The murine subventricular zone (SVZ) in a brain contains a population of stem cells and daily produces tens of thousands of neurons throughout lifetime. However, the mechanisms of SVZ neural stem/progenitor cell (NSPC) maintenance, differentiation and cell-fate specification are still not clear. To understand these parameters via histone methylations with bivalent mechanism, the SVZ NSPCs were first isolated by using a culture technique called neurosphere assay (NSA). It has been a challenge to culture pure cell populations of SVZ subtypes, so the NSA was initially validated. The H3K27me3 mark, which has a dominant role in the bivalent mechanism, has not been studied in postnatal and adult SVZ in vivo, yet their role has been implicated to control the shift of embryonic cortical neurogenesis to gliogenesis. Therefore, we have first investigated whether H3K27me3 marks are present in the postnatal and adult SVZ NSPC population and whether their marks have been changed after stroke or demyelination in central nervous system (CNS) by immunohistrochemistry. With the confirmation of H3K27me3 mark present in SVZ NSPCs, the presence of H3K27me3 catalyzer, called polycomb repressive complex 2 (PRC2) core components (Eed, Ezh2, Suz12) including Jarid2, was investigated and confirmed in postnatal SVZ in vitro by qRT-PCR and Western blot. To understand the role of PRC2 enzymatic activity in postnatal SVZ neurosphere self-renewal and multipotency, Eed was down-regulated by using lentiviral mediated delivery of shRNA. Also, PRC2 dependent or independent function of Jarid2 was examined via knockdown approach. The lack of Eed in the neurospheres resulted the attenuation of self-renewal and oligodendrogenesis, whereas the Jarid2 knockdown neurospheres showed the decreased proliferation with no SVZ NSPC differentiation. Based on these knockdown studies, it suggests Eed and Jarid2 might not share their function in the postnatal SVZ NSPCs to govern postnatal SVZ NSPC self-renewal and multipotency.

Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2-/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.

Neural stem/progenitor cells (NSPCs) play a crucial part in the evolutionary development of the human neocortex. During early postnatal development, NSPCs give rise to immature neurons called neuroblasts within the subventricular zone (SVZ) that utilize unique migratory streams to integrate widely in the cerebral cortex. However, the cellular mechanisms enabling these unique migratory routes through the compacted cellular landscape remain unknown. Special emphasis has been placed on understanding the susceptibility of these brain regions to severe conditions such as congenital heart disease (CHD), resulting in poor neurological outcomes. Owing to its reminiscent complexity to humans, the neonatal piglet (Sus scrofa domesticus), which possesses a highly evolved gyrencephalic neocortex and an expansive outer SVZ, provides a powerful translational model system for the study of how heart dysfunction impacts cortical development from both a modern and evolutionary perspective. The present study provides a detailed characterization of neuroblast migration along their associate substrates in the piglet cortex under normal physiological conditions and how reduced oxygenation (i.e., hypoxia) can impact their vulnerability and/or resistance to injury during a critical period of postnatal development. In this thesis, I investigated the spatiotemporal distribution and developmental origin of SVZ-derived neuroblasts. Following BrdU tracing, multiplex labeling, and confocal microscopy, I show that the porcine brain contains populations of newly generated (BrdU+/DCX+) neurons in the prefrontal cortex that are produced postnatally. Regional analyses using immunohistochemical staining for doublecortin (DCX), a marker expressed by immature neurons, revealed that DCX+ clusters co-express markers of neuronal cell migration (PSA-NCAM), GABAergic interneuron marker (GABA+), and specific transcription factors (SCGN+SP8+) associated with the caudal- and lateral ganglionic eminence progenitor domains in the ventral forebrain. Moreover, I found that DCX+ neuroblasts are encased by astrocytic processes and tightly associated with blood vessels in the SVZ. Additionally, this thesis describes the use of chronic hypoxia as a model to profile neuroblast migration along associated substrates in pathological conditions related to CHD. Together, this work serves as a framework for the functional utilization of the neonatal piglet to understand the impact of substrate-dependent neuronal migration on brain maturation and neurodevelopmental diseases. / Doctor of Philosophy / Congenital heart disease (CHD) remains a significant cause of abnormal fetal brain development, affecting 1-2% of live births per year. Although many surgical strategies have shown promise in increasing quality of life, the current challenges remain the long-term cognitive deficits and diverse neurodevelopmental disabilities due to CHD. Recent studies suggest that dysregulated neurogenesis, which is associated with impaired neocortical development in human fetuses of CHD, may be influenced by altered brain circulation of blood and oxygen deliverance during critical periods of prenatal cortical growth. The brain's subventricular zone (SVZ) niche is essential for producing new neurons following birth to restore, repair, and replace existing neurons in the developing brain. In addition, these newborn neurons undergo long-distance migration from the SVZ to reach their final cortical destinations and ultimately contribute to brain development/plasticity. This study seeks to characterize the migration patterns of newborn neurons and the substrates (e.g., blood vessels or astrocytes), enabling the movement along the unique migratory routes under normal and pathological (i.e., hypoxia) conditions. In short, we found that the vast majority of the SVZ-derived newborn neurons are inhibitory neurons (i.e., interneurons) that originate in the deep region of the brain called the telencephalon and migrate tangentially utilizing blood vessels as scaffolds to the cortex, which is likely to contribute to cortical plasticity. These postnatal piglet findings demonstrate that swine represent a powerful translational model system to study large-brained mammalian cortical development and neuronal migration as it correlates to humans in normal and diseased states.

