disease. Evidently, the mechanisms of NPC production as well as their true effect on the brain pathology and function need to be carefully investigated before any therapeutic use or clinical trials could be considered. The purpose of this thesis was to provide new information about the mechanisms of differentiation of NPCs in healthy brain as well as in neurological diseases of FXS and AD. The results are discussed in more detail below. 6.1 FMRP DEFICIENCY HAS EFFECT ON DIFFERENTIATION OF NPCs (I) In study I, we carried out experiments to investigate whether FMRP deficiency has an impact on NPC differentiation. The effects of FMRP deficiency during the early stage of neuronal differentiation have been poorly understood so far. We showed that FMRP deficiency alters the morphology, differentiation and the functions of both cultured human and mouse NPCs, indicating a profound role of FMRP in development of NPCs. This conclusion was further supported by the finding that FMRP deficient neurospheres differentiated more into Tuj-1 positive neurons and less into GFAP positive astrocytes compared to controls. Furthermore, FMRPdeficient neurons had shorter primary neurites and their number was reduced compared to controls. The Ca2+ imaging data showed functional differences between wt and FMRP during differentiation of the NPC population, which was characterized by a clear metabotropic glutamate response and small response to elevated K+. Interestingly, most of the FMRP deficient NPCs responded to Ach stimulation with an intense oscillation. The number of these oscillating cells was significantly higher in FMRP deficient NPCs compared to wt NPCs. FMRP deficient NPCs showed oscillatory response also to other stimuli such as ATP or NE indicating that the alteration is caused by Ca 2+ signaling rather than specific Ach receptor. Both the morphological and functional alterations indicate that the absence of FMRP plays a crucial role in early neuronal development. In previous studies abnormal dedritic spines have been found in several cerebral cortical regions in both

46

FXS patients and FMRP deficient mice (Comery et al., 1997; Irwin et al., 2000; Nimchinsky et al., 2001; Galvez et al., 2003). Also, altered synaptic structures and functions have been reported in cultured hippocampal neurons of FMRP deficient mouse (Braun and Segal 2000). It has been suggested that the altered Ca2+ signaling at the early stages of neuronal maturation correlates with changes in neuronal morphology and neurotransmitter expression (Ciccolini et al., 2003). This association between altered Ca2+ signaling and abnormal NPC morphology could well be causal since the increase in Ca2+ oscillation frequency is known to dramatically increase expression of certain genes (Dolmetch et al., 1998). Taken together, these data suggest that altered Ca2+ signaling in the FMRP deficient NPC population with a robust mGluR response is likely to contribute to disturbed neurogenesis, synapse formation, and neural maturation in FXS. Stimulation of type I mGluRs results in FMRP synthesis, which then leads to translation-dependent elongation of dendritic spines (Vanderklish et al., 2002). Importantly, a main function of FMRP is to act as a translational suppressor (Bagni and Greenough 2005; Zalfa et al., 2003). Without FMRP expression, neurons are known to have abnormal dendritic spines because of the lack of FMRP’s suppressive effects on protein translation (Vanderklish and Edelman 2005). In addition, the translation-dependent form of LTD, which is known to be induced by mGluR, has also been shown to be enhanced in Fmr1 KO mice. Altogether, these findings indicate that mGluR-dependent translational changes may be exaggerated when FMRP is not present. In agreement with this hypothesis, Weiler and coworkers (2004) demonstrated that the overall group I mGluR-stimulated translation is significantly depressed in synaptoneurosomes of the Fmr1 KO mice. The high number of cells with oscillatory response in both human and mouse FMRP deficient NPCs may indicate that the cellular plasticity is altered, particularly in those cell types which also respond to mGluR activation. Based on our results obtained by using specific antagonists of glutamate receptors, we suggest that these cells mainly express mGluR5. This finding is of interest, since disturbances in type I mGluR signaling has been associated with pathophysiological changes such as epilepsy, cognitive impairment, and abnormal dendritic spines which all are found in FXS (Bear et al., 2004). In addition, a systemic administration of mGluR5 antagonist has been shown to prevent audiogenic seizures in fmr1 KO mice (Yan et al., 2005). In the future, the type I mGluR antagonists might offer a potential pharmacological treatment for neurological and psychiatric symptoms in FXS (Bear et al., 2004). Our study increases the evidence that disruptions in signaling mediated by group 1 mGluRs are associated with the pathogenesis of FXS. The generation of oscillatory signals is a complex phenomenon and is regulated by various messengers (Taylor and Thorn 2001) such as G protein signaling (RGS) protein (Luo et al., 2001). However, the exact mechanisms behind the transmitter mediated oscillatory responses are not well known. It has been previously shown that mRNA levels of RGS4 are specifically altered in the brain of Fmr1 KO mice (Tervonen et al., 2005). This change might be involved in the oscillatory responses.

47

As a translational regulator, FMRP (Li et al., 2001; Mazroui et al., 2002; Miyashiro et al., 2003; Zalfa et al., 2003; Bagni & Greenough 2005) could thus regulate the expression of the proteins involved in the generation of Ca2+ oscillations, which in turn modify gene expression and direct cells along specific developmental pathways (Dolmetsch et al. 1998; Taylor and Thorn 2001). We also found that the number of newborn cells in the SVZ was significantly increased in Fmr1 KO brain compared to wt brain. We therefore suggest that the FMRP deficiency alters the differentiation only in particular cell populations. Actually, this finding is in line with our observations in Ca2+ imaging studies. This study demonstrated that NPCs represent a potential model cell type to study the mechanisms behind the FXS when using FMRP deficient mouse cells. In addition, by using of human NPCs isolated from aborted FXS fetuses it was possible to study human cells, too. Together with morphological methods, we used Ca2+ imaging to study the functional alterations and again, for the first time showed functional alteration in developing neurons from human origin. Our study shows that FMRP is important in neuronal development and by using NPCs it is possible to learn more about the FXS.

6.2 THE ROLE OF NEUROTRANSMITTERS IN DIFFERENTIATION OF NPCs (II) A body of evidences indicates that NTs have an important role in the regulation of NPC differentiation (Hagg, 2005) and thus an impact on the regenerative capacity of the CNS. Several NTs typically act via GPCRs, and hence are involved in neuronal plasticity and development. In particular, receptor-mediated changes in intracellular Ca2+ represent important signaling mechanism in plasticity responses (Ciccolini et al., 2003). Briefly, this pathway is initiated by the binding of NT to a specific GPCR, which is coupled with the Gq subtype of G proteins. The resulting activation of phospholipase C leads to release of DAG, which then turns on specific isoform(s) of PKC and IP3. The released IP3 causes Ca2+ mobilization from intracellular stores (Berridge et al., 2000). This PKC - Ca2+ signaling may take place via MAP-kinase cascades through several mechanisms, such as the activation of RAF-1, PYK2, SRC, or transactivation of EGF receptor (Cullen and Lockyer 2002; Pierce et al., 2002). In study I, differentiated NPCs were classified into two functional groups based on their NT responsiveness. Type I NPCs showed robust mGluR5, but small Ca2+ response to elevated K+, while type II cells showed a robust response to elevated K+ and only iGluR glutamate responses instead of mGluR5 response. These results indicate that NTs may have an important role in the early state of NPCs differentiation. To elucidate NT responsiveness in more detail, in study II, we challenged 1 – 8 days differentiated mouse wt NPCs with various neurotransmitters and measured their Ca2+ responses.

48

During the first differentiation day the NPC population was quite homogenous. Most of NPCs responded to NE, ATP, ACh, and muscarinic agonist OxoM. In earlier studies, both purinergic and muscarinic ACh signaling have been shown to enhance the proliferation of NPCs (Mishra et al., 2006; Ma et al., 2000; Williams et al., 2004; Kotani et al., 2006; Lin et al., 2007) and NE depletion to reduce the proliferation of NPCs (Kulkarni et al., 2002). However, muscarinic stimulation has been shown to promote neurogenesis and molecular processes that mediate the early steps of NPCs specification to neuronal cell lineage, too (Zhao et al., 2003). We suggest that during the first differentiation days NPCs still have their self-renewal properties and respond to NE and ACh stimulations. The positive correlation between ACh/OxoM responding cells and cells with elevated potassium chloride responses is also in agreement with the earlier finding. However, there were cell populations which still responded to ACh after 8 days of differentiation and simultanously showed neuronal characteristics, which is in line with previous studies which showed that Ach may promote neurogenesis (Zhao et al., 2003). When stimulated with neuropeptides (SP, NT, AngII, and Ox-A), a substantial proportion of cells responded only to SP stimulation with fast and spiky responses during the first days of difefrentiation. In previous studies SP has been shown to be expressed in EGF-generated embryonic mouse striatal primordial cells (Reynolds et al., 1992). SP has been shown to interact with neurokinin receptor and thus may have implications in many physiological and pathological conditions such as learning, memory and pain (Langosch et al., 2005). Interestingly, SP has been speculated to be involved in the regulation of neuronal maturation (Jacquin et al., 1992). SP is also known to be released from axons in the bone marrow and stimulate haematopoietic stem/progenitor cells to proliferate (Rameshwar et al., 2001). In our experiment, the number of SP responding cells declined during differentiation. A similar trend was seen in the population of NE responding cells. The positive correlation between SP and NE responding cells suggests that SP responses occur in cells that also respond to NE. Previously, both SP and NE have been shown to control the fate of hematopoietic stem cells (Katayama et al., 2006). The negative correlation between SP responding cells and ACh/OxoM responding cells indicates that in the early stage of differentiation there are two different cellular subpopulations. This further suggests that receptor expression patterns are important in the fate determination of NPCs towards different cell lineages and contribute to the generation of neuronal diversity in the mature brain (Muotri and Gage, 2006; Kronenberg et al., 2003). The more detailed analysis of glutamate responses showed that a significant proportion of NPCs displayed mGluR5 responses and the trend was similar in response to NE and SP. Also the number of mGluR5 responding cells declines during differentiation. In previous studies, mGluR5 receptor has been shown to be involved in synaptic plasticity and learning (Simonyi et al., 2005). MGluR5 has also a role in the proliferation and survival of NPCs in vivo (Di Giorgi Gerevini et al., 2004; Gandhi et al., 2008). Previous findings that adult mice lacking mGluR5 have a reduced number of dividing progenitors in SVZ and that embryonic stem cells (ES)

49

express mGluR5 support the hypothesis that mGluR5 regulates the early cell fate determination (Di Giorgi Gerevini et al., 2004). NPCs showing pure ionotropic responses to glutamate were also common. The same cells had typical robust responses to elevated potassium. These cells with pure ionotropic response and potassium response were denoted type II cells which appeared later during the early differentiation period. Spatial analysis showed that type II cells were most abundantly located in the outer periphery of the neurosphere migration area. There was a time-dependent inverse correlation between type I and II cells, which may indicate that type I cells converted to type II cells and then further to neurons. Our data suggest that prior to and during early differentiation, NPCs show metabotropic responses to a variety of NTs, but in particular through mGluR5 receptor. We also further suggest that withdrawal of mitogens causes cell migration from neurospheres intitiating thus the differentiation program. The progenitor characteristics progressively decreased and neuronal characteristics increased during the migration. We suggest that NTs have an important role at this stage on the fate determination. When the differentiating cells obtain more neuronal characteristics, they lose mGluR5 responses (and other metabotropic responses), and start responding through ionotropic glutamate receptors and gain the ability to respond to depolarization. Taken together, our data show that the differentiation of NPCs is a dynamic process and NTs have an important role. Cells denoted type I NPCs showed both radial glial and neuronal markers depending on differentiation time and spatial location relative to the neurosphere. Our results may indicate that metabotropic glutamate responses and ionotropic responses may be key events in the differention process. We also suggest that other receptors such as of ACh and NE play an important role in neuronal differentiation. The finding that differentiating NPCs responded to various NTs in the early stage of differentiation may indicate that the fate of cells could be influenced by changes in levels of NTs in that particular differentiation stage. Our method differentiating NPCs with Ca2+ -imaging let us to characterize the combinations of neurotransmitter receptors which were present within a single cell in this study. Compared to immunostaining methods, Ca 2+ imaging can gather more information about the combination of neurotransmitter receptors in individual cells.

6.3 THE EFFECTS OF AD GENOTYPE AND AD-LIKE NEURAL ENVIRONMENT ON DIFFERENTIATION OF CULTURED OR TRANSPLANTED NPCs (III) In animal models of AD, stem cell transplantation and enhancement of endogenous neurogenesis have been shown to be beneficial in terms of improved cognitive functions (Blurton-Jones et al. 2009). Therefore, cell transplantation or treatment with neurogenesis stimulating compounds could be an alternative future therapeutic

50

approach also for AD patients. However, AD brain is challenging as an environment for NPCs and the interplay between transplanted or newly born neurons and the AD brain environment should be carefully analyzed. As the source of NPCs for transplantation, iPS-derived NPCs are a very potent alternative. However, in the case the patient carries a disease causing mutation, also iPS-derived NPCs with the same mutation may display neuronal dysfunction. Such mutations may have an impact on behavior and fate of transplanted cells and thus the research on the effect of AD-genotype on transplanted cells is of utmost importance. In study III, we elucidated the impact of both the brain A -burdened environment and AD-genotype on NPCs in vivo and in vitro. In in vivo experiments, we transplanted GFP-positive NPCs into the mouse brain and followed the survival, migration, and differentiation of the transplanted NPC as well as their effects on hippocampal neurogenesis. Transplanted NPCs survived throughout the study period of 8 weeks, but importantly, both intrinsic and extrinsic AD-like pathology changed their differentiation towards a neuronal fate. Three major findings support this notion. First, a smaller portion of the NPCs transplanted into APdE9-mutant mice differentiated into astrocytes when compared to NPCs transplanted into wt mouse brain. Second, APdE9-mutant NPCs differentiated more into Tuj-1 positive neurons in vitro when compared to wt controls. Third, higher numbers of NPCs differentiated into GFAP positive astrocytes in wt mouse brain compared to APdE9 mouse brain. Our results are opposite to previous findings which showed that human NPCs exposed to exogenous APP and transplanted into APP23 mice favor glial differentiation (Kwak et al., 2006; Marutle et al., 2007). The exact reason for this discrepancy is not known but different mouse strains may partly explain it. In addition, human NPCs transplanted into mouse brain may react differently compared to mouse NPCs and cause inflammatory reactions. Previous studies have shown variable in vitro results on the effects of A exposure on neuronal differentiation (Haughney et al., 2002; Lopez-Toledano and Shelanski 2004; Chen and Dong 2009), which may probably be due to differences in A peptide prepations applied (Lopez-Toledano and Shelanski 2004). In our in vitro studies, we used different A peptide forms to thoroughly explore the effect of A on NPCs. We showed that different A preparations have distinct effects on differentiating NPCs. A 1-42 decreased the amount of Tuj-1 positive neurons in APdE9 NPCs cultures, but not in wt NPCs cultures. Low A 1-40 concentration increased the number of Tuj-1 positive neurons in NPCs regardless of their genotype. The effect of A 1-40 that we found is in agreement with an earlier report (Chen and Dong 2009). Also, our finding that prefibrillized A 1-42 increased the number of Tuj-1 positive cells, again regardless of cell genotype, is in agreement with the report by Lopez-Toledano and coworkers (2004). Overall, our results indicate that mutant APP and PS1 expressed by NPCs alter the differentiation potential as well and that different forms of A peptide have a potential to guide the differentiation of the NPCs into opposite directions.

