Die Bildung neuen Nervenzellen im erwachsenen Gehirn—adulte Neurogenese—ist bei Säugetieren auf spezifische Regionen beschränkt. Eine der beiden bekannten ist der Hippokampus, eine Gehirnstruktur, die eine wichtige Rolle beim Lernen sowie der Gedächtnisbildung spielt. Ein Reservoir von neuralen Stammzellen befindet sich in der subgranulären Zone des hippokampalen Gyrus dentatus. Diese Zellen teilen sich fortwährend und bilden neue Nervenzellen. Die Regulation adulter hippokampaler Neurogenese wird sowohl von der Umgebung beeinflusst als auch von mehreren Genen gesteuert. In der vorliegenden Arbeit wurden mittels Hochdurchsatz- Genexpressionsverfahren die an der Neurogenese beteiligten Gene identifiziert und ihr Zusammenspiel untersucht. Anhand von genetischen, umgebungsbedingten und zeitlichen Angaben und Variationen wurde ein vielseitiger Datensatz erstellt, der einen multidimensionalen Blick auf den proliferativen Phänotyp verschafft. Netzwerke aus Gen-Gen und Gen-Phänotyp Interaktionen wurden beschrieben und in einer mehrschichtigen Ressource zusammengefasst. Ein Kern-Netzwerk bestehend aus immerwiederkehrenden Modulen aus verschiedenen Ebenen wurde anhand von Proliferation als Keim-Phänotyp identifiziert. Aus diesem Kern-Netzwerk sind neue Gene und ihre Interaktionen hervorgegangen, die potentiell bei der Regulierung adulter Neurogenesis beteiligt sind. / Neurogenesis, the production of new neurons, is restricted in the adult brain of mammals to only a few regions. One of these sites of adult neurogenesis is the hippocampus, a structure essential for many types of learning. A pool of stem cells is maintained in the subgranular zone of the hippocampal dentate gyrus which proliferate and can differentiate into new neurons, astrocytes and oligodendroctytes. Regulation of adult hippocampal neurogenesis occurs in response to en- vironmental stimuli and is under the control of many genes. This work employs high-throughput gene expression technologies to identify these genes and their interactions with each other and the neurogenesis phenotype. Harnessing variation from genetic, environmental and temporal sources, a multi-faceted dataset has been generated which offers a multidimensional view of the neural precursor proliferation phenotype. Networks of gene-gene and gene-phenotype interac- tions have been described and merged into a multilayer resource. A core subnetwork derived from modules recurring in the different layers has been identified using the proliferation phenotype as a seed. This subnetwork has suggested novel genes and interactions potentially involved in the regulation of adult hippocampal neurogenesis.

O exercício físico vem sendo cada vez mais utilizado como uma intervenção terapêutica para a depressão. Diversos estudos mostram a sua eficácia em episódios depressivos mais leves. Sua eficácia em episódios mais graves como em indivíduos hospitalizados ainda não foi suficientemente estudada. Faltam informações também sobre a existência de fatores que possam predizer ou moderar o efeito antidepressivo do exercício como também informações sobre os potenciais mecanismos ou correlatos biológicos que possam estar associados aos efeitos antidepressivos do exercício em indivíduos deprimidos. A presente tese consiste de quatro artigos, sendo o primeiro uma revisão sistemática sobre os potenciais fatores preditores ou moderadores do efeito antidepressivo do exercício, o segundo, uma revisão sistemática sobre os potenciais mecanismos ou correlatos biológicos do exercício, o terceiro, um ensaio clínico randomizado avaliando os efeitos do exercício físico em pacientes internados com depressão grave, e o ultimo, uma análise dos efeitos do exercício em marcadores de neurogênese e estresse oxidativo. Os resultados encontrados na primeira revisão sugerem que existem vários potenciais candidatos a preditor ou moderador do efeito antidepressivo do exercício físico; entretanto, a literatura não é suficientemente robusta para sugerir de forma consistente sua existência. De acordo com a segunda revisão, o exercício pode potencialmente promover respostas agudas em diversos biomarcadores. No entanto, as respostas crônicas em adaptação ao treinamento parecem ser menos consistentes. Cabe ressaltar que a literatura apresenta diversas limitações importantes, impedindo conclusões mais sólidas, tanto para as respostas agudas quanto para as crônicas. Com o ensaio clinico, evidenciamos que o exercício físico pode ser uma intervenção terapêutica eficaz na redução dos sintomas depressivos e na melhora de alguns domínios da qualidade de vida de participantes hospitalizados com depressão grave. Enquanto no ultimo artigo, utilizando uma amostra do ensaio clinico, foram encontradas diminuições nos níveis séricos de marcadores de stress oxidativo, porem, não houve alterações nos níveis de marcadores de neurogênese. A presente tese avança no entendimento do fenômeno do efeito antidepressivo do exercício, achando pontos a serem explorados em futuras