Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.

Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.

Adult hippocampal neurogenesis is orchestrated by neural stem cell (NSC) activity. Some associations exist between autophagy and neurogenesis, yet much remains unknown about autophagic regulation of adult neurogenesis. This thesis interrogates the requirement and role of Beclin1 and Atg5, two regulators of autophagy, in the formation of adult hippocampal neurons. To examine adult brain NSCs, the experiments presented in the first objective of this thesis test the ability to isolate adult NSCs using flow cytometry and a DNA-binding dye, DyeCycleViolet. While adult NSCs could not be isolated from the adult neurogenic niches using this methodology, it was effective in isolating endothelial cells. This provided valuable insight on the use of DNA-binding dyes and a new method for isolation of brain endothelial cells. The next objective determines the role of Beclin1 in adult NSCs and their progeny using an inducible model. Beclin1 loss in Nestin-expressing hippocampal NSCs resulted in reduced proliferation, autophagy, and adult neurogenesis within one month. Single-cell RNA sequencing and other methods illuminated that loss of Beclin1 resulted in mitosis reduction, disrupted mitotic regulation of chromatin maintenance, and induction of DNA damage. The final objective first tests whether Beclin1 loss results in similar deficits within GLAST-expressing NSCs and progeny. This model mirrored neurogenesis deficits and requirement of Beclin1 in mitosis and DNA maintenance. Next, to test whether this phenotype occurs with other autophagy proteins, Atg5 was removed from GLAST NSCs. This resulted in reduced autophagy and a transient decrease in neurons in the absence of any effect on NSC proliferation. Thus, proliferation deficits are unique to Beclin1 loss and do not underlie reduced adult hippocampal neurogenesis after Atg5 removal. This work demonstrates a novel discovery of mitosis regulation in adult NSCs by Beclin1, and individual roles of Beclin1 and Atg5 in neurogenesis.

Ce travail de thèse a permis de caractériser avec succès des analogues structuraux de l´allopregnanolone présentant pour certains d'entre eux des effets bénéfiques et des avantages par rapport à la molécule de référence. En particulier, l'analogue O-allyl-AP, qui stimule in vitro la prolifération des cellules progénitrices, la différenciation neuronale et protège les cellules souches neurales adultes contre l'apoptose induite par le peptide Aβ42, est aussi efficace in vivo pour contrecarrer le déclin de la neurogenèse lié à l'âge et améliorer les performances cognitives au cours du vieillissement. De façon intéressante, les effets proliférateur et neuroprotecteur de l´O-allyl-AP semblent impliquer différents mécanismes d'action. Des expériences supplémentaires sont nécessaires pour conclure sur la capacité de l´O-allyl-AP à stabiliser le déclin de l'activité neurologique et à réduire les caractéristiques physiopathologiques de la Maladie d'Alzheimer (MA) chez les souris Tg2576. Nos résultats ouvrent des perspectives intéressantes pour l'application de l´O-allyl-AP dans le développement de stratégies thérapeutiques contre la MA et les maladies neurodégénératives. / This PhD work allowed us to successfully characterize structural analogs of allopregnanolone. Some of these analogs showed beneficial effects and advantages with respect to the molecule of reference. In particular, the analog O-allyl-AP stimulates proliferation of progenitor cells in different neural in vitro models, neuronal differentiation and protects adult neural stem cells against Aβ-induced apoptosis. In addition, O-allyl-AP is effective in counteracting the decline in neurogenesis related to age and in improving cognitive performance during aging. Interestingly, proliferative and neuroprotective effects seem to involve different mechanisms of action. Additional experiments are needed to confirm our preliminary data about the ability of O-allyl-AP to attenuate the decrease of neurogenic activity and to reduce pathophysiological hallmarks of Alzheimer disease (AD) in Tg2576 mice. Our findings provide interesting perspectives for using O-allyl-AP in the development of therapeutic strategies against AD and other neurodegenerative diseases.