51

We also explored the effect of A 1-40 and A 1-42 peptides alone or in combination on NPCs, and investigated the role of AD-like environment and genotype on the proliferation and migration of NPCs in vitro and in vivo. In in vitro experiments, A 142 alone inhibited both proliferation and migration of wt NPCs, which is in agreement with previous studies (Bondolfi et al., 2002; Haughey et al., 2002). Also, migration of APdE9 mutant NPCs was reduced after exposure to A 1-42, whereas exposure to A 1-40 showed opposite effects. In fact, migration of NPCs was increased in both genotypes after exposure to A 1-40. Furthermore, the addition of both A 1-40 and A 1-42 together partially reversed the A 1-42 inhibitory effect. Our in vivo experiment showed that not only the AD-like environment but also the genotype of transplanted cells alters the survival and migration of NPCs. Interestingly, the highest number of transplanted NPCs was found in aged APdE9 mice transplanted with APdE9 NPCs. Neither increased apoptosis in younger wt mice, nor enhanced proliferation in aged APdE9 mice explained these findings. Similarly, wt NPCs migrated longer distances transversally when transplanted into APdE9 mice and the migration distance was further increased when APdE9 NPCs were transplanted. We speculated that overexpression of APP, both in cells and in mouse together with brain endogenous microenvironment, may stimulate migration of APdE9 NPCs transplanted into APdE9 mice. In the literature, APP has been shown to be involved in the migration, neurite outgrowth, proliferation, and differentiation of neurons (Salinero et al., 2000; Caille et al., 2004; De Strooper and Annaert 2000; Ando et al., 1999; Marutle et al., 2007). In vitro, A 1-42 alone decreased both proliferation and migration of NPCs, which is not in line with our in vivo findings. Probably, the brain endogenous signals enhanced cell mobility and thus higher migration activity was seen in the AD brain. In several previous studies, AD-mimicking brain conditions and secreted chemokines have been shown to stimulate and guide progenitor cell migration (Ryu et al., 2009; Tran 2007; Miller et al., 2008; Bhattacharyya et al., 2008b). plaques could induce a mileau with a local enhancing effect on migration. For example, NPCs may be attracted by A deposits. However, in agreement with previous studies by Blurton-Jones et al., (2009), we found limited or negligible chemotaxis of NPCs towards the A deposits. On the other hand, the fact that A 1-40 partially reversed the inhibitory effect of A 1-42 on the migrating NPCs in vitro suggests that A 1-40 is one of the factors contributing to enhanced migration of NPCs in the APdE9 mouse brain. This study combined in vivo and in vitro experiments, which actually demonstrated quite opposite effects. In the in vitro study, synthetic A 1-42 clearly inhibited cell migration and cell survival of differentiating NPCs, while in the in vivo experiment, the AD-mimicking brain environment increased survival and migration of transplanted NPCs. Variable results between simple in vitro models and in vivo experiments indicated that for the wider understanding of AD environment, the use of mouse or other in vivo or ex vivo models are crucially important. Here, we used double transgenic AD mice which only increase the A 1-42/A 1-40 ratio. An improved

52

mouse model for these purposes could be a triple transgenic AD mouse model. These mice develop both A plaques and neurofibrillary tangles and therefore more closely mimic AD pathology (Oddo et al., 2003). Our hypotheses were that the ADenvironment might be toxic for transplanted NPCs and therefore will reduce cell survival. Actually, our in vivo study shows that AD brain was not as unfavorable an environment for transplanted NPCs as we thought and provides hope that stem cell transplantation could be one alternative therapeutic approach also for AD patients. 6.4 TRANSPLANTED NEUROGENESIS (III)

NPCs

STIMULATE

ENDOGENOUS

Transplanted NPCs are thought not only to replace lost neurons and connections, but also to enhance a brain’s own endogenous repair mechanisms. New neurons are produced throughout life; however, several factors such as aging and disease affect endogenous neurogenesis (Lazarov et al., 2010). APdE9 mice have been shown to have impaired neurogenesis (Taniuchi et al., 2007; Demars et al., 2010). Poorly functioning neurogenesis has been suggested to contribute to clinical AD (Lazarov and Marr, 2010). Therefore, supporting the endogenous neurogenesis may be an attractive target for therapeutic interventions. In our experiment, endogenous neurogenesis gradually decreased upon aging. At 6 months of age, significant neurogenesis was seen in both wt and APdE9 mice, but no difference in the amount of endogenous DCX-positive newly born neurons was detected between the genotypes. In 9-month-old APdE9 mice, neurogenesis has been shown to be decreased (Taniuchi et al., 2007); and thereafter, neurogenesis decreases rapidly with aging. We found few new DCX-positive neurons in wt or APdE9 mice older than 9 months. Interestingly, a higher amount of DCX-positive cells were found in hippocampi of the mice where NPCs were injected compared to PBS. There was not only an increase in the number of DCX-positive cells, but also the neurite outgrowth of the DCX-positive cells was increased. PBS-injected control mice were practically devoid of both DCX-positive cells as well as neurite outgrowth. Even though we did not include behavioral analysis in our experiment we suggest that the NPC transplantation stimulates endogenous neurogenesis, differentiation of NPCs and might thus have beneficial effects on cognitive functions. In an earlier study, NPC transplantation has been shown to rescue the memory deficits in triple transgenic AD mice (Blurton-Jones et al., 2009) via elevation of BDNF levels. BDNF has also been shown to increase neurogenesis in many studies (Rossi et al., 2006; Choi et al., 2009; Leng et al., 2009). Our finding that NPC transplantation stimulated endogenous neurogenesis even in old AD mouse brain was unexpected. These results may indicate that even in old brain, endogenous NPCs still retain their differentiation potential and the reason why they do not differentiate is the changed brain environment, not the disruption of the NPCs. Thus, these results support the accepted view of how dominant a role environmental factors are in the regulation of NPCs and also give hope that by using

53

external stimulus it could be possible to activate a patient’s own NPCs for therapeutic purposes. 6.5 NRF2 MEDIATES THE PROTECTION OF NPCs AGAINST A TOXICITY AND PROMOTES ENDOGENOUS NEUROGENESIS (IV) One of the main mechanisms of neurotoxicity in AD is thought to be OS, which may damage cellular macromolecules, eventually leading to cell death (Halliwell 2006). Together with inflammatory processes, OS renders the AD brain quite an unfavorable environment for transplanted NPCs. Nrf2 is a transcription factor which is known to induce expression of hundreds of cytoprotective and detoxication genes. Both in cell culture and in animal models the induction or overexpression of Nrf2 has been shown to be protective against neurodegeneration (Shih et al., 2003; Kanninen et al., 2008; Kanninen et al., 2009). Also vice versa, neurons isolated from Nrf2 KO mice show increased sensitivity to OS (Lee et al., 2003). One way to alleviate the effects of OS could be to increase the expression of Nrf2-responsive genes and thus increase the tolerance against OS. Interestingly, Nrf2 is known to promote neuronal differentiation of neuroblastoma cells (Zhao et al., 2009) suggesting that in addition to its protective function, Nrf2 may have a role in neuronal differentiation. This study also focused on the investigation of possible impacts of Nrf2 on proliferation, migration, and differentiation of NPCs as well as its possible protective effects against A toxicity. In earlier studies, it has been shown that both endogenous neurogenesis as well as Nrf2-pathway is impaired in the APdE9 mouse model of AD (Taniuchi et al., 2007; Kanninen et al., 2008; Choudhry et al., 2010). We thus hypothesize that loss of these two factors together can have negative impacts on endogenous neurogenesis and cognitive functions. Thus, supporting the patient´s own neurogenesis might alleviate memory loss in AD. Our results are in strong agreement with previous results showing that Nrf2 overepression protects against A 1-42 toxicity, and that loss of Nrf2 (utilizing Nrf2 -/cells) makes the cells more vulnerable against A 1-42 toxicity. As a conclusion, all of our A exposure experiments indicated that Nrf2 has a protective role against A toxicity. Nrf2 overexpression protected NPCs against A 1-42 toxicity and Nrf2 deficency diminished survival, proliferation, migration, and differentiation of NPCs. Our results also support the previous findings that Nrf2 plays a role in nerve cell differentiation. We showed that KA stimulated the neurogenesis with proliferation and differentiation of NPCs in both wt and Nrf2-/- mice. However, the number of Ki67-positive proliferating cells and DCX-positive newly formed neurons was significantly higher in wt mice compared to Nrf2-/- mice. Similar results were also detected in in vitro proliferation and differentiation experiments, as Nrf2 overexpressing cells proliferated faster and differentiated more into Tuj-1 positive neurons compared to either wt or Nrf2 -/- NPCs. These in vivo and in vitro results

54

suggest that Nrf2 has a crucial role in proliferation and differentiation. However, we did not detect an ability of Nrf2 to affect cell migration. In this study, we also combined both in vivo and in vitro experiments. These experiments clearly indicate that Nrf2 has a novel role to stimulate proliferation and differentiation of NPCs. In the in vitro study, we showed that Nrf2 overexpression had advantageous effects for survival, proliferation, and neuronal differentiation of NPCs and that the deficiency of Nrf2 exacerbated A toxicity. The knowledge that Nrf2 has both protective and neurogenic function raises the idea that NPCs with boosted Nrf2 could be beneficial for cell transplantation in the AD brain. NPCs with boosted Nrf2 could have a protective role against A toxicity, and alleviate OS leading to increased cell survival, and make the brain environment more favorable for endogenous NPCs. In addition, the use of the NPCs with boosted Nrf2 pathway for transplantation might result in enhanced neuronal differentiation and improved neuronal recovery. Another therapeutic option to obtain the same benefits could be the stimulation of endogenous neurogenesis by using small doses of Nrf2 by introducing small molecules directly into the brain.

55

7 Conclusions and summary The following conclusions can be made based on the presented results. I In the first study, we hypothesized that FMRP could be involved in differentiation of NPCs. Our results show that FMRP deficiency in FXS altered differentiation and function of NPCs during the early maturation. We demonstrated that FMRPdeficient neurospheres generate more Tuj-1 positive neurons with fewer, shorter neurites and smaller cell body volume compared to control. In addition, the number of GFAP positive astrocytes was decreased due to increased apoptotic cell death in a FMRP deficient cell population. Functional studies showed that a higher proportion of FMRP deficient cells responded to neurotransmitters with intense oscillatory Ca2+ response. We also detected a higher proportion of BrdU positive proliferating cells in SVZ of FMRP deficient mouse brain. II In the second study, our hypothesis was that neurotransmitters (NTs) are important regulators of proliferation and differentiation of NPCs. Several NTs typically activate ionotropic or G protein-coupled receptors and change the intracellular Ca2+ levels. Therefore, we used Ca2+ -imaging to monitor neurotransmitter responsiveness at certain stages of NPC differentiation. Our data indicate that differentiation of NPCs is a dynamic process and the immature and more mature NPCs can be divided into sub-groups based on their NT responsiveness. At the first differentiation day, most of the cells showed mGluR5, NE, Ach, and ATP responses. A small proportion of cells responded also to SP. These NT responses gradually disappeared when the NPCs matured. After 8 days of differentiation, the cells showed very robust responses to NMDA involving ionotropic glutamate receptors and depolarization indicating that the NPCs have differentiated into neurons. III In the third study, we investigated whether AD-like environment and AD-genotype have impacts on the cell fate upon NPC transplantation. Therefore, we dissected out the contributions of transplanted cell genotype on the behavior of NPCs in AD-like mouse brain. We showed in in vivo experiments that AD brain environment enhanced the transplanted NPC survival, migration, and neuronal differentiation. Furthermore, if APdE9 NPCs were transplanted into the AD brain they survived and migrated longer distances compared to wt NPCs. We also showed that transplanted cells enhanced a mouse’s own neurogenesis even in old AD brain and probably this could be a way to improve memory deficits. These results suggest that NPC

56

transplantation and enhanced neurogenesis could be beneficial also for AD patients even if using autologous NPCs isolated from patients suffering familiar form of AD. IV In the fourth study, we hypothesized that Nrf2 could protect differentiating NPCs against A toxicity as well as have a role in NPC proliferation, migration, and maturation. To examine the possible roles of Nrf2, we cultured wt, Nrf2 overexpressing, and Nrf2 -/- NPCs and perfomed survival, proliferation, differentiation, and migration studies with and without A treatment. In addition, to examine the role of Nrf2 in physiology of NPCs we did in vivo experiment by stimulating wt and Nrf2 -/- mice hippocampal neurogenesis by KA. Our results are in strong agreement with earlier studies where overexpression of Nrf2 protected NPCs against A 1-42 toxicity and the deficiency of Nrf2 made the cells more sensitive to A 1-42 toxicity. Furthermore, both in vitro and in vivo experiments showed that Nrf2 plays a role in nerve cell differentiation and proliferation. NPCs overexpressing Nrf2 proliferated better and produced a higher number of Tuj-1 positive neurons compared to wt and Nrf2 -/- NPCs. After KA stimulation, a higher number of proliferated (Ki67 positive) and DCX positive neurons were seen in wt mice compared to Nrf2 -/- mice. To summarize, the presented results demonstrate that NPCs are excellent tools to study the mechanisms of neuronal differentiation. Culturing NPCs isolated from normal or genetically modified mice allows studying the mechanisms behind the normal neurogenesis as well as the effects of genetic mutation. Here, we show that FMRP has an important role in neuronal development and the deficiency of FMRP causes the changes in the nerve cells at the early developmental stage. We also demonstrate that several neurotransmitters have important role during the neuronal differentiation. For therapeutic purposes, cell replacement therapy by using NPCs gives hope to recover damages or alleviate the symptoms of various neurodegenerative diseases. Our results show that AD-linked genotype and environment have strong impacts on behavior of NPCs. Despite this, transplanted NPCs survived and migrated better in AD mouse brain compared to normal brain. We also demonstrate that transplanted NPCs could stimulate endogenous neurogenesis even in the brains of old AD mice. Furhermore, we show that Nrf2 overexpression could protect differentiating NPCs against A toxicity and even promote neuronal differentiation. All of these findings provide hope that cell replacement therapy could be a suitable treatment also for AD patients.

57

8 References Abitbol, M., Menini, C., Delezoide, A.L., Rhyner, T., Vekemans, M. & Mallet, J. 1993, "Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain", Nature genetics, vol. 4, no. 2, pp. 147-153. Aizenstein, H.J., Nebes, R.D., Saxton, J.A., Price, J.C., Mathis, C.A., Tsopelas, N.D., Ziolko, S.K., James, J.A., Snitz, B.E., Houck, P.R., Bi, W., Cohen, A.D., Lopresti, B.J., DeKosky, S.T., Halligan, E.M. & Klunk, W.E. 2008, "Frequent amyloid deposition without significant cognitive impairment among the elderly", Archives of Neurology, vol. 65, no. 11, pp. 1509-1517. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I.R., McGeer, P.L., O'Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G. & Wyss-Coray, T. 2000, "Inflammation and Alzheimer's disease", Neurobiology of aging, vol. 21, no. 3, pp. 383-421. Allingham-Hawkins, D.J., Babul-Hirji, R., Chitayat, D., Holden, J.J., Yang, K.T., Lee, C., Hudson, R., Gorwill, H., Nolin, S.L., Glicksman, A., Jenkins, E.C., Brown, W.T., Howard-Peebles, P.N., Becchi, C., Cummings, E., Fallon, L., Seitz, S., Black, S.H., Vianna-Morgante, A.M., Costa, S.S., Otto, P.A., Mingroni-Netto, R.C., Murray, A., Webb, J. & Vieri, F. 1999, "Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data", American Journal of Medical Genetics, vol. 83, no. 4, pp. 322-325. Altman, J. 1969, "Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb", The Journal of comparative neurology, vol. 137, no. 4, pp. 433-457. Altman, J. 1966, "Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis", Experimental neurology, vol. 16, no. 3, pp. 263-278. ALTMAN, J. 1963, "Autoradiographic investigation of cell proliferation in the brains of rats and cats", The Anatomical Record, vol. 145, pp. 573-591. ALTMAN, J. 1962, "Are new neurons formed in the brains of adult mammals?", Science (New York, N.Y.), vol. 135, no. 3509, pp. 1127-1128. Altman, J. & Das, G.D. 1967, "Postnatal neurogenesis in the guinea-pig", Nature, vol. 214, no. 5093, pp. 1098-1101. Altman, J. & Das, G.D. 1966, "Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions", The Journal of comparative neurology, vol. 126, no. 3, pp. 337-389. Altman, J. & Das, G.D. 1965, "Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats", The Journal of comparative neurology, vol. 124, no. 3, pp. 319-335. Alvarez-Buylla, A., Herrera, D.G. & Wichterle, H. 2000, "The subventricular zone: source of neuronal precursors for brain repair", Progress in brain research, vol. 127, pp. 1-11. Alvarez-Buylla, A. & Lim, D.A. 2004, "For the long run: maintaining germinal niches in the adult brain", Neuron, vol. 41, no. 5, pp. 683-686.