investigações. Demonstra, também, a eficácia do exercício em pacientes hospitalizados com depressão grave. Por ultimo, mostra que existem diversos potenciais mecanismos e correlatos biológicos que possam vir a explicar ou estar associados a este efeito antidepressivo. / Physical exercise has been increasingly exploited as a therapeutic intervention for depression. Several studies have shown its effectiveness in milder severity of depression, however, their effectiveness in severe episodes in hospitalized individuals still needs further investigation. There is also a lack of information about factors that can predict or moderate the antidepressant effect of exercise and the potential mechanisms or biological correlates that are associated with antidepressant effects of exercise in depressed individuals. This thesis consists of four articles. The first is a systematic review of potential predictors or antidepressant effect of exercise moderators. The second is a systematic review of the potential biological mechanisms or correlates of exercise. The third is a randomized clinical trial evaluating the effects of physical exercise in patients hospitalized with severe depression. The last one, is an analysis of the effects of exercise on neurogenesis markers and oxidative stress. The findings of the first review suggest that there are several potential candidates predictors and moderators. However, the literature is not robust enough to suggest any predictor or moderator with greater consistency. According to the second review, exercise can potentially promote acute responses in several biomarkers. On the other hand, the chronic adaptations to exercise training appear to be less consistent. The literature contains several important limitations, preventing more solid conclusions regards the acute and chronic responses. According to the clinical trial, we observed that exercise can be an effective therapeutic intervention in reducing depressive symptoms and improving some domains of quality of life of participants hospitalized with severe depression. Finally, in the fourth article, using a sample of the clinical trial, serum levels of oxidative stress markers decreases were found., However, no changes in the levels of neurogenesis markers were found. This thesis advances in the understanding of the antidepressant effect of exercise phenomenon, finding points to be further explored regarding the predictors and moderators. It also demonstrates the antidepressant effectiveness of physical exercise in hospitalized patients with severe depression. Finally, shows that there are several potential mechanisms and biological correlates that may explain or be associated with this antidepressant effect.

O exercício físico vem sendo cada vez mais utilizado como uma intervenção terapêutica para a depressão. Diversos estudos mostram a sua eficácia em episódios depressivos mais leves. Sua eficácia em episódios mais graves como em indivíduos hospitalizados ainda não foi suficientemente estudada. Faltam informações também sobre a existência de fatores que possam predizer ou moderar o efeito antidepressivo do exercício como também informações sobre os potenciais mecanismos ou correlatos biológicos que possam estar associados aos efeitos antidepressivos do exercício em indivíduos deprimidos. A presente tese consiste de quatro artigos, sendo o primeiro uma revisão sistemática sobre os potenciais fatores preditores ou moderadores do efeito antidepressivo do exercício, o segundo, uma revisão sistemática sobre os potenciais mecanismos ou correlatos biológicos do exercício, o terceiro, um ensaio clínico randomizado avaliando os efeitos do exercício físico em pacientes internados com depressão grave, e o ultimo, uma análise dos efeitos do exercício em marcadores de neurogênese e estresse oxidativo. Os resultados encontrados na primeira revisão sugerem que existem vários potenciais candidatos a preditor ou moderador do efeito antidepressivo do exercício físico; entretanto, a literatura não é suficientemente robusta para sugerir de forma consistente sua existência. De acordo com a segunda revisão, o exercício pode potencialmente promover respostas agudas em diversos biomarcadores. No entanto, as respostas crônicas em adaptação ao treinamento parecem ser menos consistentes. Cabe ressaltar que a literatura apresenta diversas limitações importantes, impedindo conclusões mais sólidas, tanto para as respostas agudas quanto para as crônicas. Com o ensaio clinico, evidenciamos que o exercício físico pode ser uma intervenção terapêutica eficaz na redução dos sintomas depressivos e na melhora de alguns domínios da qualidade de vida de participantes hospitalizados com depressão grave. Enquanto no ultimo artigo, utilizando uma amostra do ensaio clinico, foram encontradas diminuições nos níveis séricos de marcadores de stress oxidativo, porem, não houve alterações nos níveis de marcadores de neurogênese. A presente tese avança no entendimento do fenômeno do efeito