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Ischaemic stroke is a major cause of mortality and chronic disability for which there is no effective treatment. The subventricular zone (SVZ) is an adult neurogenic niche which mediates limited endogenous repair following stroke. To harness this phenomenon for therapy, it is important to understand how the SVZ niche is altered in stroke, and the processes that recruit neural precursors to the site of injury, which becomes a de facto neurogenic niche. Galectin-3 (Gal-3) is a β-galactoside binding protein involved in cellular adhesion, inflammation and tumour metastasis. Gal-3 is specifically expressed in the SVZ and maintains neuroblast migration to the olfactory bulb, although its role in post-stroke neurogenesis is not well-understood. Therefore, this project aimed to (1) characterise the cytoarchitecture of the SVZ in response to stroke, and (2) examine the role of Gal-3 in stroke outcome and tissue remodelling, and test the hypothesis that Gal-3 is required for neuroblast ectopic migration into the ischaemic striatum. Using the intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice, and whole mounts of the lateral ventricular wall, significant SVZ reactive astrocytosis and increased vascular branching were observed, thereby disrupting the neuroblast migratory scaffold. Stroke increased SVZ cell proliferation without increase in cell death. Post-stroke ependymal cells were enlarged and non-proliferative, and assumed a reactive astroglial phenotype, expressing de novo high levels of glial fibrillary acidic protein. This was associated with focal planar cell polarity misalignment, and turbulent and decreased rate of cerebrospinal fluid flow. These findings demonstrate significant changes in multiple SVZ cell types which are positioned to influence post-stroke neurogenesis and regulation of the neural stem cell niche Gal-3 was up-regulated in the ischaemic brain and ipsilateral SVZ. To elucidate the role of Gal-3 after stroke, MCAO was performed in wildtype and Gal-3 null (Gal-3^-/-) mice, and parameters of stroke outcome and post-stroke neurogenesis compared. The deletion of Gal-3 did not affect infarct volumes or neurological outcomes, although neuroblast migration into the ischaemic striatum was increased in Gal-3^-/- brains. Gal-3^-/- mice failed to mount an angiogenic response in the ischaemic striatum, and this was associated with lower levels of vascular endothelial growth factor (VEGF) and increased anti-angiogenic protein levels. Loss of Gal-3 further disrupted the pro-proliferative neural-vascular interaction at the basement membrane. The current data indicate that Gal-3 is a pleiotropic molecule which has distinct roles in both the SVZ and the post-stroke striatum as niches of adult neurogenesis.

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements. / Neurogenesis persists throughout the adulthood in two regions of the mammalian central nervous system (SNC): the sub-ventricular zone (SVZ) of the forebrain and the sub-granular zone (SGZ) of the hippocampus. Neurogenesis is possible due to the proliferation capacity of stem cells present within both the SVZ and SGZ niches, but with aging, the forebrain undergoes a drastic reduction in its number of adult neural stem cells (aNSCs), a decrease of cell proliferation and an alteration of the neurogenic niches. However, a key unresolved question remains: how the onset of aNSC loss is temporally related to changes of proliferating activity and to structural alterations within the principal stem cell niche (the SVZ)? To gain insights into the initial events leading to aging-associated aNSC loss, we investigated the changes occurring to aNSCs and the SVZ niche between young adulthood and middle-age. The SVZ niche of middle-aged mice (12-months-old) was found to display reduced expression of markers for multiple neural precursor sub-populations when compared to young adult mice (2-months-old). Anatomically, this was associated with significant cytological aberrations, including an overall atrophy of the SVZ, loss of sub-ependymal cells, and accumulation of large lipid droplets within the ependyma. Functionally, these changes correlated with diminished SVZ activity and reduced number of newly born neurons reaching the principal target tissue: the olfactory bulbs. To determine whether changes were evident at the level of the SVZ stem cells, we evaluated key in vitro and in vivo parameters of aNSCs. Tissue culture experiments showed that equal numbers of neurosphere-forming aNSCs could be isolated from young adult and middle-aged forebrains. However, at middle-age, neural precursors seemed to be less sensitive to growth factors during their in vitro differentiation and displayed signs of increased quiescence in vivo. Collectively, these findings demonstrate that, with early aging, aNCS and their SVZ niche go through significant changes, and suggest that aging-associated aNSC loss is secondary to these events.