58 Ando, K., Oishi, M., Takeda, S., Iijima, K., Isohara, T., Nairn, A.C., Kirino, Y., Greengard, P. & Suzuki, T. 1999, "Role of phosphorylation of Alzheimer's amyloid precursor protein during neuronal differentiation", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 19, no. 11, pp. 4421-4427. Antar, L.N., Afroz, R., Dictenberg, J.B., Carroll, R.C. & Bassell, G.J. 2004, "Metabotropic glutamate receptor activation regulates fragile x mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 24, no. 11, pp. 2648-2655. Antar, L.N. & Bassell, G.J. 2003, "Sunrise at the synapse: the FMRP mRNP shaping the synaptic interface", Neuron, vol. 37, no. 4, pp. 555-558. Arce, M.P., Rodriguez-Franco, M.I., Gonzalez-Munoz, G.C., Perez, C., Lopez, B., Villarroya, M., Lopez, M.G., Garcia, A.G. & Conde, S. 2009, "Neuroprotective and cholinergic properties of multifunctional glutamic acid derivatives for the treatment of Alzheimer's disease", Journal of medicinal chemistry, vol. 52, no. 22, pp. 7249-7257. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & Lindvall, O. 2002, "Neuronal replacement from endogenous precursors in the adult brain after stroke", Nature medicine, vol. 8, no. 9, pp. 963-970. Ashford, J.W. 2004, "APOE genotype effects on Alzheimer's disease onset and epidemiology", Journal of molecular neuroscience : MN, vol. 23, no. 3, pp. 157-165. Ashley, C.T., Sutcliffe, J.S., Kunst, C.B., Leiner, H.A., Eichler, E.E., Nelson, D.L. & Warren, S.T. 1993, "Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat", Nature genetics, vol. 4, no. 3, pp. 244-251. Bagni, C. & Greenough, W.T. 2005, "From mRNP trafficking to spine dysmorphogenesis: the roots of fragile X syndrome", Nature reviews.Neuroscience, vol. 6, no. 5, pp. 376-387. Baird, L. & Dinkova-Kostova, A.T. 2011, "The cytoprotective role of the Keap1-Nrf2 pathway", Archives of Toxicology, vol. 85, no. 4, pp. 241-272. Baker, S.A., Baker, K.A. & Hagg, T. 2004, "Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone", The European journal of neuroscience, vol. 20, no. 2, pp. 575-579. Bakker, C.E., Verheij, C., Willemsen, R. van der Helm, R., Oerlemans, F., Vermeij, M., Bygrave, A., Hoogeveen, A., Oostra, B.A., Reyniers, E., De Boulle, K., D´Hooge, R., Cras, P., van Velzen, D., Nagels, G., Marti, J.J., De Deyn, P., Darby, J.K. & Willems, P.J. 1994, “ Fmr1 knockout mice: a model to study Fragile X mental retardation”, Cell, no. 78, pp. 23-33. Ballard, C., Gauthier, S., Corbett, A., Brayne, C., Aarsland, D. & Jones, E. 2011, "Alzheimer's disease", Lancet, vol. 377, no. 9770, pp. 1019-1031. Ballatore, C., Lee, V.M. & Trojanowski, J.Q. 2007, "Tau-mediated neurodegeneration in Alzheimer's disease and related disorders", Nature reviews.Neuroscience, vol. 8, no. 9, pp. 663-672. Banasr, M., Hery, M., Printemps, R. & Daszuta, A. 2004, "Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone", Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, vol. 29, no. 3, pp. 450-460. Bandyopadhyay, S., Goldstein, L.E., Lahiri, D.K. & Rogers, J.T. 2007, "Role of the APP nonamyloidogenic signaling pathway and targeting alpha-secretase as an alternative drug target

59 for treatment of Alzheimer's disease", Current medicinal chemistry, vol. 14, no. 27, pp. 28482864. Barkho, B.Z., Song, H., Aimone, J.B., Smrt, R.D., Kuwabara, T., Nakashima, K., Gage, F.H. & Zhao, X. 2006, "Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation", Stem cells and development, vol. 15, no. 3, pp. 407421. Bassell, G.J. & Warren, S.T. 2008, "Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function", Neuron, vol. 60, no. 2, pp. 201-214. Bear, M.F., Huber, K.M. & Warren, S.T. 2004, "The mGluR theory of fragile X mental retardation", Trends in neurosciences, vol. 27, no. 7, pp. 370-377. Belluzzi, O., Benedusi, M., Ackman, J. & LoTurco, J.J. 2003, "Electrophysiological differentiation of new neurons in the olfactory bulb", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 32, pp. 10411-10418. Berridge, M.J. 2004, "Calcium signal transduction and cellular control mechanisms", Biochimica et biophysica acta, vol. 1742, no. 1-3, pp. 3-7. Berridge, M.J., Bootman, M.D. & Roderick, H.L. 2003, "Calcium signalling: dynamics, homeostasis and remodelling", Nature reviews.Molecular cell biology, vol. 4, no. 7, pp. 517-529. Berridge, M.J., Lipp, P. & Bootman, M.D. 2000, "The versatility and universality of calcium signalling", Nature reviews.Molecular cell biology, vol. 1, no. 1, pp. 11-21. Beydoun, M.A., Beydoun, H.A. & Wang, Y. 2008, "Obesity and central obesity as risk factors for incident dementia and its subtypes: a systematic review and meta-analysis", Obesity reviews : an official journal of the International Association for the Study of Obesity, vol. 9, no. 3, pp. 204-218. Bhattacharyya, A., McMillan, E., Wallace, K., Tubon, T.C.,Jr, Capowski, E.E. & Svendsen, C.N. 2008, "Normal Neurogenesis but Abnormal Gene Expression in Human Fragile X Cortical Progenitor Cells", Stem cells and development, vol. 17, no. 1, pp. 107-117. Bhattacharyya, B.J., Banisadr, G., Jung, H., Ren, D., Cronshaw, D.G., Zou, Y. & Miller, R.J. 2008, "The chemokine stromal cell-derived factor-1 regulates GABAergic inputs to neural progenitors in the postnatal dentate gyrus", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 28, no. 26, pp. 6720-6730. Bithell, A. & Williams, B.P. 2005, "Neural stem cells and cell replacement therapy: making the right cells", Clinical science (London, England : 1979), vol. 108, no. 1, pp. 13-22. Bizon, J.L. & Gallagher, M. 2005, "More is less: neurogenesis and age-related cognitive decline in Long-Evans rats", Science of aging knowledge environment : SAGE KE, vol. 2005, no. 7, pp. re2. Blackwell, E., Zhang, X. & Ceman, S. 2010, "Arginines of the RGG box regulate FMRP association with polyribosomes and mRNA", Human molecular genetics, vol. 19, no. 7, pp. 1314-1323. Blurton-Jones, M., Kitazawa, M., Martinez-Coria, H., Castello, N.A., Muller, F.J., Loring, J.F., Yamasaki, T.R., Poon, W.W., Green, K.N. & LaFerla, F.M. 2009, "Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 32, pp. 13594-13599. Boekhoorn, K., Joels, M. & Lucassen, P.J. 2006, "Increased proliferation reflects glial and vascularassociated changes, but not neurogenesis in the presenile Alzheimer hippocampus", Neurobiology of disease, vol. 24, no. 1, pp. 1-14.

60 Bondolfi, L., Calhoun, M., Ermini, F., Kuhn, H.G., Wiederhold, K.H., Walker, L., Staufenbiel, M. & Jucker, M. 2002, "Amyloid-associated neuron loss and gliogenesis in the neocortex of amyloid precursor protein transgenic mice", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 22, no. 2, pp. 515-522. Borta, A. & Hoglinger, G.U. 2007, "Dopamine and adult neurogenesis", Journal of neurochemistry, vol. 100, no. 3, pp. 587-595. Braun, K. & Segal, M. 2000, "FMRP involvement in formation of synapses among cultured hippocampal neurons", Cerebral cortex (New York, N.Y.: 1991), vol. 10, no. 10, pp. 10451052. Brazel, C.Y., Nunez, J.L., Yang, Z. & Levison, S.W. 2005, "Glutamate enhances survival and proliferation of neural progenitors derived from the subventricular zone", Neuroscience, vol. 131, no. 1, pp. 55-65. Brennan, F.X., Albeck, D.S. & Paylor, R. 2006, "Fmr1 knockout mice are impaired in a leverpress escape/avoidance task", Genes, brain, and behavior, vol. 5, no. 6, pp. 467-471. Brezun, J.M. & Daszuta, A. 1999, "Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats", Neuroscience, vol. 89, no. 4, pp. 999-1002. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O'Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wilkinson, K.D., Keene, J.D., Darnell, R.B. & Warren, S.T. 2001, "Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome", Cell, vol. 107, no. 4, pp. 477-487. Bull, N.D. & Bartlett, P.F. 2005, "The adult mouse hippocampal progenitor is neurogenic but not a stem cell", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 25, no. 47, pp. 10815-10821. Burbank, K.S. & Mitchison, T.J. 2006, "Microtubule dynamic instability", Current biology : CB, vol. 16, no. 14, pp. R516-7. Burton, N.C., Kensler, T.W. & Guilarte, T.R. 2006, "In vivo modulation of the Parkinsonian phenotype by Nrf2", Neurotoxicology, vol. 27, no. 6, pp. 1094-1100. Caille, I., Allinquant, B., Dupont, E., Bouillot, C., Langer, A., Muller, U. & Prochiantz, A. 2004, "Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone", Development (Cambridge, England), vol. 131, no. 9, pp. 2173-2181. Calkins, M.J., Jakel, R.J., Johnson, D.A., Chan, K., Kan, Y.W. & Johnson, J.A. 2005, "Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription", Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 1, pp. 244-249. Calkins, M.J., Vargas, M.R., Johnson, D.A. & Johnson, J.A. 2010, "Astrocyte-specific overexpression of Nrf2 protects striatal neurons from mitochondrial complex II inhibition", Toxicological sciences : an official journal of the Society of Toxicology, vol. 115, no. 2, pp. 557-568. Callan, M.A., Cabernard, C., Heck, J., Luois, S., Doe, C.Q. & Zarnescu, D.C. 2010, "Fragile X protein controls neural stem cell proliferation in the Drosophila brain", Human molecular genetics, vol. 19, no. 15, pp. 3068-3079. Cameron, H.A., McEwen, B.S. & Gould, E. 1995, "Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 15, no. 6, pp. 4687-4692.

61 Cameron, H.A. & McKay, R.D. 2001, "Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus", The Journal of comparative neurology, vol. 435, no. 4, pp. 406417. Caporaso, G.L., Takei, K., Gandy, S.E., Matteoli, M., Mundigl, O., Greengard, P. & De Camilli, P. 1994, "Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 14, no. 5 Pt 2, pp. 3122-3138. Chan, J.Y. & Kwong, M. 2000, "Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein", Biochimica et biophysica acta, vol. 1517, no. 1, pp. 19-26. Chan, K. & Kan, Y.W. 1999, "Nrf2 is essential for protection against acute pulmonary injury in mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 22, pp. 12731-12736. Chen, L. & Toth, M. 2001, "Fragile X mice develop sensory hyperreactivity to auditory stimuli", Neuroscience, vol. 103, no. 4, pp. 1043-1050. Chen, W., Sun, Z., Wang, X.J., Jiang, T., Huang, Z., Fang, D. & Zhang, D.D. 2009, "Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response", Molecular cell, vol. 34, no. 6, pp. 663-673. Chen, Y. & Dong, C. 2009, "Abeta40 promotes neuronal cell fate in neural progenitor cells", Cell death and differentiation, vol. 16, no. 3, pp. 386-394. Choi, S.H., Li, Y., Parada, L.F. & Sisodia, S.S. 2009, "Regulation of hippocampal progenitor cell survival, proliferation and dendritic development by BDNF", Molecular neurodegeneration, vol. 4, pp. 52. Choi, S.H., Veeraraghavalu, K., Lazarov, O., Marler, S., Ransohoff, R.M., Ramirez, J.M. & Sisodia, S.S. 2008, "Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation", Neuron, vol. 59, no. 4, pp. 568580. Choi, Y.S., Cho, K.O. & Kim, S.Y. 2007, "Asymmetry in enhanced neurogenesis in the rostral dentate gyrus following kainic acid-induced status epilepticus in adult rats", Archives of Pharmacal Research, vol. 30, no. 5, pp. 646-652. Chonchaiya, W., Schneider, A. & Hagerman, R.J. 2009, "Fragile X: a family of disorders", Advances in Pediatrics, vol. 56, pp. 165-186. Choudhry, F., Howlett, D.R., Richardson, J.C., Francis, P.T. & Williams, R.J. 2010, "Pro-oxidant diet enhances beta/gamma secretase-mediated APP processing in APP/PS1 transgenic mice", Neurobiology of aging, . Chow, V.W., Mattson, M.P., Wong, P.C. & Gleichmann, M. 2010, "An overview of APP processing enzymes and products", Neuromolecular medicine, vol. 12, no. 1, pp. 1-12. Ciccolini, F., Collins, T.J., Sudhoelter, J., Lipp, P., Berridge, M.J. & Bootman, M.D. 2003, "Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 1, pp. 103-111. Clapham, D.E. 2007, "Calcium signaling", Cell, vol. 131, no. 6, pp. 1047-1058. Clapham, D.E. 1995, "Calcium signaling", Cell, vol. 80, no. 2, pp. 259-268.

62 Clelland, C.D., Choi, M., Romberg, C., Clemenson, G.D.,Jr, Fragniere, A., Tyers, P., Jessberger, S., Saksida, L.M., Barker, R.A., Gage, F.H. & Bussey, T.J. 2009, "A functional role for adult hippocampal neurogenesis in spatial pattern separation", Science (New York, N.Y.), vol. 325, no. 5937, pp. 210-213. Colak, D., Mori, T., Brill, M.S., Pfeifer, A., Falk, S., Deng, C., Monteiro, R., Mummery, C., Sommer, L. & Gotz, M. 2008, "Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 28, no. 2, pp. 434-446. Comery, T.A., Harris, J.B., Willems, P.J., Oostra, B.A., Irwin, S.A., Weiler, I.J. & Greenough, W.T. 1997, "Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits", Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 10, pp. 5401-5404. Cooper-Kuhn, C.M., Winkler, J. & Kuhn, H.G. 2004, "Decreased neurogenesis after cholinergic forebrain lesion in the adult rat", Journal of neuroscience research, vol. 77, no. 2, pp. 155165. Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. & Pericak-Vance, M.A. 1993, "Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families", Science (New York, N.Y.), vol. 261, no. 5123, pp. 921-923. Coronas, V., Bantubungi, K., Fombonne, J., Krantic, S., Schiffmann, S.N. & Roger, M. 2004, "Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the postnatal subventricular zone", Journal of neurochemistry, vol. 91, no. 6, pp. 1292-1301. Cova, L., Armentero, M.T., Zennaro, E., Calzarossa, C., Bossolasco, P., Busca, G., Lambertenghi Deliliers, G., Polli, E., Nappi, G., Silani, V. & Blandini, F. 2010, "Multiple neurogenic and neurorescue effects of human mesenchymal stem cell after transplantation in an experimental model of Parkinson's disease", Brain research, vol. 1311, pp. 12-27. Cullen, P.J. & Lockyer, P.J. 2002, "Integration of calcium and Ras signalling", Nature reviews.Molecular cell biology, vol. 3, no. 5, pp. 339-348. Darnell, J.C., Van Driesche, S.J., Zhang, C., Hung, K.Y., Mele, A., Fraser, C.E., Stone, E.F., Chen, C., Fak, J.J., Chi, S.W., Licatalosi, D.D., Richter, J.D. & Darnell, R.B. 2011, "FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism", Cell, vol. 146, no. 2, pp. 247-261. Das, S. & Basu, A. 2008, "Inflammation: a new candidate in modulating adult neurogenesis", Journal of neuroscience research, vol. 86, no. 6, pp. 1199-1208. De Strooper, B. 2010, "Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process", Physiological Reviews, vol. 90, no. 2, pp. 465-494. De Strooper, B. & Annaert, W. 2000, "Proteolytic processing and cell biological functions of the amyloid precursor protein", Journal of cell science, vol. 113 ( Pt 11), no. Pt 11, pp. 18571870. de Vries, H.E., Witte, M., Hondius, D., Rozemuller, A.J., Drukarch, B., Hoozemans, J. & van Horssen, J. 2008, "Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease?", Free radical biology & medicine, vol. 45, no. 10, pp. 1375-1383. Deisseroth, K., Singla, S., Toda, H., Monje, M., Palmer, T.D. & Malenka, R.C. 2004, "Excitationneurogenesis coupling in adult neural stem/progenitor cells", Neuron, vol. 42, no. 4, pp. 535552.

