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Glia, once considered as mere ‘glue’ for the central nervous system (CNS), have now emerged as active participants in almost every aspect of nervous system development, homeostasis, and even disease. Among these, oligodendroglia, comprising of oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) are responsible for myelinating the CNS. Additionally, recent discoveries have implicated these cells in other processes including phagocytosis, synaptogenesis, ability to influence neural activity, and even animal behaviour. OPCs originate during embryogenesis from neural stem cells, establish a non-overlapping grid-like pattern across CNS, and persist throughout life. They are also one of the most proliferative cell types within the brain, which differentiate into OLs. Given their widespread presence and multifaceted functions, it is not surprising that oligodendroglia are implicated in the pathogenesis of diseases such as Multiple Sclerosis (MS). MS is a highly prevalent demyelinating disease, characterised by a severe loss of OLs, neuronal atrophy, and disrupted neural circuits. Furthermore, the endogenous mechanisms of repair and regeneration fail, leading to progressive deterioration, including motor deficits and cognitive decline. Current clinical therapies mainly focus on slowing disease progression and alleviating symptoms. Therefore, there is an urgent need for the development of novel and improved regenerative therapies. My doctoral research focused on OPCs as a therapeutic avenue due to their stem-cell-like properties. By leveraging established links between cell cycle regulation and proliferation, my study aimed to specifically target G1 phase shortening through Cdk4 and CyclinD1 (4D) overexpression. To first evaluate its effect under physiological conditions, I employed a sophisticated triple transgenic mouse line that allows for spatiotemporal control of 4D overexpression in oligodendroglia. This approach led to an increase in OPC proliferation in the white and grey matter of the brain, effectively enhancing oligodendrogenesis. Subsequently, I tested the efficacy of 4D in a preclinical model of MS using cuprizone-induced demyelination. While no significant improvements in learning and memory functions were evident, a comprehensive analysis of cellular and functional effects of 4D will shed light on its mechanisms of action. Additionally, it is plausible that 4D might have positive outcomes on other aspects of behaviour; however, this requires further investigation. Altogether, the findings presented in this thesis introduce a novel tool aimed at augmenting endogenous oligodendrogenesis under physiological conditions and represent a significant step toward developing innovative therapeutic strategies for demyelinating disorders.:Table of Contents CHAPTER 1: INTRODUCTION 1.1. HISTORY OF OLIGODENDROGLIA 1 1.2. OLIGODENDROGLIA DURING DEVELOPMENT 4 1.3. OLIGODENDROGLIA IN ADULTHOOD 7 1.3.1. OPCS – DENSITY AND FUNCTIONS 7 1.3.2. OLS – DENSITY AND FUNCTIONS 8 1.4. OLIGODENDROGLIAL HETEROGENEITY 11 1.4.1. OPCS 11 1.4.2. OLS 12 1.5. OPC CELL CYCLE DYNAMICS 14 1.5.1. QUANTIFICATION OF OPC CELL CYCLE LENGTH 15 1.5.2. FACTORS INFLUENCING OPC CELL CYCLE 16 1.6. MYELIN AND MYELINATION 19 1.6.1. STRUCTURE AND COMPOSITION 19 1.6.2. FUNCTIONS 20 1.7. OLIGODENDROGENESIS AND BEHAVIOUR 21 1.7.1. LEARNING AND MEMORY 21 1.7.2. OTHERS 23 1.8. OLIGODENDROGLIA IN DISEASE AND REGENERATION 24 1.9. MS 26 1.9.1. MOUSE MODELS OF MS 28 1.10. CURRENT THERAPIES FOR DEMYELINATING DISEASES 31 1.11. AIM OF THE PROJECT 33 CHAPTER 2: MATERIALS AND METHODS 2.1. MATERIALS 36 2.1.1. MOUSE STRAINS 36 2.1.2. GENOTYPING PRIMERS 36 2.1.3. BUFFERS AND SOLUTIONS 37 2.1.4. CHEMICALS AND KITS 38 2.1.5. ANTIBODIES 39 2.2. METHODS 40 2.2.1. ANIMALS 40 2.2.2. GENOTYPING 40 2.2.3. DRUG TREATMENTS 40 2.2.4. BEHAVIOUR TESTS 41 2.2.4.1. OFT 41 2.2.4.2. EPM 42 2.2.4.3. ROTAROD 42 2.2.4.4. RW/CW 42 2.2.4.5. MWM 43 2.2.4.6. BM 44 2.2.5. IMMUNOHISTOCHEMISTRY 46 2.2.6. IMAGE ACQUISITION AND CELLULAR QUANTIFICATION 46 2.2.8. STATISTICS 47 CHAPTER 3: RESULTS - PART I CELLULAR AND BEHAVIOURAL EFFECTS OF GENETIC MANIPULATION OF CELL CYCLE OF OLIGODENDROCYTE PROGENITORS VIA CDK4/CYCLIND1 (4D) OVEREXPRESSION 3.1. CHARACTERISATION OF 4D OVEREXPRESSION MEDIATED BY TRIPLE TRANSGENIC MICE 48 3.2. 