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