63 Demars, M., Hu, Y.S., Gadadhar, A. & Lazarov, O. 2010, "Impaired neurogenesis is an early event in the etiology of familial Alzheimer's disease in transgenic mice", Journal of neuroscience research, vol. 88, no. 10, pp. 2103-2117. Demars, M.P., Bartholomew, A., Strakova, Z. & Lazarov, O. 2011, "Soluble amyloid precursor protein: a novel proliferation factor of adult progenitor cells of ectodermal and mesodermal origin", Stem cell research & therapy, vol. 2, no. 4, pp. 36. Deng, W., Saxe, M.D., Gallina, I.S. & Gage, F.H. 2009, "Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 29, no. 43, pp. 1353213542. Devys, D., Lutz, Y., Rouyer, N., Bellocq, J.P. & Mandel, J.L. 1993, "The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation", Nature genetics, vol. 4, no. 4, pp. 335-340. Di Giorgi Gerevini, V.D., Caruso, A., Cappuccio, I., Ricci Vitiani, L., Romeo, S., Della Rocca, C., Gradini, R., Melchiorri, D. & Nicoletti, F. 2004, "The mGlu5 metabotropic glutamate receptor is expressed in zones of active neurogenesis of the embryonic and postnatal brain", Brain research.Developmental brain research, vol. 150, no. 1, pp. 17-22. Di Giorgi-Gerevini, V., Melchiorri, D., Battaglia, G., Ricci-Vitiani, L., Ciceroni, C., Busceti, C.L., Biagioni, F., Iacovelli, L., Canudas, A.M., Parati, E., De Maria, R. & Nicoletti, F. 2005, "Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells", Cell death and differentiation, vol. 12, no. 8, pp. 11241133. Dickson, D.W. 1997, "The pathogenesis of senile plaques", Journal of neuropathology and experimental neurology, vol. 56, no. 4, pp. 321-339. Dinkova-Kostova, A.T., Holtzclaw, W.D., Cole, R.N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M. & Talalay, P. 2002, "Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants", Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 18, pp. 11908-11913. Dobkin, C., Rabe, A., Dumas, R., El Idrissi, A., Haubenstock, H. & Brown, W.T. 2000, "Fmr1 knockout mouse has a distinctive strain-specific learning impairment", Neuroscience, vol. 100, no. 2, pp. 423-429. Doetsch, F. & Alvarez-Buylla, A. 1996, "Network of tangential pathways for neuronal migration in adult mammalian brain", Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 25, pp. 14895-14900. Doetsch, F., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. 1997, "Cellular composition and threedimensional organization of the subventricular germinal zone in the adult mammalian brain", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 17, no. 13, pp. 5046-5061. Doetsch, F. & Hen, R. 2005, "Young and excitable: the function of new neurons in the adult mammalian brain", Current opinion in neurobiology, vol. 15, no. 1, pp. 121-128. Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. 2002, "EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells", Neuron, vol. 36, no. 6, pp. 1021-1034. Dolmetsch, R.E., Xu, K. & Lewis, R.S. 1998, "Calcium oscillations increase the efficiency and specificity of gene expression", Nature, vol. 392, no. 6679, pp. 933-936.

64 Duan, W., Peng, Q., Masuda, N., Ford, E., Tryggestad, E., Ladenheim, B., Zhao, M., Cadet, J.L., Wong, J. & Ross, C.A. 2008, "Sertraline slows disease progression and increases neurogenesis in N171-82Q mouse model of Huntington's disease", Neurobiology of disease, vol. 30, no. 3, pp. 312-322. Eberhart, D.E., Malter, H.E., Feng, Y. & Warren, S.T. 1996, "The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals", Human molecular genetics, vol. 5, no. 8, pp. 1083-1091. Einstein, O. & Ben-Hur, T. 2008, "The changing face of neural stem cell therapy in neurologic diseases", Archives of Neurology, vol. 65, no. 4, pp. 452-456. Eisch, A.J. & Harburg, G.C. 2006, "Opiates, psychostimulants, and adult hippocampal neurogenesis: Insights for addiction and stem cell biology", Hippocampus, vol. 16, no. 3, pp. 271-286. Ekdahl, C.T., Claasen, J.H., Bonde, S., Kokaia, Z. & Lindvall, O. 2003, "Inflammation is detrimental for neurogenesis in adult brain", Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 23, pp. 13632-13637. El Khoury, J., Hickman, S.E., Thomas, C.A., Cao, L., Silverstein, S.C. & Loike, J.D. 1996, "Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils", Nature, vol. 382, no. 6593, pp. 716-719. Emerit, M.B., Riad, M. & Hamon, M. 1992, "Trophic effects of neurotransmitters during brain maturation", Biology of the neonate, vol. 62, no. 4, pp. 193-201. Ermini, F.V., Grathwohl, S., Radde, R., Yamaguchi, M., Staufenbiel, M., Palmer, T.D. & Jucker, M. 2008, "Neurogenesis and alterations of neural stem cells in mouse models of cerebral amyloidosis", The American journal of pathology, vol. 172, no. 6, pp. 1520-1528. Farris, W., Mansourian, S., Chang, Y., Lindsley, L., Eckman, E.A., Frosch, M.P., Eckman, C.B., Tanzi, R.E., Selkoe, D.J. & Guenette, S. 2003, "Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo", Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 7, pp. 4162-4167. Farris, W., Mansourian, S., Leissring, M.A., Eckman, E.A., Bertram, L., Eckman, C.B., Tanzi, R.E. & Selkoe, D.J. 2004, "Partial loss-of-function mutations in insulin-degrading enzyme that induce diabetes also impair degradation of amyloid beta-protein", The American journal of pathology, vol. 164, no. 4, pp. 1425-1434. Feng, Y., Gutekunst, C.A., Eberhart, D.E., Yi, H., Warren, S.T. & Hersch, S.M. 1997, "Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 17, no. 5, pp. 1539-1547. Floyd, R.A. & Hensley, K. 2002, "Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases", Neurobiology of aging, vol. 23, no. 5, pp. 795-807. Frankland, P.W., Wang, Y., Rosner, B., Shimizu, T., Balleine, B.W., Dykens, E.M., Ornitz, E.M. & Silva, A.J. 2004, "Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice", Molecular psychiatry, vol. 9, no. 4, pp. 417-425. Freundlieb, N., Francois, C., Tande, D., Oertel, W.H., Hirsch, E.C. & Hoglinger, G.U. 2006, "Dopaminergic substantia nigra neurons project topographically organized to the subventricular zone and stimulate precursor cell proliferation in aged primates", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 26, no. 8, pp. 23212325.

65 Fu, Y.H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkerk, A.J., Holden, J.J., Fenwick, R.G.,Jr & Warren, S.T. 1991, "Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox", Cell, vol. 67, no. 6, pp. 1047-1058. Fukuda, S., Kato, F., Tozuka, Y., Yamaguchi, M., Miyamoto, Y. & Hisatsune, T. 2003, "Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 28, pp. 93579366. Furukawa, K., Sopher, B.L., Rydel, R.E., Begley, J.G., Pham, D.G., Martin, G.M., Fox, M. & Mattson, M.P. 1996, "Increased activity-regulating and neuroprotective efficacy of alphasecretase-derived secreted amyloid precursor protein conferred by a C-terminal heparinbinding domain", Journal of neurochemistry, vol. 67, no. 5, pp. 1882-1896. Gage, F.H. 2004, "Structural plasticity of the adult brain", Dialogues in clinical neuroscience, vol. 6, no. 2, pp. 135-141. Gage, F.H. 2002, "Neurogenesis in the adult brain", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 22, no. 3, pp. 612-613. Galli, R., Gritti, A., Bonfanti, L. & Vescovi, A.L. 2003, "Neural stem cells: an overview", Circulation research, vol. 92, no. 6, pp. 598-608. Galli, R., Gritti, A. & Vescovi, A.L. 2008, "Adult neural stem cells", Methods in molecular biology (Clifton, N.J.), vol. 438, pp. 67-84. Gantois, I., Bakker, C.E., Reyniers, E., Willemsen, R., D'Hooge, R., De Deyn, P.P., Oostra, B.A. & Kooy, R.F. 2001, "Restoring the phenotype of fragile X syndrome: insight from the mouse model", Current Molecular Medicine, vol. 1, no. 4, pp. 447-455. Gascon, E., Dayer, A.G., Sauvain, M.O., Potter, G., Jenny, B., De Roo, M., Zgraggen, E., Demaurex, N., Muller, D. & Kiss, J.Z. 2006, "GABA regulates dendritic growth by stabilizing lamellipodia in newly generated interneurons of the olfactory bulb", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 26, no. 50, pp. 1295612966. Gether, U. 2000, "Uncovering molecular mechanisms involved in activation of G protein-coupled receptors", Endocrine reviews, vol. 21, no. 1, pp. 90-113. Glenner, G.G., Wong, C.W., Quaranta, V. & Eanes, E.D. 1984, "The amyloid deposits in Alzheimer's disease: their nature and pathogenesis", Applied Pathology, vol. 2, no. 6, pp. 357-369. Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N. & James, L. 1991, "Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease", Nature, vol. 349, no. 6311, pp. 704-706. Gonzalez-Billault, C., Jimenez-Mateos, E.M., Caceres, A., Diaz-Nido, J., Wandosell, F. & Avila, J. 2004, "Microtubule-associated protein 1B function during normal development, regeneration, and pathological conditions in the nervous system", Journal of neurobiology, vol. 58, no. 1, pp. 48-59. Gould, E., Cameron, H.A. & McEwen, B.S. 1994, "Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus", The Journal of comparative neurology, vol. 340, no. 4, pp. 551-565. Gould, E., Tanapat, P., Hastings, N.B. & Shors, T.J. 1999, "Neurogenesis in adulthood: a possible role in learning", Trends in cognitive sciences, vol. 3, no. 5, pp. 186-192.

66 Grote, H.E. & Hannan, A.J. 2007, "Regulators of adult neurogenesis in the healthy and diseased brain", Clinical and experimental pharmacology & physiology, vol. 34, no. 5-6, pp. 533-545. Gueneau, G., Privat, A., Drouet, J. & Court, L. 1982, "Subgranular zone of the dentate gyrus of young rabbits as a secondary matrix. A high-resolution autoradiographic study", Developmental neuroscience, vol. 5, no. 4, pp. 345-358. Guo, W., Allan, A.M., Zong, R., Zhang, L., Johnson, E.B., Schaller, E.G., Murthy, A.C., Goggin, S.L., Eisch, A.J., Oostra, B.A., Nelson, D.L., Jin, P. & Zhao, X. 2011, "Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning", Nature medicine, vol. 17, no. 5, pp. 559-565. Guo, W., Murthy, A.C., Zhang, L., Johnson, E.B., Schaller, E.G., Allan, A.M. & Zhao, X. 2012, "Inhibition of GSK3beta improves hippocampus-dependent learning and rescues neurogenesis in a mouse model of fragile X syndrome", Human molecular genetics, vol. 21, no. 3, pp. 681691. Hagg, T. 2009, "From neurotransmitters to neurotrophic factors to neurogenesis", The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, vol. 15, no. 1, pp. 20-27. Hagg, T. 2005, "Molecular regulation of adult CNS neurogenesis: an integrated view", Trends in neurosciences, vol. 28, no. 11, pp. 589-595. Halliwell, B. 2006, "Oxidative stress and neurodegeneration: where are we now?", Journal of neurochemistry, vol. 97, no. 6, pp. 1634-1658. Hanger, D.P., Seereeram, A. & Noble, W. 2009, "Mediators of tau phosphorylation in the pathogenesis of Alzheimer's disease", Expert review of neurotherapeutics, vol. 9, no. 11, pp. 1647-1666. Hartmann, T., Bieger, S.C., Bruhl, B., Tienari, P.J., Ida, N., Allsop, D., Roberts, G.W., Masters, C.L., Dotti, C.G., Unsicker, K. & Beyreuther, K. 1997, "Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides", Nature medicine, vol. 3, no. 9, pp. 1016-1020. Haughey, N.J., Liu, D., Nath, A., Borchard, A.C. & Mattson, M.P. 2002a, "Disruption of neurogenesis in the subventricular zone of adult mice, and in human cortical neuronal precursor cells in culture, by amyloid beta-peptide: implications for the pathogenesis of Alzheimer's disease", Neuromolecular medicine, vol. 1, no. 2, pp. 125-135. Haughey, N.J., Nath, A., Chan, S.L., Borchard, A.C., Rao, M.S. & Mattson, M.P. 2002b, "Disruption of neurogenesis by amyloid beta-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's disease", Journal of neurochemistry, vol. 83, no. 6, pp. 1509-1524. Hensley, K., Carney, J.M., Mattson, M.P., Aksenova, M., Harris, M., Wu, J.F., Floyd, R.A. & Butterfield, D.A. 1994, "A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 8, pp. 3270-3274. Hinds, H.L., Ashley, C.T., Sutcliffe, J.S., Nelson, D.L., Warren, S.T., Housman, D.E. & Schalling, M. 1993, "Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome", Nature genetics, vol. 3, no. 1, pp. 36-43. Hitoshi, S., Maruta, N., Higashi, M., Kumar, A., Kato, N. & Ikenaka, K. 2007, "Antidepressant drugs reverse the loss of adult neural stem cells following chronic stress", Journal of neuroscience research, vol. 85, no. 16, pp. 3574-3585.