antidepressivo do exercício, achando pontos a serem explorados em futuras investigações. Demonstra, também, a eficácia do exercício em pacientes hospitalizados com depressão grave. Por ultimo, mostra que existem diversos potenciais mecanismos e correlatos biológicos que possam vir a explicar ou estar associados a este efeito antidepressivo. / Physical exercise has been increasingly exploited as a therapeutic intervention for depression. Several studies have shown its effectiveness in milder severity of depression, however, their effectiveness in severe episodes in hospitalized individuals still needs further investigation. There is also a lack of information about factors that can predict or moderate the antidepressant effect of exercise and the potential mechanisms or biological correlates that are associated with antidepressant effects of exercise in depressed individuals. This thesis consists of four articles. The first is a systematic review of potential predictors or antidepressant effect of exercise moderators. The second is a systematic review of the potential biological mechanisms or correlates of exercise. The third is a randomized clinical trial evaluating the effects of physical exercise in patients hospitalized with severe depression. The last one, is an analysis of the effects of exercise on neurogenesis markers and oxidative stress. The findings of the first review suggest that there are several potential candidates predictors and moderators. However, the literature is not robust enough to suggest any predictor or moderator with greater consistency. According to the second review, exercise can potentially promote acute responses in several biomarkers. On the other hand, the chronic adaptations to exercise training appear to be less consistent. The literature contains several important limitations, preventing more solid conclusions regards the acute and chronic responses. According to the clinical trial, we observed that exercise can be an effective therapeutic intervention in reducing depressive symptoms and improving some domains of quality of life of participants hospitalized with severe depression. Finally, in the fourth article, using a sample of the clinical trial, serum levels of oxidative stress markers decreases were found., However, no changes in the levels of neurogenesis markers were found. This thesis advances in the understanding of the antidepressant effect of exercise phenomenon, finding points to be further explored regarding the predictors and moderators. It also demonstrates the antidepressant effectiveness of physical exercise in hospitalized patients with severe depression. Finally, shows that there are several potential mechanisms and biological correlates that may explain or be associated with this antidepressant effect.

O exercício físico vem sendo cada vez mais utilizado como uma intervenção terapêutica para a depressão. Diversos estudos mostram a sua eficácia em episódios depressivos mais leves. Sua eficácia em episódios mais graves como em indivíduos hospitalizados ainda não foi suficientemente estudada. Faltam informações também sobre a existência de fatores que possam predizer ou moderar o efeito antidepressivo do exercício como também informações sobre os potenciais mecanismos ou correlatos biológicos que possam estar associados aos efeitos antidepressivos do exercício em indivíduos deprimidos. A presente tese consiste de quatro artigos, sendo o primeiro uma revisão sistemática sobre os potenciais fatores preditores ou moderadores do efeito antidepressivo do exercício, o segundo, uma revisão sistemática sobre os potenciais mecanismos ou correlatos biológicos do exercício, o terceiro, um ensaio clínico randomizado avaliando os efeitos do exercício físico em pacientes internados com depressão grave, e o ultimo, uma análise dos efeitos do exercício em marcadores de neurogênese e estresse oxidativo. Os resultados encontrados na primeira revisão sugerem que existem vários potenciais candidatos a preditor ou moderador do efeito antidepressivo do exercício físico; entretanto, a literatura não é suficientemente robusta para sugerir de forma consistente sua existência. De acordo com a segunda revisão, o exercício pode potencialmente promover respostas agudas em diversos biomarcadores. No entanto, as respostas crônicas em adaptação ao treinamento parecem ser menos consistentes. Cabe ressaltar que a literatura apresenta diversas limitações importantes, impedindo conclusões mais sólidas, tanto para as respostas agudas quanto para as crônicas. Com o ensaio clinico, evidenciamos que o exercício físico pode ser uma intervenção terapêutica eficaz na redução dos sintomas depressivos e na melhora de alguns domínios da qualidade de vida de participantes hospitalizados com depressão grave. Enquanto no ultimo artigo, utilizando uma amostra do ensaio clinico, foram encontradas diminuições nos níveis séricos de marcadores de stress oxidativo, porem, não houve alterações nos níveis de marcadores de