La migration des cellules souches / progénitrices neurales (CSPN) dans le cerveau adulte est cruciale pour la réparation cérébrale. Reeline (Rln) est une protéine de la matrice extracellulaire, régulant le positionnement des neurones pendant la croticogénèse. Nous révélons un rôle nouveau de Rln chez l'adulte. In vitro, Rln est chémocinétique mais pas chémoattractante. In vivo, Rln induit le détachement et la dispersion des CSNP de la zone sousventriculaire (SVZ) hors du courant rostral de migration (RMS) où elles sont sinon confinées. Rln potentialise le recrutement spontané des CSPN vers les lésions démyélinisantes où un tiers deviennent oligodendrocytaires. L'expression endogène de Rln est stimulée après lésion. Les animaux sans voie de signalisation Rln ont un recrutement réduit des CSPN vers les lésions.Ces résultats révèlent que Rln est un arbitre clef de la migration post-lésionnelle des CSPN et que permettre au CSPN de sortir du RMS est une stratégie thérapeutique prometteuse. / Neural stem/ progenitor cell (NSPC) migration in the adult brain is crucial for brain repair. Reelin (Rln) is an extracellular matrix protein regulating neuron positioning during coricogenesis. We reveal new roles of Rln in adult NSPC migration. In vitro, Rln promotes detachment, is chemokinetic but not chemoattractant. After Rln ectopic overexpression in the healthly brain, subventricular zone (SVZ) NSPC detach from the rostal migratory stream (RMS) in which they are normally restricted, and disperse in adjacent fiber tracts. Rln over-expression potentiates spontaneous cell recruitment to demyelinated lesion and one third of the NSPC recruited adopt an oligodendrocytic phenotype. Rln expression is spontaneously upregulated after lesion, and disruption of its signaling pathway results in reduced NSPC recruitment toward lesion. Our study reveals that Rln is a key player of post-lesional NSPC migration and that allowing NSPC to escape from RMS is a promising therapeutic approach

Parkinson’s disease (PD) has been reported to negatively affect adult neurogenesis. Mitochondrial dysfunction associated with PD may be involved, given that recent studies have identified mitochondria to be central regulators of neural stem cell (NSC) fate decisions. For this thesis, we sought to characterize adult neurogenesis in PINK1 and parkin knockout (KO) mouse models of PD. Immunohistochemical staining of subventricular zone (SVZ) and subgranular zone (SGZ) tissue sections from 6 month old mice was performed in order to identify and quantify changes in specific cell populations involved with adult neurogenesis. The loss of PINK1 or parkin was found to cause aberrant changes in adult neurogenesis, particularly in the SGZ. Going forward, it would be interesting to determine if the observed changes in adult neurogenesis were the result of mitochondrial dysfunction.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.

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There is a basic rule to mammalian neocortical expansion: as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.