4D OVEREXPRESSION IN ADULT MICE INCREASES OPC PROLIFERATION IN CC AND CTX 49 3.3. 4D-INDUCED INCREASE IN OPC PROLIFERATION IS AGE-DEPENDENT 51 3.4. 4D OVEREXPRESSION INCREASES DENSITY OF OLS AND MYELIN IN CC AND CTX 52 3.5. 4D-INDUCED INCREASE IN OPC PROLIFERATION IS TEMPORALLY CORRELATED TO ACTIVATION OF 4D 53 3.6. 4D OVEREXPRESSION DOES NOT AFFECT ANXIETY-LIKE BEHAVIOUR ON THE OPEN FIELD AND ELEVATED PLUS MAZE TEST 55 3.7. 4D OVEREXPRESSION LEADS TO IMPAIRED LEARNING ON THE MORRIS WATER MAZE TEST 57 3.8. 4D OVEREXPRESSION NEGATIVELY IMPACTS RUNNING SPEEDS ON THE RUNNING/COMPLEX WHEEL TEST 59 3.9. 4D OVEREXPRESSION HAS A LONG-TERM NEGATIVE EFFECT ON RUNNING SPEEDS ON THE RUNNING/COMPLEX WHEEL TEST 61 CHAPTER 4: RESULTS - PART II CELLULAR AND BEHAVIOURAL CHARACTERISATION OF CUPRIZONE-INDUCED DEMYELINATION MODEL OF MULTIPLE SCLEROSIS 4.1. CUPRIZONE DIET LEADS TO OLIGODENDROCYTE LOSS AND DEMYELINATION ACROSS BRAIN REGIONS 64 4.2. TERMINATION OF CUPRIZONE DIET TRIGGERS SPONTANEOUS REGENERATION ACROSS BRAIN REGIONS 66 4.3. CUPRIZONE-INDUCED DEMYELINATION IMPAIRS LEARNING ON THE MORRIS WATER MAZE TEST 68 4.4. CUPRIZONE-INDUCED DEMYELINATION ADVERSELY AFFECTS BODY WEIGHT AND PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 70 CHAPTER 5: RESULTS - PART III BEHAVIOURAL EFFECT OF 4D-INDUCED OLIGODENDROGENESIS IN THE MODEL OF CUPRIZONE-INDUCED DEMYELINATION 5.1. 4D OVEREXPRESSION BEFORE THE ONSET OF CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE COGNITIVE PERFORMANCE ON BARNES MAZE 73 5.2. 4D OVEREXPRESSION BEFORE THE ONSET OF CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE MOTOR PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 75 5.3. SIMULTANEOUS 4D OVEREXPRESSION AND CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE MOTOR PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 78 CHAPTER 6: DISCUSSION 6.1. CELLULAR IMPLICATIONS OF 4D OVEREXPRESSION UNDER PHYSIOLOGICAL CONDITIONS 81 6.2. BEHAVIOURAL IMPLICATIONS OF 4D OVEREXPRESSION UNDER PHYSIOLOGICAL CONDITIONS 85 6.3. 4D AS A THERAPEUTIC TOOL 88 6.4. CONCLUSIONS AND OUTLOOK 90 REFERENCES 93 ACKNOWLEDGEMENTS 124 APPENDIX I 125 APPENDIX II 126

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In the present study 5-hydroxytryptamine (5-HT) 1A receptor knockout mice (KO), mice overexpressing the 5-HT1A receptor (OE), and wild-type (WT) mice were used to investigate the influence of 5-HT1A receptor on the development of the serotonergic system in the brain, from the embryonic day 12.5 to the postnatal day 15.5. Neither the absence nor the overexpression of 5-HT1A receptor influenced the development and differentiation of serotonergic neurons in the raphe area of the mouse brain. However, a delay in the initial development of the serotonergic projections to the mesencephalic tegmentum, cerebral cortex and hypothalamus was observed in both transgenic mice lines. The brain levels of 5-HT and 5-hydroxyindoleacetic acid were significantly higher in both transgenic mice lines during the late embryonic and early postnatal periods as compared to WT mice. An increase in the turnover of 5-HT was not observed before the early postnatal period. Both the absence and the overexpression of 5-HT1A receptor delayed the development of the dopaminergic system of the mesencephalic tegmentum in the early embryonic period. In OE mice the postnatal development of the noradrenergic system appeared to be exaggerated. The immunoreactivity for the neurotrophic protein S100ß was higher in the cerebral cortex, striatum and hippocampus of OE mice as compare to WT and KO mice. The expression of synaptic proteins, such as synapatobrevin and synaptotagmin was reduced in KO and OE mice during the early embryonic period. This reduction may be linked to the delayed development of the serotonergic projections and the dopaminergic system. In addition, no influence of 5-HT1A receptor mutations on the myelination of the brain was observed. Zusammenfassung In der vorliegenden Arbeit wurden die 5-Hydroxytryptamin (5-HT)1A Rezeptor Knockout (KO), überexprimierenden (ÜE) Mäuse und die Wild-Typ (WT) Mäuse, in den Entwicklungsperioden vom embryonalen Tag 12,5 bis postnatalen Tag 15,5 untersucht, um weitere Informationen über den Einfluss vom 5-HT1A Rezeptor auf die Entwicklung des serotonergen