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements. / Neurogenesis persists throughout the adulthood in two regions of the mammalian central nervous system (SNC): the sub-ventricular zone (SVZ) of the forebrain and the sub-granular zone (SGZ) of the hippocampus. Neurogenesis is possible due to the proliferation capacity of stem cells present within both the SVZ and SGZ niches, but with aging, the forebrain undergoes a drastic reduction in its number of adult neural stem cells (aNSCs), a decrease of cell proliferation and an alteration of the neurogenic niches. However, a key unresolved question remains: how the onset of aNSC loss is temporally related to changes of proliferating activity and to structural alterations within the principal stem cell niche (the SVZ)? To gain insights into the initial events leading to aging-associated aNSC loss, we investigated the changes occurring to aNSCs and the SVZ niche between young adulthood and middle-age. The SVZ niche of middle-aged mice (12-months-old) was found to display reduced expression of markers for multiple neural precursor sub-populations when compared to young adult mice (2-months-old). Anatomically, this was associated with significant cytological aberrations, including an overall atrophy of the SVZ, loss of sub-ependymal cells, and accumulation of large lipid droplets within the ependyma. Functionally, these changes correlated with diminished SVZ activity and reduced number of newly born neurons reaching the principal target tissue: the olfactory bulbs. To determine whether changes were evident at the level of the SVZ stem cells, we evaluated key in vitro and in vivo parameters of aNSCs. Tissue culture experiments showed that equal numbers of neurosphere-forming aNSCs could be isolated from young adult and middle-aged forebrains. However, at middle-age, neural precursors seemed to be less sensitive to growth factors during their in vitro differentiation and displayed signs of increased quiescence in vivo. Collectively, these findings demonstrate that, with early aging, aNCS and their SVZ niche go through significant changes, and suggest that aging-associated aNSC loss is secondary to these events.

Title: The subnuclear localisation of Notch responsive genes. Candidate Name: Matthew Jones Notch signalling is a highly conserved cell-cell communication pathway with critical roles in metazoan development and mutations in Notch pathway components are implicated in many types of cancer. Notch is an excellent and well-studied model of biological signalling and gene regulation, with a single intracellular messenger, one receptor and two ligands in Drosophila. However, despite the limited number of chemical players involved, a striking number of different outcomes arise. Molecular studies have shown that Notch activates different targets in different cell types and it is well known that Notch is important for maintaining a stem cell fate in some situations and driving differentiation in others. Thus some of the factors affecting the regulation of Notch target genes are yet to be discovered. Previous studies in various organisms have found that the location of a gene within the nucleus is important for its regulation and genome reorganisation can occur following gene activation or during development. Therefore this project aimed to label individual Notch responsive loci and determine their subnuclear localisation. In order to tag loci of interest a CRISPR/Cas9 genome-editing method was established that enabled the insertion of locus tags at Notch targets, namely the well-characterized Enhancer of split locus and also dpn and Hey, two transcription factors involved in neural cell fate decisions. The ParB/Int system is a recently developed locus tagging system and is not well characterised in Drosophila. It has a number of advantages over the traditional LacO/LacI-GFP locus tagging system as it does not rely on binding site repeats for signal amplification and can label two loci simultaneously in different colours. This thesis characterised the ParB/Int system in the Drosophila salivary gland and larval L3 neuroblast. Using 3D image segmentation hundreds of nuclei were reconstructed and a volume based normalisation method was applied to determine the subnuclear localisation of several Notch targets with and without genetic manipulations of the Notch pathway.