67 Hoglinger, G.U., Rizk, P., Muriel, M.P., Duyckaerts, C., Oertel, W.H., Caille, I. & Hirsch, E.C. 2004, "Dopamine depletion impairs precursor cell proliferation in Parkinson disease", Nature neuroscience, vol. 7, no. 7, pp. 726-735. Hollmann, M. & Heinemann, S. 1994, "Cloned glutamate receptors", Annual Review of Neuroscience, vol. 17, pp. 31-108. Holscher, C. 2011, "Diabetes as a risk factor for Alzheimer's disease: insulin signalling impairment in the brain as an alternative model of Alzheimer's disease", Biochemical Society transactions, vol. 39, no. 4, pp. 891-897. Huber, K.M., Gallagher, S.M., Warren, S.T. & Bear, M.F. 2002, "Altered synaptic plasticity in a mouse model of fragile X mental retardation", Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 11, pp. 7746-7750. Hur, E.M. & Zhou, F.Q. 2010, "GSK3 signalling in neural development", Nature reviews.Neuroscience, vol. 11, no. 8, pp. 539-551. Imayoshi, I., Sakamoto, M., Ohtsuka, T., Takao, K., Miyakawa, T., Yamaguchi, M., Mori, K., Ikeda, T., Itohara, S. & Kageyama, R. 2008, "Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain", Nature neuroscience, vol. 11, no. 10, pp. 11531161. Iqbal, K., Alonso Adel, C., Chen, S., Chohan, M.O., El-Akkad, E., Gong, C.X., Khatoon, S., Li, B., Liu, F., Rahman, A., Tanimukai, H. & Grundke-Iqbal, I. 2005, "Tau pathology in Alzheimer disease and other tauopathies", Biochimica et biophysica acta, vol. 1739, no. 2-3, pp. 198210. Iqbal, K., Liu, F., Gong, C.X., Alonso Adel, C. & Grundke-Iqbal, I. 2009, "Mechanisms of tauinduced neurodegeneration", Acta Neuropathologica, vol. 118, no. 1, pp. 53-69. Irwin, S.A., Galvez, R. & Greenough, W.T. 2000, "Dendritic spine structural anomalies in fragile-X mental retardation syndrome", Cerebral cortex (New York, N.Y.: 1991), vol. 10, no. 10, pp. 1038-1044. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M. & Nabeshima, Y. 1997, "An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements", Biochemical and biophysical research communications, vol. 236, no. 2, pp. 313322. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., O'Connor, T. & Yamamoto, M. 2003, "Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles", Genes to cells : devoted to molecular & cellular mechanisms, vol. 8, no. 4, pp. 379-391. Itou, Y., Nochi, R., Kuribayashi, H., Saito, Y. & Hisatsune, T. 2011, "Cholinergic activation of hippocampal neural stem cells in aged dentate gyrus", Hippocampus, vol. 21, no. 4, pp. 446459. Iwata, N., Tsubuki, S., Takaki, Y., Shirotani, K., Lu, B., Gerard, N.P., Gerard, C., Hama, E., Lee, H.J. & Saido, T.C. 2001, "Metabolic regulation of brain Abeta by neprilysin", Science (New York, N.Y.), vol. 292, no. 5521, pp. 1550-1552. Jaako, K., Zharkovsky, T. & Zharkovsky, A. 2009, "Effects of repeated citalopram treatment on kainic acid-induced neurogenesis in adult mouse hippocampus", Brain research, vol. 1288, pp. 18-28.

68 Jablonska, B., Aguirre, A., Vandenbosch, R., Belachew, S., Berthet, C., Kaldis, P. & Gallo, V. 2007, "Cdk2 is critical for proliferation and self-renewal of neural progenitor cells in the adult subventricular zone", The Journal of cell biology, vol. 179, no. 6, pp. 1231-1245. Jacquemont, S., Farzin, F., Hall, D., Leehey, M., Tassone, F., Gane, L., Zhang, L., Grigsby, J., Jardini, T., Lewin, F., Berry-Kravis, E., Hagerman, P.J. & Hagerman, R.J. 2004, "Aging in individuals with the FMR1 mutation", American Journal of Mental Retardation : AJMR, vol. 109, no. 2, pp. 154-164. Jacquin, T.D., Denizot, J.P. & Denavit-Saubie, M. 1992, "Substance P immunoreactivity of rat brain stem neurons in primary culture", Journal of neuroscience research, vol. 31, no. 1, pp. 131135. Jakel, R.J., Kern, J.T., Johnson, D.A. & Johnson, J.A. 2005, "Induction of the protective antioxidant response element pathway by 6-hydroxydopamine in vivo and in vitro", Toxicological sciences : an official journal of the Society of Toxicology, vol. 87, no. 1, pp. 176-186. Jakel, R.J., Schneider, B.L. & Svendsen, C.N. 2004, "Using human neural stem cells to model neurological disease", Nature reviews.Genetics, vol. 5, no. 2, pp. 136-144. Jakel, R.J., Townsend, J.A., Kraft, A.D. & Johnson, J.A. 2007, "Nrf2-mediated protection against 6hydroxydopamine", Brain research, vol. 1144, pp. 192-201. Jankowsky, J.L., Fadale, D.J., Anderson, J., Xu, G.M., Gonzales, V., Jenkins, N.A., Copeland, N.G., Lee, M.K., Younkin, L.H., Wagner, S.L., Younkin, S.G. & Borchelt, D.R. 2004, "Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase", Human molecular genetics, vol. 13, no. 2, pp. 159-170. Jessberger, S., Romer, B., Babu, H. & Kempermann, G. 2005, "Seizures induce proliferation and dispersion of doublecortin-positive hippocampal progenitor cells", Experimental neurology, vol. 196, no. 2, pp. 342-351. Jhaveri, D.J., Mackay, E.W., Hamlin, A.S., Marathe, S.V., Nandam, L.S., Vaidya, V.A. & Bartlett, P.F. 2010, "Norepinephrine directly activates adult hippocampal precursors via beta3adrenergic receptors", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 30, no. 7, pp. 2795-2806. Jin, K., Galvan, V., Xie, L., Mao, X.O., Gorostiza, O.F., Bredesen, D.E. & Greenberg, D.A. 2004, "Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Ind) mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 36, pp. 13363-13367. Johnstone, M., Gearing, A.J. & Miller, K.M. 1999, "A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced", Journal of neuroimmunology, vol. 93, no. 1-2, pp. 182-193. Kanemitsu, H., Tomiyama, T. & Mori, H. 2003, "Human neprilysin is capable of degrading amyloid beta peptide not only in the monomeric form but also the pathological oligomeric form", Neuroscience letters, vol. 350, no. 2, pp. 113-116. Kanninen, K., Heikkinen, R., Malm, T., Rolova, T., Kuhmonen, S., Leinonen, H., Yla-Herttuala, S., Tanila, H., Levonen, A.L., Koistinaho, M. & Koistinaho, J. 2009, "Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 38, pp. 16505-16510. Kanninen, K., Malm, T.M., Jyrkkänen, H.K., Goldsteins, G., Keksa-Goldsteine, V., Tanila, H., Yamamoto, M., Yla-Herttuala, S., Levonen, A.L. & Koistinaho, J. 2008, "Nuclear factor

69 erythroid 2-related factor 2 protects against beta amyloid", Molecular and cellular neurosciences, vol. 39, no. 3, pp. 302-313. Kaplan, M.S. 1981, "Neurogenesis in the 3-month-old rat visual cortex", The Journal of comparative neurology, vol. 195, no. 2, pp. 323-338. Kaplan, M.S. & Bell, D.H. 1984, "Mitotic neuroblasts in the 9-day-old and 11-month-old rodent hippocampus", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 4, no. 6, pp. 1429-1441. Kaplan, M.S. & Bell, D.H. 1983, "Neuronal proliferation in the 9-month-old rodent-radioautographic study of granule cells in the hippocampus", Experimental brain research.Experimentelle Hirnforschung.Experimentation cerebrale, vol. 52, no. 1, pp. 1-5. Kaplan, M.S. & Hinds, J.W. 1977, "Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs", Science (New York, N.Y.), vol. 197, no. 4308, pp. 1092-1094. Karnik, S.S., Gogonea, C., Patil, S., Saad, Y. & Takezako, T. 2003, "Activation of G-proteincoupled receptors: a common molecular mechanism", Trends in endocrinology and metabolism: TEM, vol. 14, no. 9, pp. 431-437. Kaspar, J.W., Niture, S.K. & Jaiswal, A.K. 2009, "Nrf2:INrf2 (Keap1) signaling in oxidative stress", Free radical biology & medicine, vol. 47, no. 9, pp. 1304-1309. Katayama, Y., Battista, M., Kao, W.M., Hidalgo, A., Peired, A.J., Thomas, S.A. & Frenette, P.S. 2006, "Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow", Cell, vol. 124, no. 2, pp. 407-421. Katzman, R. 1986, "Alzheimer's disease", The New England journal of medicine, vol. 314, no. 15, pp. 964-973. Kazanis, I. 2011, "Can Adult Neural Stem Cells Create New Brains? Plasticity in the Adult Mammalian Neurogenic Niches: Realities and Expectations in the Era of Regenerative Biology", The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, . Kempermann, G. & Gage, F.H. 2002, "Genetic determinants of adult hippocampal neurogenesis correlate with acquisition, but not probe trial performance, in the water maze task", The European journal of neuroscience, vol. 16, no. 1, pp. 129-136. Kempermann, G., Jessberger, S., Steiner, B. & Kronenberg, G. 2004, "Milestones of neuronal development in the adult hippocampus", Trends in neurosciences, vol. 27, no. 8, pp. 447452. Kempermann, G., Kuhn, H.G. & Gage, F.H. 1997, "More hippocampal neurons in adult mice living in an enriched environment", Nature, vol. 386, no. 6624, pp. 493-495. Kenney, A.M. & Rowitch, D.H. 2000, "Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors", Molecular and cellular biology, vol. 20, no. 23, pp. 9055-9067. Khor, T.O., Huang, M.T., Kwon, K.H., Chan, J.Y., Reddy, B.S. & Kong, A.N. 2006, "Nrf2-deficient mice have an increased susceptibility to dextran sulfate sodium-induced colitis", Cancer research, vol. 66, no. 24, pp. 11580-11584. Kim, H.M., Hwang, D.H., Lee, J.E., Kim, S.U. & Kim, B.G. 2009, "Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue sparing after spinal cord injury", PloS one, vol. 4, no. 3, pp. e4987.

70 Kitazawa, M., Yamasaki, T.R. & LaFerla, F.M. 2004, "Microglia as a potential bridge between the amyloid beta-peptide and tau", Annals of the New York Academy of Sciences, vol. 1035, pp. 85-103. Klyubin, I., Betts, V., Welzel, A.T., Blennow, K., Zetterberg, H., Wallin, A., Lemere, C.A., Cullen, W.K., Peng, Y., Wisniewski, T., Selkoe, D.J., Anwyl, R., Walsh, D.M. & Rowan, M.J. 2008, "Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 28, no. 16, pp. 4231-4237. Koh, K., Cha, Y., Kim, S. & Kim, J. 2009, "tBHQ inhibits LPS-induced microglial activation via Nrf2mediated suppression of p38 phosphorylation", Biochemical and biophysical research communications, vol. 380, no. 3, pp. 449-453. Koistinaho, M., Lin, S., Wu, X., Esterman, M., Koger, D., Hanson, J., Higgs, R., Liu, F., Malkani, S., Bales, K.R. & Paul, S.M. 2004, "Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides", Nature medicine, vol. 10, no. 7, pp. 719726. Kojima, T., Hirota, Y., Ema, M., Takahashi, S., Miyoshi, I., Okano, H. & Sawamoto, K. 2010, "Subventricular zone-derived neural progenitor cells migrate along a blood vessel scaffold toward the post-stroke striatum", Stem cells (Dayton, Ohio), vol. 28, no. 3, pp. 545-554. Kokaia, Z. & Lindvall, O. 2003, "Neurogenesis after ischaemic brain insults", Current opinion in neurobiology, vol. 13, no. 1, pp. 127-132. Kooy, R.F., D'Hooge, R., Reyniers, E., Bakker, C.E., Nagels, G., De Boulle, K., Storm, K., Clincke, G., De Deyn, P.P., Oostra, B.A. & Willems, P.J. 1996, "Transgenic mouse model for the fragile X syndrome", American Journal of Medical Genetics, vol. 64, no. 2, pp. 241-245. Kornblum, H.I. 2007, "Introduction to neural stem cells", Stroke; a journal of cerebral circulation, vol. 38, no. 2 Suppl, pp. 810-816. Kosik, K.S., Joachim, C.L. & Selkoe, D.J. 1986, "Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 83, no. 11, pp. 40444048. Kotani, S., Yamauchi, T., Teramoto, T. & Ogura, H. 2006, "Pharmacological evidence of cholinergic involvement in adult hippocampal neurogenesis in rats", Neuroscience, vol. 142, no. 2, pp. 505-514. Kotlo, K.U., Yehiely, F., Efimova, E., Harasty, H., Hesabi, B., Shchors, K., Einat, P., Rozen, A., Berent, E. & Deiss, L.P. 2003, "Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells", Oncogene, vol. 22, no. 6, pp. 797-806. Kraft, A.D., Lee, J.M., Johnson, D.A., Kan, Y.W. & Johnson, J.A. 2006, "Neuronal sensitivity to kainic acid is dependent on the Nrf2-mediated actions of the antioxidant response element", Journal of neurochemistry, vol. 98, no. 6, pp. 1852-1865. Kriegstein, A. & Alvarez-Buylla, A. 2009, "The glial nature of embryonic and adult neural stem cells", Annual Review of Neuroscience, vol. 32, pp. 149-184. Kronenberg, G., Reuter, K., Steiner, B., Brandt, M.D., Jessberger, S., Yamaguchi, M. & Kempermann, G. 2003, "Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli", The Journal of comparative neurology, vol. 467, no. 4, pp. 455-463.

71 Kulkarni, V.A., Jha, S. & Vaidya, V.A. 2002, "Depletion of norepinephrine decreases the proliferation, but does not influence the survival and differentiation, of granule cell progenitors in the adult rat hippocampus", The European journal of neuroscience, vol. 16, no. 10, pp. 2008-2012. Kwak, M.K., Wakabayashi, N., Greenlaw, J.L., Yamamoto, M. & Kensler, T.W. 2003, "Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway", Molecular and cellular biology, vol. 23, no. 23, pp. 8786-8794. Kwak, Y.D., Brannen, C.L., Qu, T., Kim, H.M., Dong, X., Soba, P., Majumdar, A., Kaplan, A., Beyreuther, K. & Sugaya, K. 2006, "Amyloid precursor protein regulates differentiation of human neural stem cells", Stem cells and development, vol. 15, no. 3, pp. 381-389. Kwak, Y.D., Dantuma, E., Merchant, S., Bushnev, S. & Sugaya, K. 2010, "Amyloid-beta precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway", Neurotoxicity research, vol. 18, no. 3-4, pp. 328-338. Kwong, M., Kan, Y.W. & Chan, J.Y. 1999, "The CNC basic leucine zipper factor, Nrf1, is essential for cell survival in response to oxidative stress-inducing agents. Role for Nrf1 in gamma-gcs(l) and gss expression in mouse fibroblasts", The Journal of biological chemistry, vol. 274, no. 52, pp. 37491-37498. Laggerbauer, B., Ostareck, D., Keidel, E.M., Ostareck-Lederer, A. & Fischer, U. 2001, "Evidence that fragile X mental retardation protein is a negative regulator of translation", Human molecular genetics, vol. 10, no. 4, pp. 329-338. Langosch, J.M., Kupferschmid, S., Heinen, M., Walden, J., Herpfer, I., Fiebich, B.L. & Lieb, K. 2005, "Effects of substance P and its antagonist L-733060 on long term potentiation in guinea pig hippocampal slices", Progress in neuro-psychopharmacology & biological psychiatry, vol. 29, no. 2, pp. 315-319. Lazarov, O., Demars, M.P., Da Tommy Zhao, K., Ali, H.M., Grauzas, V., Kney, A. & Larson, J. 2011, "Impaired survival of neural progenitor cells in dentate gyrus of adult mice lacking FMRP", Hippocampus, . Lazarov, O. & Marr, R.A. 2010, "Neurogenesis and Alzheimer's disease: at the crossroads", Experimental neurology, vol. 223, no. 2, pp. 267-281. Lazarov, O., Mattson, M.P., Peterson, D.A., Pimplikar, S.W. & van Praag, H. 2010, "When neurogenesis encounters aging and disease", Trends in neurosciences, vol. 33, no. 12, pp. 569-579. Lee, J.M., Calkins, M.J., Chan, K., Kan, Y.W. & Johnson, J.A. 2003, "Identification of the NF-E2related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis", The Journal of biological chemistry, vol. 278, no. 14, pp. 12029-12038. Lee, J.M., Li, J., Johnson, D.A., Stein, T.D., Kraft, A.D., Calkins, M.J., Jakel, R.J. & Johnson, J.A. 2005, "Nrf2, a multi-organ protector?", The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 19, no. 9, pp. 1061-1066. Lee, Y.J., Han, S.B., Nam, S.Y., Oh, K.W. & Hong, J.T. 2010, "Inflammation and Alzheimer's disease", Archives of Pharmacal Research, vol. 33, no. 10, pp. 1539-1556. Leissring, M.A., Farris, W., Chang, A.Y., Walsh, D.M., Wu, X., Sun, X., Frosch, M.P. & Selkoe, D.J. 2003, "Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death", Neuron, vol. 40, no. 6, pp. 10871093.