neurogênese. A presente tese avança no entendimento do fenômeno do efeito antidepressivo do exercício, achando pontos a serem explorados em futuras investigações. Demonstra, também, a eficácia do exercício em pacientes hospitalizados com depressão grave. Por ultimo, mostra que existem diversos potenciais mecanismos e correlatos biológicos que possam vir a explicar ou estar associados a este efeito antidepressivo. / Physical exercise has been increasingly exploited as a therapeutic intervention for depression. Several studies have shown its effectiveness in milder severity of depression, however, their effectiveness in severe episodes in hospitalized individuals still needs further investigation. There is also a lack of information about factors that can predict or moderate the antidepressant effect of exercise and the potential mechanisms or biological correlates that are associated with antidepressant effects of exercise in depressed individuals. This thesis consists of four articles. The first is a systematic review of potential predictors or antidepressant effect of exercise moderators. The second is a systematic review of the potential biological mechanisms or correlates of exercise. The third is a randomized clinical trial evaluating the effects of physical exercise in patients hospitalized with severe depression. The last one, is an analysis of the effects of exercise on neurogenesis markers and oxidative stress. The findings of the first review suggest that there are several potential candidates predictors and moderators. However, the literature is not robust enough to suggest any predictor or moderator with greater consistency. According to the second review, exercise can potentially promote acute responses in several biomarkers. On the other hand, the chronic adaptations to exercise training appear to be less consistent. The literature contains several important limitations, preventing more solid conclusions regards the acute and chronic responses. According to the clinical trial, we observed that exercise can be an effective therapeutic intervention in reducing depressive symptoms and improving some domains of quality of life of participants hospitalized with severe depression. Finally, in the fourth article, using a sample of the clinical trial, serum levels of oxidative stress markers decreases were found., However, no changes in the levels of neurogenesis markers were found. This thesis advances in the understanding of the antidepressant effect of exercise phenomenon, finding points to be further explored regarding the predictors and moderators. It also demonstrates the antidepressant effectiveness of physical exercise in hospitalized patients with severe depression. Finally, shows that there are several potential mechanisms and biological correlates that may explain or be associated with this antidepressant effect.

Die Bildung neuen Nervenzellen im erwachsenen Gehirn—adulte Neurogenese—ist bei Säugetieren auf spezifische Regionen beschränkt. Eine der beiden bekannten ist der Hippokampus, eine Gehirnstruktur, die eine wichtige Rolle beim Lernen sowie der Gedächtnisbildung spielt. Ein Reservoir von neuralen Stammzellen befindet sich in der subgranulären Zone des hippokampalen Gyrus dentatus. Diese Zellen teilen sich fortwährend und bilden neue Nervenzellen. Die Regulation adulter hippokampaler Neurogenese wird sowohl von der Umgebung beeinflusst als auch von mehreren Genen gesteuert. In der vorliegenden Arbeit wurden mittels Hochdurchsatz- Genexpressionsverfahren die an der Neurogenese beteiligten Gene identifiziert und ihr Zusammenspiel untersucht. Anhand von genetischen, umgebungsbedingten und zeitlichen Angaben und Variationen wurde ein vielseitiger Datensatz erstellt, der einen multidimensionalen Blick auf den proliferativen Phänotyp verschafft. Netzwerke aus Gen-Gen und Gen-Phänotyp Interaktionen wurden beschrieben und in einer mehrschichtigen Ressource zusammengefasst. Ein Kern-Netzwerk bestehend aus immerwiederkehrenden Modulen aus verschiedenen Ebenen wurde anhand von Proliferation als Keim-Phänotyp identifiziert. Aus diesem Kern-Netzwerk sind neue Gene und ihre Interaktionen hervorgegangen, die potentiell bei der Regulierung adulter Neurogenesis beteiligt sind.:Zusammenfassung i Abstract iii Acknowledgements vii Contents ix Preface xiii General Introduction 1 Adult Neurogenesis 1 Historical setting 1 Neurogenesis exists in two regions of the adult mammalian brain 1 Implications of neurogenesis in the hippocampus 1 The Hippocampal Formation 2 Function of the hippocampus in learning and memory 2 The functional role of adult neurogenesis 2 Anatomy of the hippocampal formation 2 Neural Precursor Biology 3 The subgranular zone as a neurogenic niche 3 Neuronal maturation is a multi-step pathway 3 Regulation of Adult Neurogenesis 3 Neurogenesis is modulated by age 3 Neurogenesis is modulated by environmental factors 4 Neurogenesis is modulated by genetic background 4 Genetics of the BXD RI Cross 5 C57BL/6 and DBA/2 