Systems im Gehirn zu erhalten. Sowohl das Fehlen des 5-HT1A Rezeptors als auch dessen Überexpression hatten zwar keinen Einfluss auf die Entwicklung und Differenzierung der serotonergen Neurone in den Raphe Regionen, verzögerte aber die erste Entwicklung der serotonergen Innervierungen im mesencephalen Tegmentum, Hypothalamus und cerebralen Cortex. In den späten embryonalen und insbesondere frühpostnatalen Perioden waren die 5-HT- und 5-HIAA-Spiegel bei KO und ÜE Mäusen im Vergleich zu WT Mäusen signifikant erhöht. Eine Erhöhung des 5-HT Turnovers wurde erst in der frühpostnatalen Periode beobachtet. Auch die Entwicklung des dopaminergen Systems im Mesencephalon war in der frühen embryonalen Periode sowohl bei KO als auch bei ÜE Mäusen verlangsamt. Die Überexpression des 5-HT1A Rezeptors begünstigte möglicherweise die postnatale Entwicklung des noradrenergen Systems. Bei ÜE Mäusen war die Immunreaktivität des neurotrophen Proteins S100? im cerebralen Cortex, Hippocampus und Striatum stärker als bei WT und KO Mäusen. Die Expression der synaptischen Proteine wie Synaptobrevin und Synaptotagmin war sowohl bei KO als auch bei ÜE Mäusen in der frühen embryonalen Periode verzögert. Dies könnte mit der verzögerten Entwicklung der serotonergen Projektionen und des dopaminergen Systems in Zusammenhang stehen. Darüber hinaus hatten transgene Veränderungen am 5-HT1A Rezeptor keinen Einfluss auf die Myelinisierung im Gehirn der Maus. Schlagwörter: serotonerges System, Entwicklung des Gehirns, 5-HT1A Rezeptor, transgene Mäuse, dopaminerges System, noradrenerges System, S100ß, Synaptisches Protein, Myelinisierung / In the present study 5-hydroxytryptamine (5-HT) 1A receptor knockout mice (KO), mice overexpressing the 5-HT1A receptor (OE), and wild-type (WT) mice were used to investigate the influence of 5-HT1A receptor on the development of the serotonergic system in the brain, from the embryonic day 12.5 to the postnatal day 15.5. Neither the absence nor the overexpression of 5-HT1A receptor influenced the development and differentiation of serotonergic neurons in the raphe area of the mouse brain. However, a delay in the initial development of the serotonergic projections to the mesencephalic tegmentum, cerebral cortex and hypothalamus was observed in both transgenic mice lines. The brain levels of 5-HT and 5-hydroxyindoleacetic acid were significantly higher in both transgenic mice lines during the late embryonic and early postnatal periods as compared to WT mice. An increase in the turnover of 5-HT was not observed before the early postnatal period. Both the absence and the overexpression of 5-HT1A receptor delayed the development of the dopaminergic system of the mesencephalic tegmentum in the early embryonic period. In OE mice the postnatal development of the noradrenergic system appeared to be exaggerated. The immunoreactivity for the neurotrophic protein S100ß was higher in the cerebral cortex, striatum and hippocampus of OE mice as compare to WT and KO mice. The expression of synaptic proteins, such as synapatobrevin and synaptotagmin was reduced in KO and OE mice during the early embryonic period. This reduction may be linked to the delayed development of the serotonergic projections and the dopaminergic system. In addition, no influence of 5-HT1A receptor mutations on the myelination of the brain was observed.

BACE1, die beta-Sekretase, spielt eine zentrale Rolle bei der Entstehung von Amyloid, einem charakteristischen histopathologischen Merkmal der Alzheimer-Demenz. Die physiologische Funktion von BACE1 ist unklar. Neuere Studien zeigten eine Rolle bei der Myelinisierung. Die vorliegende Arbeit untersucht die Rolle von BACE1 bei der Myelinisierung im Zentralen Nervensystem. Zusammenfassend zeigt die Studie keinen Einfluss einer BACE1-Inhibiton auf die primäre Ausprägung der Myelinscheiden im Corpus callosum. Sie widerspricht damit der Hypothese, dass BACE1 via Neuregulin-1-Prozessierung notwendig für die Myelinisierung im ZNS ist. Ob es sich dabei um lokale Differenzen einzelner anatomischer Regionen handelt muss in weiteren Studien untersucht werden. Zudem zeigt diese Arbeit einen kleinen, aber signifikanten Einfluss von BACE1 bei der Remyelinisierung im Corpus callosum nach Cuprizonebehandlung auf.

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