72 Lemaire, V., Koehl, M., Le Moal, M. & Abrous, D.N. 2000, "Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus", Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 20, pp. 1103211037. Leng, J., Jiang, L., Chen, H. & Zhang, X. 2009, "Brain-derived neurotrophic factor and electrophysiological properties of voltage-gated ion channels during neuronal stem cell development", Brain research, vol. 1272, pp. 14-24. Li, W., Khor, T.O., Xu, C., Shen, G., Jeong, W.S., Yu, S. & Kong, A.N. 2008, "Activation of Nrf2antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis", Biochemical pharmacology, vol. 76, no. 11, pp. 1485-1489. Li, Z., Zhang, Y., Ku, L., Wilkinson, K.D., Warren, S.T. & Feng, Y. 2001, "The fragile X mental retardation protein inhibits translation via interacting with mRNA", Nucleic acids research, vol. 29, no. 11, pp. 2276-2283. Lie, D.C., Dziewczapolski, G., Willhoite, A.R., Kaspar, B.K., Shults, C.W. & Gage, F.H. 2002, "The adult substantia nigra contains progenitor cells with neurogenic potential", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 22, no. 15, pp. 66396649. Lie, D.C., Song, H., Colamarino, S.A., Ming, G.L. & Gage, F.H. 2004, "Neurogenesis in the adult brain: new strategies for central nervous system diseases", Annual Review of Pharmacology and Toxicology, vol. 44, pp. 399-421. Lim, D.A., Suarez-Farinas, M., Naef, F., Hacker, C.R., Menn, B., Takebayashi, H., Magnasco, M., Patil, N. & Alvarez-Buylla, A. 2006, "In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis", Molecular and cellular neurosciences, vol. 31, no. 1, pp. 131-148. Lin, C.W., Sim, S., Ainsworth, A., Okada, M., Kelsch, W. & Lois, C. 2010, "Genetically increased cell-intrinsic excitability enhances neuronal integration into adult brain circuits", Neuron, vol. 65, no. 1, pp. 32-39. Lin, J.H., Takano, T., Arcuino, G., Wang, X., Hu, F., Darzynkiewicz, Z., Nunes, M., Goldman, S.A. & Nedergaard, M. 2007, "Purinergic signaling regulates neural progenitor cell expansion and neurogenesis", Developmental biology, vol. 302, no. 1, pp. 356-366. Lin, M.T. & Beal, M.F. 2006, "Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases", Nature, vol. 443, no. 7113, pp. 787-795. Lindvall, O. & Kokaia, Z. 2006, "Stem cells for the treatment of neurological disorders", Nature, vol. 441, no. 7097, pp. 1094-1096. Liu, X., Wang, Q., Haydar, T.F. & Bordey, A. 2005, "Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors", Nature neuroscience, vol. 8, no. 9, pp. 1179-1187. Lledo, P.M., Alonso, M. & Grubb, M.S. 2006, "Adult neurogenesis and functional plasticity in neuronal circuits", Nature reviews.Neuroscience, vol. 7, no. 3, pp. 179-193. Lois, C. & Alvarez-Buylla, A. 1994, "Long-distance neuronal migration in the adult mammalian brain", Science (New York, N.Y.), vol. 264, no. 5162, pp. 1145-1148. Lopez-Toledano, M.A. & Shelanski, M.L. 2004, "Neurogenic effect of beta-amyloid peptide in the development of neural stem cells", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 24, no. 23, pp. 5439-5444.

73 Lossinsky, A.S. & Shivers, R.R. 2004, "Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review", Histology and histopathology, vol. 19, no. 2, pp. 535-564. Lu, R., Wang, H., Liang, Z., Ku, L., O'donnell, W.T., Li, W., Warren, S.T. & Feng, Y. 2004, "The fragile X protein controls microtubule-associated protein 1B translation and microtubule stability in brain neuron development", Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 42, pp. 15201-15206. Lu, Z., Elliott, M.R., Chen, Y., Walsh, J.T., Klibanov, A.L., Ravichandran, K.S. & Kipnis, J. 2011, "Phagocytic activity of neuronal progenitors regulates adult neurogenesis", Nature cell biology, vol. 13, no. 9, pp. 1076-1083. Lue, L.F., Kuo, Y.M., Roher, A.E., Brachova, L., Shen, Y., Sue, L., Beach, T., Kurth, J.H., Rydel, R.E. & Rogers, J. 1999, "Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease", The American journal of pathology, vol. 155, no. 3, pp. 853862. Luo, X., Popov, S., Bera, A.K., Wilkie, T.M. & Muallem, S. 2001, "RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations", Molecular cell, vol. 7, no. 3, pp. 651-660. Luo, Y., Shan, G., Guo, W., Smrt, R.D., Johnson, E.B., Li, X., Pfeiffer, R.L., Szulwach, K.E., Duan, R., Barkho, B.Z., Li, W., Liu, C., Jin, P. & Zhao, X. 2010, "Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells", PLoS genetics, vol. 6, no. 4, pp. e1000898. Ma, D.K., Bonaguidi, M.A., Ming, G.L. & Song, H. 2009, "Adult neural stem cells in the mammalian central nervous system", Cell research, vol. 19, no. 6, pp. 672-682. Ma, D.K., Kim, W.R., Ming, G.L. & Song, H. 2009, "Activity-dependent extrinsic regulation of adult olfactory bulb and hippocampal neurogenesis", Annals of the New York Academy of Sciences, vol. 1170, pp. 664-673. Ma, W., Maric, D., Li, B.S., Hu, Q., Andreadis, J.D., Grant, G.M., Liu, Q.Y., Shaffer, K.M., Chang, Y.H., Zhang, L., Pancrazio, J.J., Pant, H.C., Stenger, D.A. & Barker, J.L. 2000, "Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation", The European journal of neuroscience, vol. 12, no. 4, pp. 12271240. Mak, G.K. & Weiss, S. 2010, "Paternal recognition of adult offspring mediated by newly generated CNS neurons", Nature neuroscience, vol. 13, no. 6, pp. 753-758. Malm, T., Koistinaho, M., Muona, A., Magga, J. & Koistinaho, J. 2010, "The role and therapeutic potential of monocytic cells in Alzheimer's disease", Glia, vol. 58, no. 8, pp. 889-900. Mandelkow, E.M., Stamer, K., Vogel, R., Thies, E. & Mandelkow, E. 2003, "Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses", Neurobiology of aging, vol. 24, no. 8, pp. 1079-1085. Mandrekar, S., Jiang, Q., Lee, C.Y., Koenigsknecht-Talboo, J., Holtzman, D.M. & Landreth, G.E. 2009, "Microglia mediate the clearance of soluble Abeta through fluid phase macropinocytosis", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 29, no. 13, pp. 4252-4262. Mark, R.J., Keller, J.N., Kruman, I. & Mattson, M.P. 1997, "Basic FGF attenuates amyloid betapeptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+ATPase activity in hippocampal neurons", Brain research, vol. 756, no. 1-2, pp. 205-214.

74 Marques, S.C., Oliveira, C.R., Outeiro, T.F. & Pereira, C.M. 2010, "Alzheimer's disease: the quest to understand complexity", Journal of Alzheimer's disease : JAD, vol. 21, no. 2, pp. 373-383. Marutle, A., Ohmitsu, M., Nilbratt, M., Greig, N.H., Nordberg, A. & Sugaya, K. 2007, "Modulation of human neural stem cell differentiation in Alzheimer (APP23) transgenic mice by phenserine", Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 30, pp. 12506-12511. Masuda, T., Nakagawa, S., Boku, S., Nishikawa, H., Takamura, N., Kato, A., Inoue, T. & Koyama, T. 2012, "Noradrenaline increases neural precursor cells derived from adult rat dentate gyrus through beta2 receptor", Progress in neuro-psychopharmacology & biological psychiatry, vol. 36, no. 1, pp. 44-51. Mattson, M.P. 2004, "Pathways towards and away from Alzheimer's disease", Nature, vol. 430, no. 7000, pp. 631-639. Mattson, M.P., Cheng, B., Culwell, A.R., Esch, F.S., Lieberburg, I. & Rydel, R.E. 1993, "Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the betaamyloid precursor protein", Neuron, vol. 10, no. 2, pp. 243-254. Mazroui, R., Huot, M.E., Tremblay, S., Filion, C., Labelle, Y. & Khandjian, E.W. 2002, "Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression", Human molecular genetics, vol. 11, no. 24, pp. 3007-3017. Mazur-Kolecka, B., Golabek, A., Nowicki, K., Flory, M. & Frackowiak, J. 2006, "Amyloid-beta impairs development of neuronal progenitor cells by oxidative mechanisms", Neurobiology of aging, vol. 27, no. 9, pp. 1181-1192. McNaull, B.B., Todd, S., McGuinness, B. & Passmore, A.P. 2010, "Inflammation and antiinflammatory strategies for Alzheimer's disease--a mini-review", Gerontology, vol. 56, no. 1, pp. 3-14. Merkle, F.T., Mirzadeh, Z. & Alvarez-Buylla, A. 2007, "Mosaic organization of neural stem cells in the adult brain", Science (New York, N.Y.), vol. 317, no. 5836, pp. 381-384. Miller, R.J., Banisadr, G. & Bhattacharyya, B.J. 2008, "CXCR4 signaling in the regulation of stem cell migration and development", Journal of neuroimmunology, vol. 198, no. 1-2, pp. 31-38. Min, W.W., Yuskaitis, C.J., Yan, Q., Sikorski, C., Chen, S., Jope, R.S. & Bauchwitz, R.P. 2009, "Elevated glycogen synthase kinase-3 activity in Fragile X mice: key metabolic regulator with evidence for treatment potential", Neuropharmacology, vol. 56, no. 2, pp. 463-472. Ming, G.L. & Song, H. 2005, "Adult neurogenesis in the mammalian central nervous system", Annual Review of Neuroscience, vol. 28, pp. 223-250. Mishra, S.K., Braun, N., Shukla, V., Fullgrabe, M., Schomerus, C., Korf, H.W., Gachet, C., Ikehara, Y., Sevigny, J., Robson, S.C. & Zimmermann, H. 2006, "Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation", Development (Cambridge, England), vol. 133, no. 4, pp. 675-684. Miyashiro, K.Y., Beckel-Mitchener, A., Purk, T.P., Becker, K.G., Barret, T., Liu, L., Carbonetto, S., Weiler, I.J., Greenough, W.T. & Eberwine, J. 2003, "RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice", Neuron, vol. 37, no. 3, pp. 417-431. Mohapel, P., Leanza, G., Kokaia, M. & Lindvall, O. 2005, "Forebrain acetylcholine regulates adult hippocampal neurogenesis and learning", Neurobiology of aging, vol. 26, no. 6, pp. 939-946. Moore, A.H. & O'Banion, M.K. 2002, "Neuroinflammation and anti-inflammatory therapy for Alzheimer's disease", Advanced Drug Delivery Reviews, vol. 54, no. 12, pp. 1627-1656.

75 Morrens, J., Van Den Broeck, W. & Kempermann, G. 2012, "Glial cells in adult neurogenesis", Glia, vol. 60, no. 2, pp. 159-174. Mu, Y. & Gage, F.H. 2011, "Adult hippocampal neurogenesis and its role in Alzheimer's disease", Molecular neurodegeneration, vol. 6, pp. 85. Muotri, A.R. & Gage, F.H. 2006, "Generation of neuronal variability and complexity", Nature, vol. 441, no. 7097, pp. 1087-1093. Murakami, K., Irie, K., Ohigashi, H., Hara, H., Nagao, M., Shimizu, T. & Shirasawa, T. 2005, "Formation and stabilization model of the 42-mer Abeta radical: implications for the longlasting oxidative stress in Alzheimer's disease", Journal of the American Chemical Society, vol. 127, no. 43, pp. 15168-15174. Murray, A. 2000, "Premature ovarian failure and the FMR1 gene", Seminars in reproductive medicine, vol. 18, no. 1, pp. 59-66. Nakatomi, H., Kuriu, T., Okabe, S., Yamamoto, S., Hatano, O., Kawahara, N., Tamura, A., Kirino, T. & Nakafuku, M. 2002, "Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors", Cell, vol. 110, no. 4, pp. 429441. Narimatsu, N., Harada, N., Kurihara, H., Nakagata, N., Sobue, K. & Okajima, K. 2009, "Donepezil improves cognitive function in mice by increasing the production of insulin-like growth factor-I in the hippocampus", The Journal of pharmacology and experimental therapeutics, vol. 330, no. 1, pp. 2-12. Nguyen, L., Malgrange, B., Breuskin, I., Bettendorff, L., Moonen, G., Belachew, S. & Rigo, J.M. 2003, "Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 8, pp. 3278-3294. Nguyen, L., Rigo, J.M., Rocher, V., Belachew, S., Malgrange, B., Rogister, B., Leprince, P. & Moonen, G. 2001, "Neurotransmitters as early signals for central nervous system development", Cell and tissue research, vol. 305, no. 2, pp. 187-202. Nguyen, T., Nioi, P. & Pickett, C.B. 2009, "The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress", The Journal of biological chemistry, vol. 284, no. 20, pp. 13291-13295. Nielsen, D.M., Derber, W.J., McClellan, D.A. & Crnic, L.S. 2002, "Alterations in the auditory startle response in Fmr1 targeted mutant mouse models of fragile X syndrome", Brain research, vol. 927, no. 1, pp. 8-17. Nikolaev, A., McLaughlin, T., O'Leary, D.D. & Tessier-Lavigne, M. 2009, "APP binds DR6 to trigger axon pruning and neuron death via distinct caspases", Nature, vol. 457, no. 7232, pp. 981989. Nimchinsky, E.A., Oberlander, A.M. & Svoboda, K. 2001, "Abnormal development of dendritic spines in FMR1 knock-out mice", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 21, no. 14, pp. 5139-5146. Nissant, A., Bardy, C., Katagiri, H., Murray, K. & Lledo, P.M. 2009, "Adult neurogenesis promotes synaptic plasticity in the olfactory bulb", Nature neuroscience, vol. 12, no. 6, pp. 728-730. Nistal, M., Santamaria, L. & Paniagua, R. 1992, "The ampulla of the ductus deferens in man: morphological and ultrastructural aspects", Journal of anatomy, vol. 180 ( Pt 1), no. Pt 1, pp. 97-104.