5 Recombinant Inbred Lines 5 The BXD panel 6 Quantitative genetics 6 Microarray Analysis 7 The concept of ‘whole genome’ expression analysis 7 Technical considerations 8 Theoretical considerations 9 Current Analytical Methods 9 Network Analysis 10 Network Description and Terminology 10 Graph Theory 10 Multiple-Network Comparison 11 Biological networks 11 Types of Biological Network 11 Sources of Network Data 12 Biological Significance of Networks 12 Aim of the current work 13 Methods and Materials 15 Animals 15 BXD panel 15 Progenitor strains 15 Animal behaviour 15 Running wheel activity 15 Enriched environment 16 Morris water maze 16 Open field test 16 Corticosterone assay 16 Histology 17 Tissue collection 17 BrdU staining 17 Statistics 17 Cell culture 18 Maintenance and differentiation 18 Immunostaining 18 RNA isolation 18 Microarray processing 18 Affymetrix arrays 18 M430v2 probe reannotation 19 Illumina arrays 19 Illumina probe reannotation 19 Bioinformatics 19 Translating the STRING network 19 QTL mapping 20 Network graph layout 20 Triplot 20 Enrichment analysis 20 Mammalian Adult Neurogenesis Gene Ontology 21 Introduction 21 Results 25 The cell stage ontology 25 The process ontology 25 Genes known to regulate hippocampal adult neurogenesis 26 Enrichment analysis 27 The MANGO gene network 27 Discussion 28 Hippocampal Coexpression Networks from the BXD Panel 31 Introduction 31 Results 32 Variation and covariation of gene expression across a panel of inbred lines 32 A hippocampal expression correlation network 32 Diverse neurogenesis phenotypes associate with discrete transcript networks 34 Discussion 34 Interactions Between Gene Expression Phenotypes and Genotype 37 Introduction 37 Results 39 QTL analysis and interval definitions 39 Pleiotropic loci and ‘trans-bands’ 39 Transcript expression proxy-QTLs can help in dissection of complex phenotypes 41 Interaction network 43 Discussion 43 Strain-Dependent Effects of Environment 47 Introduction 47 Results 48 Effects of strain and environment on precursor cell proliferation 48 Effects of strain and environment on learning behaviour 52 Transcript expression associated with different housing environments 53 Strain differences in transcript regulation 55 Distance-weighted coexpression networks 57 Discussion 58 Expression Time Course from Differentiating Cell Culture 61 Introduction 61 Results 63 Differentiation of proliferating precursors into neurons in vitro 63 Transcripts associated with stages of differentiation 63 Early events in NPC differentiation 64 A network of transcript coexpression during in vitro differentiation 66 Discussion 67 Integrated Gene Interaction Networks 71 Introduction 71 Results 72 Description of network layers 72 Merging of network layers to a multigraph 74 A network of genes controls neural precursor proliferation in the adult hippocampus 75 Novel candidate regulators of adult hippocampal neurogenesis 77 Novel pathways regulating adult hippocampal neurogenesis 77 Discussion 79 General Discussion 81 References 89 Selbständigkeitserklärung 107 / Neurogenesis, the production of new neurons, is restricted in the adult brain of mammals to only a few regions. One of these sites of adult neurogenesis is the hippocampus, a structure essential for many types of learning. A pool of stem cells is maintained in the subgranular zone of the hippocampal dentate gyrus which proliferate and can differentiate into new neurons, astrocytes and oligodendroctytes. Regulation of adult hippocampal neurogenesis occurs in response to en- vironmental stimuli and is under the control of many genes. This work employs high-throughput gene expression technologies to identify these genes and their interactions with each other and the neurogenesis phenotype. Harnessing variation from genetic, environmental and temporal sources, a multi-faceted dataset has been generated which offers a multidimensional view of the neural precursor proliferation phenotype. Networks of gene-gene and gene-phenotype interac- tions have been described and merged into a multilayer resource. A core subnetwork derived from modules recurring in the different layers has been identified using the proliferation phenotype as a seed. This subnetwork has suggested novel genes and interactions potentially involved in the regulation of adult hippocampal neurogenesis.:Zusammenfassung i Abstract iii Acknowledgements vii Contents ix Preface xiii General Introduction 1 Adult Neurogenesis 1 Historical setting 1 Neurogenesis exists in two regions of the adult mammalian brain 1 Implications of neurogenesis in the hippocampus 1 The Hippocampal Formation 2 Function of the hippocampus in learning and memory 2 The functional role of adult neurogenesis 2 Anatomy of the hippocampal formation 2 Neural Precursor Biology 3 The subgranular zone as a neurogenic niche 3 Neuronal maturation is a multi-step pathway 3 Regulation of Adult Neurogenesis 3 Neurogenesis is modulated by age 3 Neurogenesis is modulated by environmental factors 