In the adult mammalian brain, neural stem cells (NSCs) in the dentate gyrus (DG) of hippocampus continuously give rise to new neurons that provide structural plasticity and playing a role in learning and memory. Alterations in this process of adult neurogenesis have been linked to the pathology of various neurodegenerative diseases. Nevertheless, our knowledge regarding adult neurogenesis in the spectrum disorder of amyotrophic lateral sclerosis (ALS)/ frontotemporal dementia (FTD) remains incomplete. Therefore, I aimed to characterize how the ALS/FTD-associated mutation within the FUS protein affects adult NSCs during aging and how this may contribute to hippocampal function. For this purpose, a heterozygous knock-in ALS/FTD mouse model was used in which one copy of FUS harbors a deletion of its nuclear localization signal and their wild-type littermates were used as a control. The obtained results suggest a strong impact of the developing FUS-associated pathology on the hippocampal NSCs in the adult brain. For the first time, an age-dependent increase in NSCs proliferation and in the production of early postmitotic neurons was observed. In addition, the hippocampal network function and the response of adult neurogenesis to external stimulations seem to be altered by FUS mutation. The expected boost of NSC activity upon external stimulation appears to be blocked at particular developmental stages, however, the effect of the used strategies on the function of the hippocampal-cortical circuitry could still be detected. For further investigation, the in vitro analyses of adult NSCs were performed. Their proliferation activity was markedly increased, in comparison to wild-type cells, confirming the in vivo results. It appears that cells harboring mutated FUS are more likely to exit the quiescence state and enter the cell cycle. Furthermore, the combined cultivation of cells of both genotypes revealed that wild-type cells accelerate their proliferation to a similar level as mutant cells. Interestingly, the proliferation behavior of mutant cells remained unchanged regardless of the presence of wild-type cells. This suggests a spreading or transfer of the pathological mechanisms between the NSCs. Additionally, a commonly known hallmark of ALS/FTD pathology, the generation and accumulation of stress granules, was examined. Understanding the pathomechanisms of ALS/FTD-FUS in relation to adult hippocampal neurogenesis clearly requires further investigation. This appears to be crucial to provide the fundamental basis for new NSC-based preventative and therapeutic strategies.:1. Introduction 8 1.1. Adult hippocampal neurogenesis 9 1.1.1. The process of generating new granule neurons within the adult dentate gyrus 9 1.1.2. Regulation and function of AHN 12 1.2. Amyotrophic lateral sclerosis/ frontotemporal dementia 17 1.2.1. Amyotrophic lateral sclerosis/ frontotemporal dementia – spectrum disorder 17 1.2.2. FUS protein characterization and its contribution to ALS/FTD pathology 20 1.2.3. Knock-in mouse model of ALS/FTD-FUS 24 2. Aims 27 3. Materials and methods 28 3.1. In vivo 28 3.1.1. Animals 28 3.1.2. Physical activity 28 3.1.3. Enrichment environment 28 3.1.4. Thymidine labeling and tissue preparation 29 3.1.5. Immunohistochemistry - DAB staining 29 3.1.6. Quantification 30 3.1.7. Electrophysiology 30 3.1.8. Statistical analysis 30 3.2. In vitro 31 3.2.1. Generation of cell line 31 3.2.2. Culturing cell lines 31 3.2.3. Western Blotting 32 3.2.3.1. Collecting protein lysates 32 3.2.3.2. Bradford method 32 3.2.3.3. Electrophoresis and transfer 32 3.2.3.4. Fluorescent immunostaining 33 3.2.4. CFSE labeling and FACS analysis 33 3.2.5. Stress treatment 34 3.2.6. Fluorescent immunostaining (without BrdU) 34 3.2.7. BrdU labeling 35 3.2.8. Fluorescent immunostaining with BrdU 35 3.2.9. Cell cycle analysis 35 3.2.10. Statistical analysis 36 4. Results 37 4.1. In vivo 37 4.1.1. General characterization of the knock-in mouse model of ALS/FTD-FUS 37 4.1.2. Proliferation of hippocampal NSCs in FusDNLS/wt mice is unchanged at young age but strongly increased at old age 38 4.1.3. Response of AHN to external stimulus, such as physical activity, is altered in aged FusDNLS/wt mice 41 4.1.4. FUS mutation causes an increased generation of DCX-positive immature neurons in the DG and an altered response to the stimulatory effects of ENR in mice at 12 months of age 45 4.2. In vitro 52 4.2.1. Partial cytoplasmic mislocalization of mutant FUS protein in FusDNLS/wt cells 52 4.2.2. General level of FUS protein remains stable regardless of genotype 54 4.2.3. FusDNLS/wt cells generated from young animals exhibit distinct proliferation pattern 56 4.2.4. Fuswt/wt cells, cultured together with FusDNLS/wt ones, exhibit FusDNLS/wt-like acceleration of proliferation activity 57 4.2.5. Stress granules dynamics in cells in vitro 59 4.2.6. FusDNLS/wt cells are more likely-activated 65 5. Discussion 67 6. Bibliography 79