76 Nixon, K. & Crews, F.T. 2002, "Binge ethanol exposure decreases neurogenesis in adult rat hippocampus", Journal of neurochemistry, vol. 83, no. 5, pp. 1087-1093. Nordstedt, C., Caporaso, G.L., Thyberg, J., Gandy, S.E. & Greengard, P. 1993, "Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells", The Journal of biological chemistry, vol. 268, no. 1, pp. 608-612. Nunomura, A., Perry, G., Aliev, G., Hirai, K., Takeda, A., Balraj, E.K., Jones, P.K., Ghanbari, H., Wataya, T., Shimohama, S., Chiba, S., Atwood, C.S., Petersen, R.B. & Smith, M.A. 2001, "Oxidative damage is the earliest event in Alzheimer disease", Journal of neuropathology and experimental neurology, vol. 60, no. 8, pp. 759-767. Oddo, S., Caccamo, A., Kitazawa, M., Tseng, B.P. & LaFerla, F.M. 2003, "Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease", Neurobiology of aging, vol. 24, no. 8, pp. 1063-1070. O'Donnell, W.T. & Warren, S.T. 2002, "A decade of molecular studies of fragile X syndrome", Annual Review of Neuroscience, vol. 25, pp. 315-338. O'Keeffe, G.C., Barker, R.A. & Caldwell, M.A. 2009, "Dopaminergic modulation of neurogenesis in the subventricular zone of the adult brain", Cell cycle (Georgetown, Tex.), vol. 8, no. 18, pp. 2888-2894. O'Keeffe, G.C., Tyers, P., Aarsland, D., Dalley, J.W., Barker, R.A. & Caldwell, M.A. 2009, "Dopamine-induced proliferation of adult neural precursor cells in the mammalian subventricular zone is mediated through EGF", Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 21, pp. 8754-8759. Olcese, J.M., Cao, C., Mori, T., Mamcarz, M.B., Maxwell, A., Runfeldt, M.J., Wang, L., Zhang, C., Lin, X., Zhang, G. & Arendash, G.W. 2009, "Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease", Journal of pineal research, vol. 47, no. 1, pp. 82-96. Palmer, T.D., Markakis, E.A., Willhoite, A.R., Safar, F. & Gage, F.H. 1999, "Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 19, no. 19, pp. 8487-8497. Palmer, T.D., Ray, J. & Gage, F.H. 1995, "FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain", Molecular and cellular neurosciences, vol. 6, no. 5, pp. 474-486. Palmer, T.D., Willhoite, A.R. & Gage, F.H. 2000, "Vascular niche for adult hippocampal neurogenesis", The Journal of comparative neurology, vol. 425, no. 4, pp. 479-494. Pan, F., Aldridge, G.M., Greenough, W.T. & Gan, W.B. 2010, "Dendritic spine instability and insensitivity to modulation by sensory experience in a mouse model of fragile X syndrome", Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 41, pp. 17768-17773. Paola, D., Domenicotti, C., Nitti, M., Vitali, A., Borghi, R., Cottalasso, D., Zaccheo, D., Odetti, P., Strocchi, P., Marinari, U.M., Tabaton, M. & Pronzato, M.A. 2000, "Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells", Biochemical and biophysical research communications, vol. 268, no. 2, pp. 642-646. Paradee, W., Melikian, H.E., Rasmussen, D.L., Kenneson, A., Conn, P.J. & Warren, S.T. 1999, "Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function", Neuroscience, vol. 94, no. 1, pp. 185-192.

77 Park, D.H., Eve, D.J., Sanberg, P.R., Musso, J.,3rd, Bachstetter, A.D., Wolfson, A., Schlunk, A., Baradez, M.O., Sinden, J.D. & Gemma, C. 2010, "Increased neuronal proliferation in the dentate gyrus of aged rats following neural stem cell implantation", Stem cells and development, vol. 19, no. 2, pp. 175-180. Park, H.M., Kim, J.A. & Kwak, M.K. 2009, "Protection against amyloid beta cytotoxicity by sulforaphane: role of the proteasome", Archives of Pharmacal Research, vol. 32, no. 1, pp. 109-115. Pathania, M., Yan, L.D. & Bordey, A. 2010, "A symphony of signals conducts early and late stages of adult neurogenesis", Neuropharmacology, vol. 58, no. 6, pp. 865-876. Pfeiffer, B.E. & Huber, K.M. 2009, "The state of synapses in fragile X syndrome", The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, vol. 15, no. 5, pp. 549-567. Pierce, K.L., Premont, R.T. & Lefkowitz, R.J. 2002, "Seven-transmembrane receptors", Nature reviews.Molecular cell biology, vol. 3, no. 9, pp. 639-650. Pietrzik, C.U., Hoffmann, J., Stober, K., Chen, C.Y., Bauer, C., Otero, D.A., Roch, J.M. & Herzog, V. 1998, "From differentiation to proliferation: the secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells", Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 4, pp. 1770-1775. Pihlaja, R., Koistinaho, J., Kauppinen, R., Sandholm, J., Tanila, H. & Koistinaho, M. 2011, "Multiple cellular and molecular mechanisms are involved in human Abeta clearance by transplanted adult astrocytes", Glia, vol. 59, no. 11, pp. 1643-1657. Pihlaja, R., Koistinaho, J., Malm, T., Sikkilä, H., Vainio, S. & Koistinaho, M. 2008, "Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease", Glia, vol. 56, no. 2, pp. 154-163. Pike, K.E., Savage, G., Villemagne, V.L., Ng, S., Moss, S.A., Maruff, P., Mathis, C.A., Klunk, W.E., Masters, C.L. & Rowe, C.C. 2007, "Beta-amyloid imaging and memory in non-demented individuals: evidence for preclinical Alzheimer's disease", Brain : a journal of neurology, vol. 130, no. Pt 11, pp. 2837-2844. Platel, J.C., Dave, K.A. & Bordey, A. 2008a, "Control of neuroblast production and migration by converging GABA and glutamate signals in the postnatal forebrain", The Journal of physiology, vol. 586, no. 16, pp. 3739-3743. Platel, J.C., Dave, K.A., Gordon, V., Lacar, B., Rubio, M.E. & Bordey, A. 2010a, "NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network", Neuron, vol. 65, no. 6, pp. 859-872. Platel, J.C., Heintz, T., Young, S., Gordon, V. & Bordey, A. 2008b, "Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone", The Journal of physiology, vol. 586, no. 16, pp. 3783-3793. Platel, J.C., Lacar, B. & Bordey, A. 2007, "GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis", Journal of molecular histology, vol. 38, no. 4, pp. 303-311. Platel, J.C., Stamboulian, S., Nguyen, I. & Bordey, A. 2010b, "Neurotransmitter signaling in postnatal neurogenesis: The first leg", Brain Research Reviews, vol. 63, no. 1-2, pp. 60-71. Pratico, D. & Trojanowski, J.Q. 2000, "Inflammatory hypotheses: novel mechanisms of Alzheimer's neurodegeneration and new therapeutic targets?", Neurobiology of aging, vol. 21, no. 3, pp. 441-5; discussion 451-3.

78 Qin, M., Kang, J., Burlin, T.V., Jiang, C. & Smith, C.B. 2005, "Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 25, no. 20, pp. 50875095. Qiu, W.Q., Walsh, D.M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M.B., Rosner, M.R., Safavi, A., Hersh, L.B. & Selkoe, D.J. 1998, "Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation", The Journal of biological chemistry, vol. 273, no. 49, pp. 32730-32738. Qiu, W.Q., Ye, Z., Kholodenko, D., Seubert, P. & Selkoe, D.J. 1997, "Degradation of amyloid betaprotein by a metalloprotease secreted by microglia and other neural and non-neural cells", The Journal of biological chemistry, vol. 272, no. 10, pp. 6641-6646. Querfurth, H.W. & LaFerla, F.M. 2010, "Alzheimer's disease", The New England journal of medicine, vol. 362, no. 4, pp. 329-344. Rameshwar, P., Zhu, G., Donnelly, R.J., Qian, J., Ge, H., Goldstein, K.R., Denny, T.N. & Gascon, P. 2001, "The dynamics of bone marrow stromal cells in the proliferation of multipotent hematopoietic progenitors by substance P: an understanding of the effects of a neurotransmitter on the differentiating hematopoietic stem cell", Journal of neuroimmunology, vol. 121, no. 1-2, pp. 22-31. Ramsey, C.P., Glass, C.A., Montgomery, M.B., Lindl, K.A., Ritson, G.P., Chia, L.A., Hamilton, R.L., Chu, C.T. & Jordan-Sciutto, K.L. 2007, "Expression of Nrf2 in neurodegenerative diseases", Journal of neuropathology and experimental neurology, vol. 66, no. 1, pp. 75-85. Reiman, E.M., Chen, K., Liu, X., Bandy, D., Yu, M., Lee, W., Ayutyanont, N., Keppler, J., Reeder, S.A., Langbaum, J.B., Alexander, G.E., Klunk, W.E., Mathis, C.A., Price, J.C., Aizenstein, H.J., DeKosky, S.T. & Caselli, R.J. 2009, "Fibrillar amyloid-beta burden in cognitively normal people at 3 levels of genetic risk for Alzheimer's disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 16, pp. 6820-6825. Reynolds, B.A., Tetzlaff, W. & Weiss, S. 1992, "A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 12, no. 11, pp. 4565-4574. Reynolds, B.A. & Weiss, S. 1992, "Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system", Science (New York, N.Y.), vol. 255, no. 5052, pp. 1707-1710. Ring, S., Weyer, S.W., Kilian, S.B., Waldron, E., Pietrzik, C.U., Filippov, M.A., Herms, J., Buchholz, C., Eckman, C.B., Korte, M., Wolfer, D.P. & Muller, U.C. 2007, "The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 27, no. 29, pp. 7817-7826. Rodrigues Siqueira, I., Fochesatto, C., da Silva Torres, I.L., Dalmaz, C. & Alexandre Netto, C. 2005, "Aging affects oxidative state in hippocampus, hypothalamus and adrenal glands of Wistar rats", Life Sciences, vol. 78, no. 3, pp. 271-278. Rosenbaum, D.M., Rasmussen, S.G. & Kobilka, B.K. 2009, "The structure and function of Gprotein-coupled receptors", Nature, vol. 459, no. 7245, pp. 356-363. Rossi, C., Angelucci, A., Costantin, L., Braschi, C., Mazzantini, M., Babbini, F., Fabbri, M.E., Tessarollo, L., Maffei, L., Berardi, N. & Caleo, M. 2006, "Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment", The European journal of neuroscience, vol. 24, no. 7, pp. 18501856.

79 Roy, O.W., Cohen, N.R. & Nicoll, J.A. 2005, "Pathophysiology of dementias and implications for therapy", Indian journal of pathology & microbiology, vol. 48, no. 3, pp. 289-299. Rushmore, T.H., Morton, M.R. & Pickett, C.B. 1991, "The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity", The Journal of biological chemistry, vol. 266, no. 18, pp. 11632-11639. Ryu, J.K., Cho, T., Wang, Y.T. & McLarnon, J.G. 2009, "Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain", Journal of neuroinflammation, vol. 6, pp. 39. Sabo, S.L., Ikin, A.F., Buxbaum, J.D. & Greengard, P. 2001, "The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement", The Journal of cell biology, vol. 153, no. 7, pp. 1403-1414. Sahay, A. & Hen, R. 2007, "Adult hippocampal neurogenesis in depression", Nature neuroscience, vol. 10, no. 9, pp. 1110-1115. Sahay, A., Scobie, K.N., Hill, A.S., O'Carroll, C.M., Kheirbek, M.A., Burghardt, N.S., Fenton, A.A., Dranovsky, A. & Hen, R. 2011, "Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation", Nature, vol. 472, no. 7344, pp. 466-470. Saitoh, T., Sundsmo, M., Roch, J.M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T. & Schenk, D.B. 1989, "Secreted form of amyloid beta protein precursor is involved in the growth regulation of fibroblasts", Cell, vol. 58, no. 4, pp. 615-622. Salinero, O., Moreno-Flores, M.T. & Wandosell, F. 2000, "Increasing neurite outgrowth capacity of beta-amyloid precursor protein proteoglycan in Alzheimer's disease", Journal of neuroscience research, vol. 60, no. 1, pp. 87-97. Santoro, M.R., Bray, S.M. & Warren, S.T. 2012, "Molecular mechanisms of fragile x syndrome: a twenty-year perspective", Annual review of pathology, vol. 7, pp. 219-245. Satoh, T., Kosaka, K., Itoh, K., Kobayashi, A., Yamamoto, M., Shimojo, Y., Kitajima, C., Cui, J., Kamins, J., Okamoto, S., Izumi, M., Shirasawa, T. & Lipton, S.A. 2008, "Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1", Journal of neurochemistry, vol. 104, no. 4, pp. 1116-1131. Satoh, T., Okamoto, S.I., Cui, J., Watanabe, Y., Furuta, K., Suzuki, M., Tohyama, K. & Lipton, S.A. 2006, "Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic [correction of electrophillic] phase II inducers", Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 3, pp. 768-773. Seki, T. & Arai, Y. 1993, "Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 13, no. 6, pp. 23512358. Selkoe, D.J. 2011, "Resolving controversies on the path to Alzheimer's therapeutics", Nature medicine, vol. 17, no. 9, pp. 1060-1065. Selkoe, D.J. 2001, "Alzheimer's disease: genes, proteins, and therapy", Physiological Reviews, vol. 81, no. 2, pp. 741-766. Sengupta, A., Grundke-Iqbal, I. & Iqbal, K. 2006, "Regulation of phosphorylation of tau by protein kinases in rat brain", Neurochemical research, vol. 31, no. 12, pp. 1473-1480.

80 Seri, B., Garcia-Verdugo, J.M., Collado-Morente, L., McEwen, B.S. & Alvarez-Buylla, A. 2004, "Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus", The Journal of comparative neurology, vol. 478, no. 4, pp. 359-378. Shen, Q., Wang, Y., Kokovay, E., Lin, G., Chuang, S.M., Goderie, S.K., Roysam, B. & Temple, S. 2008, "Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions", Cell stem cell, vol. 3, no. 3, pp. 289-300. Shetty, A.K. & Hattiangady, B. 2007, "Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy", Hippocampus, vol. 17, no. 10, pp. 943-956. Shi, H., Belbin, O., Medway, C., Brown, K., Kalsheker, N., Carrasquillo, M., Proitsi, P., Powell, J., Lovestone, S., Goate, A., Younkin, S., Passmore, P., Genetic and Environmental Risk for Alzheimer's Disease (GERAD1) Consortium, Morgan, K. & Alzheimer's Research UK (ARUK) Consortium 2012, "Genetic variants influencing human aging from late-onset Alzheimer's disease (LOAD) genome-wide association studies (GWAS)", Neurobiology of aging, . Shih, A.Y., Imbeault, S., Barakauskas, V., Erb, H., Jiang, L., Li, P. & Murphy, T.H. 2005, "Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo", The Journal of biological chemistry, vol. 280, no. 24, pp. 22925-22936. Shih, A.Y., Johnson, D.A., Wong, G., Kraft, A.D., Jiang, L., Erb, H., Johnson, J.A. & Murphy, T.H. 2003, "Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 23, no. 8, pp. 3394-3406. Shih, A.Y., Li, P. & Murphy, T.H. 2005, "A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 25, no. 44, pp. 1032110335. Shihabuddin, L.S., Ray, J. & Gage, F.H. 1997, "FGF-2 is sufficient to isolate progenitors found in the adult mammalian spinal cord", Experimental neurology, vol. 148, no. 2, pp. 577-586. Sierra, A., Encinas, J.M., Deudero, J.J., Chancey, J.H., Enikolopov, G., Overstreet-Wadiche, L.S., Tsirka, S.E. & Maletic-Savatic, M. 2010, "Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis", Cell stem cell, vol. 7, no. 4, pp. 483-495. Simonyi, A., Schachtman, T.R. & Christoffersen, G.R. 2005, "The role of metabotropic glutamate receptor 5 in learning and memory processes", Drug news & perspectives, vol. 18, no. 6, pp. 353-361. Smits, H.A., Rijsmus, A., van Loon, J.H., Wat, J.W., Verhoef, J., Boven, L.A. & Nottet, H.S. 2002, "Amyloid-beta-induced chemokine production in primary human macrophages and astrocytes", Journal of neuroimmunology, vol. 127, no. 1-2, pp. 160-168. Spencer, C.M., Alekseyenko, O., Hamilton, S.M., Thomas, A.M., Serysheva, E., Yuva-Paylor, L.A. & Paylor, R. 2011, "Modifying behavioral phenotypes in Fmr1KO mice: genetic background differences reveal autistic-like responses", Autism research : official journal of the International Society for Autism Research, vol. 4, no. 1, pp. 40-56. Spitzer, N.C., Root, C.M. & Borodinsky, L.N. 2004, "Orchestrating neuronal differentiation: patterns of Ca2+ spikes specify transmitter choice", Trends in neurosciences, vol. 27, no. 7, pp. 415-421. Stanfield, B.B. & Trice, J.E. 1988, "Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections", Experimental brain research.Experimentelle Hirnforschung.Experimentation cerebrale, vol. 72, no. 2, pp. 399-406.