4 Neurogenesis is modulated by genetic background 4 Genetics of the BXD RI Cross 5 C57BL/6 and DBA/2 5 Recombinant Inbred Lines 5 The BXD panel 6 Quantitative genetics 6 Microarray Analysis 7 The concept of ‘whole genome’ expression analysis 7 Technical considerations 8 Theoretical considerations 9 Current Analytical Methods 9 Network Analysis 10 Network Description and Terminology 10 Graph Theory 10 Multiple-Network Comparison 11 Biological networks 11 Types of Biological Network 11 Sources of Network Data 12 Biological Significance of Networks 12 Aim of the current work 13 Methods and Materials 15 Animals 15 BXD panel 15 Progenitor strains 15 Animal behaviour 15 Running wheel activity 15 Enriched environment 16 Morris water maze 16 Open field test 16 Corticosterone assay 16 Histology 17 Tissue collection 17 BrdU staining 17 Statistics 17 Cell culture 18 Maintenance and differentiation 18 Immunostaining 18 RNA isolation 18 Microarray processing 18 Affymetrix arrays 18 M430v2 probe reannotation 19 Illumina arrays 19 Illumina probe reannotation 19 Bioinformatics 19 Translating the STRING network 19 QTL mapping 20 Network graph layout 20 Triplot 20 Enrichment analysis 20 Mammalian Adult Neurogenesis Gene Ontology 21 Introduction 21 Results 25 The cell stage ontology 25 The process ontology 25 Genes known to regulate hippocampal adult neurogenesis 26 Enrichment analysis 27 The MANGO gene network 27 Discussion 28 Hippocampal Coexpression Networks from the BXD Panel 31 Introduction 31 Results 32 Variation and covariation of gene expression across a panel of inbred lines 32 A hippocampal expression correlation network 32 Diverse neurogenesis phenotypes associate with discrete transcript networks 34 Discussion 34 Interactions Between Gene Expression Phenotypes and Genotype 37 Introduction 37 Results 39 QTL analysis and interval definitions 39 Pleiotropic loci and ‘trans-bands’ 39 Transcript expression proxy-QTLs can help in dissection of complex phenotypes 41 Interaction network 43 Discussion 43 Strain-Dependent Effects of Environment 47 Introduction 47 Results 48 Effects of strain and environment on precursor cell proliferation 48 Effects of strain and environment on learning behaviour 52 Transcript expression associated with different housing environments 53 Strain differences in transcript regulation 55 Distance-weighted coexpression networks 57 Discussion 58 Expression Time Course from Differentiating Cell Culture 61 Introduction 61 Results 63 Differentiation of proliferating precursors into neurons in vitro 63 Transcripts associated with stages of differentiation 63 Early events in NPC differentiation 64 A network of transcript coexpression during in vitro differentiation 66 Discussion 67 Integrated Gene Interaction Networks 71 Introduction 71 Results 72 Description of network layers 72 Merging of network layers to a multigraph 74 A network of genes controls neural precursor proliferation in the adult hippocampus 75 Novel candidate regulators of adult hippocampal neurogenesis 77 Novel pathways regulating adult hippocampal neurogenesis 77 Discussion 79 General Discussion 81 References 89 Selbständigkeitserklärung 107

Der Hippocampus ist ein Hirnareal welches fundamental für die Generierung von bewussten Erinnerungen und für die Etablierung von flexiblen Beziehungen zwischen kontextuellen Repräsentationen ist. Hippocampale Fehlfunktion während des Alterns wurde als ein Schlüsselfaktor für den kognitiven Abbau vorgeschlagen und im Kontext einer schnell alternden Bevölkerung wird es zwingend notwenig, mögliche Mechanismen zu verstehen, die diese Einschränkungen verhindern oder rückgängig machen können. Der Hippocampus ist ein von nur zwei Arealen im Gehirn, in dem neue Neuronen ständig im Erwachsenenleben erzeugt werden. Deren Rolle beim Lernen und bei Gedächtnisfunktionen ist jedoch nicht gut verstanden. Interessanterweise wurde adulte hippocampale Neurogenese als eine zelluläre Komponente eines Gehirnreservenmechanismus vorgeschlagen, mit dem Potenzial kognitive Fähigkeiten ein Leben lang zu erhalten sowie ein mögliches Ziel für therapeutische Ansätze darzustellen. In dieser Arbeit habe ich eine spezifische, genetisch-bedingte Expandierung von hippocampalen Nervenstammzellen genutzt, um deren intrinsisches Potenzial, neugeborene Neuronen zu erzeugen, auszuschöpfen, was zu einer lebenslangen erhöhten Neurogenese geführt hat. Dies hat die hippocampale Funktion auf mehreren Ebenen gefördert, vom verbesserten flexiblen Lernen in Navigationsaufgaben in der Jugend, über Kompensation des altersbedingten kognitiven Abbaus bis hin zur Verjüngung von kontextuellem Gedächtnis beim Altern. Zusammengefasst stellt meine Arbeit ein besseres Verständnis des funktionellen Beitrags der Neurogenese zu Lernen und Gedächtnis zur Verfügung and zeigt, dass kritische Aspekte hippocampaler kognitiver Beeinträchtigung im Alter rückgängig gemacht oder ein Leben lang durch extrinsische Ausnutzung der endogenen Hirnreserven kompensiert werden können.

Adulte Neurogenese steht im fokus der Neurowissenschaften. Die Neurogenese im Hippokampus spielt eine wichtige Rolle nach verschiedenen pathophysiologischen Ereignissen, z.B. nach epileptischen Anfällen. ATP ist ein wichtiger extrazellulärer Botenstoff, der auch im Gehirn verschiedene Prozesse beinflusst. Es wurden funktionelle P2X7- und P2Y1-Rezeptore an den Vorläuferzellen nachgewiesen. Unsere Hypothese lautet: nach einen epileptischen Anfall kommt es zur massiven Ausströmung von ATP, welches durch P2Y1-Rezeptore die Neurogenese fördert. Dieser Prozess wird durch die Aktivierung der P2X7-Rezeptoren entegegengesteuert, indem der Zelltod der neuen Zellen eingeleitet wird.

Embryonen von Höheren Krebsen (Malacostraca) zeigen ein stereotypes Zellteilungsmuster im Ektoderm des Rumpfes, im Verlaufe dessen paarige seitliche Reihen von Zellen und eine unpaare mittlere Reihe von Zellen gebildet werden. Das Muster der seitlichen Zellen ist von Dohle (1970, 1976) und Mitarbeitern geklärt worden. Die vorliegende Arbeit untersucht die cell lineage und Zelldifferenzierung der Mittellinienzellen im Thorax. Diese Zellen sind von besonderem Interesse, weil sie bei Insekten bereits intensiv erforscht wurden. (i) Die DiI Markierungen von Mittellinienzellen von Orchestia cavimana zeigen: Die Bildung der Mittellinie beginnt mit einer Zelle, die sich zweimal in Längsrichtung teilt. Die resultierenden vier Zellen werden mit a0, b0, c0 und d0 bezeichtet. Aus den Zellen a0, b0 und c0 gehen Paare von Gliazellen hervor. Die Tochterzellen von a0 und c0 umhüllen die Kommissuren. Die Zelle d0 ist ein medianer Neuroblast, aus dem mehrere Neurone hervorgehen, unter anderem ein unpaares Neuron im medianen Fasertrakt, interneurone und ein Motoneuron. (ii)BrdU Markierungen von Porcellio scaber zeigen: In den Ganglienanlagen liegt in der Mittellinie je eine Zelle, die größer ist als die benachbarten, schneller proliferiert und deshalb vermutlich ein medianer Neuroblast ist. (iii) Die Expression von engrailed setzt bei Orchestia und Porcellio ein in der Zelle a0 und wird in den zwei Tochterzellen fortgesetzt. Für Orchestia wird gezeigt, daß diese Expression zurückgeht und die Tochterzellen der Zelle d0 de novo mit einer Expression von engrailed beginnen.Aus den Ergebnissen kann abgeleitet werden, daß der gemeinsame Vorfahr von Insekten und Höheren Krebsen eine Mittellinie differenziert, die Vorläufer für Glia der Kommissuren und einen medianen Neuroblasten umfaßt und eine Expression von engrailed in den Tochterzellen des Neuroblasten zeigt. / Embryos of higher crustaceans (Malacostraca) show a highly stereotypic cell division pattern in the ectoderm of the trunk region while forming paired rows of lateral cells and an unpaired median row of midline cells. By using nuclear dyes, the pattern of the lateral cells has been determined by Dohle (1970, 1976) an co-workers. This study addresses the cell lineage and cell differentiation of the midline cells in the thorax. These kind of cells are of particular interest as they have been investigated extensively in insects. (i) The DiI labelling of midline cells in Orchestia cavimana reveals: Formation of the midline starts with a single midline cell that divides twice in longitudinal direction. The resulting four cells are termed a0, b0, c0, and d0. The cells a0, b0, and c0 give rise to pairs of glial cells. The progeny of a0 and c0 enwrap the commissures. The cell d0 is a median neuroblast that gives rise to several neurons, among them an unpaired neuron in the median fibre tract, interneurons and probably a single motoneuron. (ii) BrdU labelling in Porcellio scaber shows: there is a single larger and faster dividing cell in the midline in each segmental ganglion anlage that is a putative median neuroblast. (iii)The expression of engrailed starts in Orchestia and Porcellio the cell a0 and continues in the two daughter cells during segmentation. In Orchestia it can be shown that this expression ceases and progenies of the cell d0 start de novo with the expression of engrailed. From the results can be concluded that the common ancestor of insects and higher crustaceans differentiated an unpaired midline comprising precursors for glial cells enwrapping the commissures and a single median neuroblast whose derivatives express engrailed.