81 Stewart, R.R., Hoge, G.J., Zigova, T. & Luskin, M.B. 2002, "Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABA(A) receptors", Journal of neurobiology, vol. 50, no. 4, pp. 305-322. Stone, S.S., Teixeira, C.M., Devito, L.M., Zaslavsky, K., Josselyn, S.A., Lozano, A.M. & Frankland, P.W. 2011, "Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 31, no. 38, pp. 13469-13484. Streit, W.J., Walter, S.A. & Pennell, N.A. 1999, "Reactive microgliosis", Progress in neurobiology, vol. 57, no. 6, pp. 563-581. Su, J.H., Anderson, A.J., Cribbs, D.H., Tu, C., Tong, L., Kesslack, P. & Cotman, C.W. 2003, "Fas and Fas ligand are associated with neuritic degeneration in the AD brain and participate in beta-amyloid-induced neuronal death", Neurobiology of disease, vol. 12, no. 3, pp. 182-193. Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M.Z., Zubiaga, A., Hua, X., Fan, G. & Greenberg, M.E. 2001, "Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms", Cell, vol. 104, no. 3, pp. 365-376. Tabner, B.J., El-Agnaf, O.M., Turnbull, S., German, M.J., Paleologou, K.E., Hayashi, Y., Cooper, L.J., Fullwood, N.J. & Allsop, D. 2005, "Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia", The Journal of biological chemistry, vol. 280, no. 43, pp. 35789-35792. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. 2007, "Induction of pluripotent stem cells from adult human fibroblasts by defined factors", Cell, vol. 131, no. 5, pp. 861-872. Takahashi, K. & Yamanaka, S. 2006, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", Cell, vol. 126, no. 4, pp. 663-676. Taniuchi, N., Niidome, T., Goto, Y., Akaike, A., Kihara, T. & Sugimoto, H. 2007, "Decreased proliferation of hippocampal progenitor cells in APPswe/PS1dE9 transgenic mice", Neuroreport, vol. 18, no. 17, pp. 1801-1805. Tansey, M.G., McCoy, M.K. & Frank-Cannon, T.C. 2007, "Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention", Experimental neurology, vol. 208, no. 1, pp. 1-25. Tassone, F., Beilina, A., Carosi, C., Albertosi, S., Bagni, C., Li, L., Glover, K., Bentley, D. & Hagerman, P.J. 2007, "Elevated FMR1 mRNA in premutation carriers is due to increased transcription", RNA (New York, N.Y.), vol. 13, no. 4, pp. 555-562. Tavazoie, M., Van der Veken, L., Silva-Vargas, V., Louissaint, M., Colonna, L., Zaidi, B., GarciaVerdugo, J.M. & Doetsch, F. 2008, "A specialized vascular niche for adult neural stem cells", Cell stem cell, vol. 3, no. 3, pp. 279-288. Taylor, C.W. & Thorn, P. 2001, "Calcium signalling: IP3 rises again...and again", Current biology : CB, vol. 11, no. 9, pp. R352-5. Tervonen, T., Akerman, K., Oostra, B.A. & Castren, M. 2005, "Rgs4 mRNA expression is decreased in the brain of Fmr1 knockout mouse", Brain research.Molecular brain research, vol. 133, no. 1, pp. 162-165. Tomic, J.L., Pensalfini, A., Head, E. & Glabe, C.G. 2009, "Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction", Neurobiology of disease, vol. 35, no. 3, pp. 352-358.

82 Tran, P.B., Banisadr, G., Ren, D., Chenn, A. & Miller, R.J. 2007, "Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain", The Journal of comparative neurology, vol. 500, no. 6, pp. 1007-1033. Tuppo, E.E. & Arias, H.R. 2005, "The role of inflammation in Alzheimer's disease", The international journal of biochemistry & cell biology, vol. 37, no. 2, pp. 289-305. Turner, G., Daniel, A. & Frost, M. 1980, "X-linked mental retardation, macro-orchidism, and the Xq27 fragile site", The Journal of pediatrics, vol. 96, no. 5, pp. 837-841. Tuteja, N. 2009, "Signaling through G protein coupled receptors", Plant signaling & behavior, vol. 4, no. 10, pp. 942-947. Van de Bor, V. & Davis, I. 2004, "mRNA localisation gets more complex", Current opinion in cell biology, vol. 16, no. 3, pp. 300-307. Van der Borght, K., Mulder, J., Keijser, J.N., Eggen, B.J., Luiten, P.G. & Van der Zee, E.A. 2005, "Input from the medial septum regulates adult hippocampal neurogenesis", Brain research bulletin, vol. 67, no. 1-2, pp. 117-125. van Praag, H., Christie, B.R., Sejnowski, T.J. & Gage, F.H. 1999, "Running enhances neurogenesis, learning, and long-term potentiation in mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 23, pp. 13427-13431. Vanderklish, P.W. & Edelman, G.M. 2005, "Differential translation and fragile X syndrome", Genes, brain, and behavior, vol. 4, no. 6, pp. 360-384. Vanderklish, P.W. & Edelman, G.M. 2002, "Dendritic spines elongate after stimulation of group 1 metabotropic glutamate receptors in cultured hippocampal neurons", Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 3, pp. 1639-1644. Verkerk, A.J., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P., Pizzuti, A., Reiner, O., Richards, S., Victoria, M.F. & Zhang, F.P. 1991, "Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome", Cell, vol. 65, no. 5, pp. 905-914. Verret, L., Jankowsky, J.L., Xu, G.M., Borchelt, D.R. & Rampon, C. 2007, "Alzheimer's-type amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 27, no. 25, pp. 6771-6780. von Bohlen Und Halbach, O. 2010, "Dendritic spine abnormalities in mental retardation", Cell and tissue research, vol. 342, no. 3, pp. 317-323. Walsh, D.M., Klyubin, I., Shankar, G.M., Townsend, M., Fadeeva, J.V., Betts, V., Podlisny, M.B., Cleary, J.P., Ashe, K.H., Rowan, M.J. & Selkoe, D.J. 2005, "The role of cell-derived oligomers of Abeta in Alzheimer's disease and avenues for therapeutic intervention", Biochemical Society transactions, vol. 33, no. Pt 5, pp. 1087-1090. Walsh, D.M. & Selkoe, D.J. 2007, "A beta oligomers - a decade of discovery", Journal of neurochemistry, vol. 101, no. 5, pp. 1172-1184. Walsh, D.T., Montero, R.M., Bresciani, L.G., Jen, A.Y., Leclercq, P.D., Saunders, D., EL-Amir, A.N., Gbadamoshi, L., Gentleman, S.M. & Jen, L.S. 2002, "Amyloid-beta peptide is toxic to neurons in vivo via indirect mechanisms", Neurobiology of disease, vol. 10, no. 1, pp. 20-27. Wang, H., Ku, L., Osterhout, D.J., Li, W., Ahmadian, A., Liang, Z. & Feng, Y. 2004, "Developmentally-programmed FMRP expression in oligodendrocytes: a potential role of FMRP

83 in regulating translation in oligodendroglia progenitors", Human molecular genetics, vol. 13, no. 1, pp. 79-89. Wang, J., Xiong, S., Xie, C., Markesbery, W.R. & Lovell, M.A. 2005, "Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease", Journal of neurochemistry, vol. 93, no. 4, pp. 953-962. Wang, R., Sweeney, D., Gandy, S.E. & Sisodia, S.S. 1996, "The profile of soluble amyloid beta protein in cultured cell media. Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry", The Journal of biological chemistry, vol. 271, no. 50, pp. 31894-31902. Warner-Schmidt, J.L. & Duman, R.S. 2006, "Hippocampal neurogenesis: opposing effects of stress and antidepressant treatment", Hippocampus, vol. 16, no. 3, pp. 239-249. Weiler, I.J., Spangler, C.C., Klintsova, A.Y., Grossman, A.W., Kim, S.H., Bertaina-Anglade, V., Khaliq, H., de Vries, F.E., Lambers, F.A., Hatia, F., Base, C.K. & Greenough, W.T. 2004, "Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses", Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 50, pp. 17504-17509. Weiss, S., Dunne, C., Hewson, J., Wohl, C., Wheatley, M., Peterson, A.C. & Reynolds, B.A. 1996, "Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 16, no. 23, pp. 7599-7609. Wen, P.H., Hof, P.R., Chen, X., Gluck, K., Austin, G., Younkin, S.G., Younkin, L.H., DeGasperi, R., Gama Sosa, M.A., Robakis, N.K., Haroutunian, V. & Elder, G.A. 2004, "The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice", Experimental neurology, vol. 188, no. 2, pp. 224-237. Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., ConstantinePaton, M., Isacson, O. & Jaenisch, R. 2008, "Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease", Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 15, pp. 5856-5861. Wettschureck, N. & Offermanns, S. 2005, "Mammalian G proteins and their cell type specific functions", Physiological Reviews, vol. 85, no. 4, pp. 1159-1204. Willemsen, R., Levenga, J. & Oostra, B.A. 2011, "CGG repeat in the FMR1 gene: size matters", Clinical genetics, vol. 80, no. 3, pp. 214-225. Williams, B.P., Milligan, C.J., Street, M., Hornby, F.M., Deuchars, J. & Buckley, N.J. 2004, "Transcription of the M1 muscarinic receptor gene in neurons and neuronal progenitors of the embryonic rat forebrain", Journal of neurochemistry, vol. 88, no. 1, pp. 70-77. Wilson, B.M. & Cox, C.L. 2007, "Absence of metabotropic glutamate receptor-mediated plasticity in the neocortex of fragile X mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 7, pp. 2454-2459. Winner, B., Desplats, P., Hagl, C., Klucken, J., Aigner, R., Ploetz, S., Laemke, J., Karl, A., Aigner, L., Masliah, E., Buerger, E. & Winkler, J. 2009, "Dopamine receptor activation promotes adult neurogenesis in an acute Parkinson model", Experimental neurology, vol. 219, no. 2, pp. 543552. Wysocki, M., Luo, X., Schmeidler, J., Dahlman, K., Lesser, G.T., Grossman, H., Haroutunian, V. & Beeri, M.S. 2011, "Hypertension is Associated With Cognitive Decline in Elderly People at High Risk for Dementia", The American Journal of Geriatric Psychiatry : Official Journal of the American Association for Geriatric Psychiatry, .

84 Wyss-Coray, T., Loike, J.D., Brionne, T.C., Lu, E., Anankov, R., Yan, F., Silverstein, S.C. & Husemann, J. 2003, "Adult mouse astrocytes degrade amyloid-beta in vitro and in situ", Nature medicine, vol. 9, no. 4, pp. 453-457. Xu, H., Sweeney, D., Wang, R., Thinakaran, G., Lo, A.C., Sisodia, S.S., Greengard, P. & Gandy, S. 1997, "Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation", Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 8, pp. 3748-3752. Yamasaki, T.R., Blurton-Jones, M., Morrissette, D.A., Kitazawa, M., Oddo, S. & LaFerla, F.M. 2007, "Neural stem cells improve memory in an inducible mouse model of neuronal loss", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 27, no. 44, pp. 11925-11933. Yan, Q.J., Rammal, M., Tranfaglia, M. & Bauchwitz, R.P. 2005, "Suppression of two major Fragile X Syndrome mouse model phenotypes by the mGluR5 antagonist MPEP", Neuropharmacology, vol. 49, no. 7, pp. 1053-1066. Yang, P., Arnold, S.A., Habas, A., Hetman, M. & Hagg, T. 2008, "Ciliary neurotrophic factor mediates dopamine D2 receptor-induced CNS neurogenesis in adult mice", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 28, no. 9, pp. 22312241. Yoneyama, M., Shiba, T., Hasebe, S. & Ogita, K. 2011, "Adult neurogenesis is regulated by endogenous factors produced during neurodegeneration", Journal of pharmacological sciences, vol. 115, no. 4, pp. 425-432. Young, S.Z., Taylor, M.M. & Bordey, A. 2011, "Neurotransmitters couple brain activity to subventricular zone neurogenesis", The European journal of neuroscience, vol. 33, no. 6, pp. 1123-1132. Yuste, R. 2011, "Dendritic spines and distributed circuits", Neuron, vol. 71, no. 5, pp. 772-781. Zalfa, F., Adinolfi, S., Napoli, I., Kuhn-Holsken, E., Urlaub, H., Achsel, T., Pastore, A. & Bagni, C. 2005, "Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif", The Journal of biological chemistry, vol. 280, no. 39, pp. 33403-33410. Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A., Reis, S., Oostra, B. & Bagni, C. 2003, "The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses", Cell, vol. 112, no. 3, pp. 317-327. Zhang, C., McNeil, E., Dressler, L. & Siman, R. 2007, "Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease", Experimental neurology, vol. 204, no. 1, pp. 77-87. Zhao, C., Deng, W. & Gage, F.H. 2008, "Mechanisms and functional implications of adult neurogenesis", Cell, vol. 132, no. 4, pp. 645-660. Zhao, F., Wu, T., Lau, A., Jiang, T., Huang, Z., Wang, X.J., Chen, W., Wong, P.K. & Zhang, D.D. 2009, "Nrf2 promotes neuronal cell differentiation", Free radical biology & medicine, vol. 47, no. 6, pp. 867-879. Zhao, G., Mao, G., Tan, J., Dong, Y., Cui, M.Z., Kim, S.H. & Xu, X. 2004, "Identification of a new presenilin-dependent zeta-cleavage site within the transmembrane domain of amyloid precursor protein", The Journal of biological chemistry, vol. 279, no. 49, pp. 50647-50650.

85 Zhao, G., Tan, J., Mao, G., Cui, M.Z. & Xu, X. 2007, "The same gamma-secretase accounts for the multiple intramembrane cleavages of APP", Journal of neurochemistry, vol. 100, no. 5, pp. 1234-1246. Zhao, M.G., Toyoda, H., Ko, S.W., Ding, H.K., Wu, L.J. & Zhuo, M. 2005, "Deficits in trace fear memory and long-term potentiation in a mouse model for fragile X syndrome", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 25, no. 32, pp. 73857392. Zhao, W.Q., Alkon, D.L. & Ma, W. 2003, "c-Src protein tyrosine kinase activity is required for muscarinic receptor-mediated DNA synthesis and neurogenesis via ERK1/2 and c-AMPresponsive element-binding protein signaling in neural precursor cells", Journal of neuroscience research, vol. 72, no. 3, pp. 334-342. Zhou, C., Wen, Z.X., Shi, D.M. & Xie, Z.P. 2004, "Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro", Cell biology international, vol. 28, no. 1, pp. 63-67. Zhu, X., Lee, H.G., Moreira, P.I., Smith, M.A. & Perry, G. 2006, "Presenilin mutation: a deadly first hit in Alzheimer disease. A commentary on "aging sensitizes towards ROS formation and lipid peroxidation in PS1M146L transgenic mice"", Free radical biology & medicine, vol. 40, no. 5, pp. 737-739. Zlokovic, B.V. 2011, "Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders", Nature reviews.Neuroscience, vol. 12, no. 12, pp. 723-738.

86

ORIGINAL PUBLICATIONS (I-IV)

87

88

I Altered differentiation of neural stem cells in fragile X syndrome Castren, M., Tervonen, T., Kärkkäinen, V., Heinonen, S., Castren, E., Larsson, K., Bakker, C. E., Oostra, B.A. and Åkerman, K. PNAS 102:17834-17838, 2005

Reprinted with the kind permission by PNAS

89

90

II Neurotransmitter responsiveness during early maturation of neural progenitor cells Kärkkäinen, V., Louhivuori, V., Castren, M. and Åkerman, K. Differentiation 77:188-198, 2009

Reprinted with the kind permission by Elsevier

91

92

III Brain environment and Alzheimer´s disease mutations affect the survival, migration and differentiation of neural progenitor cells Kärkkäinen, V., Magga, J., Koistinaho, J. and Malm, T.

Submitted

93

94

IV Nrf2 protects neural progenitor cells against A toxicity and promotes endogenous neurogenesis Kärkkäinen, V., Savchenko, E., Pomeshchik, Y., Kurronen, A., Levonen, A-L., Magga, J., Kanninen, K. and Koistinaho, J.

Manuscript