Lensemble du travail de recherche réalisé s'est concentré sur l'évaluation du rôle fonctionnel que peut exercer le gène circadien Per2 sur les capacités de prolifération et différenciation des cellules souches/progénitrices à l'origine de la neurogenèse hippocampique. Ce travail a comporté d'une part, une cartographie phénotypique exhaustive de l'identité des cellules exprimant la protéine PER2 au sein de la structure hippocampique, et d'autre part une étude approfondie des conséquences de la l'invalidation de ce gène sur la régulation de la neurogenèse dans l'hippocampe de souris adultes. Dans la première partie de notre travail, nous avons démontré par une analyse immunohistochimique détaillée, qu'au niveau du gyrus dentelé (DG) de souris adultes, les cellules proliférantes exprimaient la protéine PER2 et que cette expression persistait dans les cellules de la lignée neuronale à différents stades de maturation. Par ailleurs, à l'inverse du noyau suprachiasmatique (centre générateur des rythmes circadiens), nous avons également pu observer une expression constante de cette protéine durant une période de 24h (Borgs et al, soumis). Dans la seconde partie de notre travail, nous nous sommes interrrogés sur le rôle fonctionnel que pouvait exercer le facteur de transcription circadien Per2 dans le DG de souris adultes. Nous avons montré que linvalidation de ce gène entraine dans le DG des souris déficientes pour la protéine PER2, une augmentation significative de la prolifération des progéntieurs neuronaux, ainsi que du nombre de neurones immatures. Cependant, nous navons observé aucune différence dans la génération de neurones matures (neurogenèse) entre le DG de souris sauvages et de souris invalidées pour Per2. Nos données ont révélé que le surplus de cellules en prolifération et de neurones immatures observés dans le DG de souris délétées pour Per2 apparaît donc totalement compensé par une augmentation de la mort cellulaire (Borgs et al, soumis). Pour étudier limplication fonctionnel de la protéine PER2 sur le contrôl de la prolifération de progéniteurs/cellules souches à lorigine de la neurogenèse adulte, nous avons mis au point la culture en suspension de cellules souches/progénitrices issues du DG post-natale de souris sauvages et déficientes pour Per2. Après 5 jours de culture, nous avons observé la formation de neurosphères dont la taille et dont la croissance était plus importante chez les souris déficientes pour Per2 que chez leurs homologues sauvages. Ce modèle de culture de DG nous a permis détudier de façon plus présice le destin cellulaire emprunté par les cellules proliférantes/souches dans le modèle muté, comparé au modèle sauvage. En condition de culture favorisant la différenciation, nous avons observé un plus grand nombre de neurones générés à partir des neurosphères issues de cellules de DG de souris mutées pour PER2. Ce modèle de culture de cellules progénitrices/souches issues du DG, confirme les résultats précédemment obtenus concernant le rôle de Per2 dans le contrôle de la prolifération et de la génération de nouveaux neurones in vivo. Parallèlement, nous avons tenté de déterminer si lexpression de Per2 pouvait exercer un rôle similaire au DG au sein de la zone sous ventriculaire antérieure (SVZ), la seconde zone où persiste de la neurogenèse tout au long de la vie. La SVZ du cerveau adulte représente un réservoir de progéniteurs proliférant qui vont cheminer le long dun courant rostral de migration pour atteindre le bulbe olfactif dans lequel ils vont se différencier en neurones. La protéine Per2 se révèle être exprimée dans les progéniteurs en prolifération exprimant Ki67. Tout comme dans le DG de souris adultes déficientes pour Per2, nous avons dénombré in vivo et in vitro une augmentation importante du nombre de cellules en prolifération comparé aux souris sauvages. Per2 semble donc être un des protagonistes impliqué dans la régulation de la prolifération et de la différenciation des progéniteurs/cellules souches à lorigine de la neurogenèse hippocampique.

Konstanz, University, Diss., 2005.