Stroke is the leading cause of long-term neurological disability worldwide, signifying the need for viable therapeutic options. Pre-clinical and post-mortem stroke studies have demonstrated that stroke increases the number of newborn progenitor cells (PCs) in the adult brain that can migrate to the site of injury. While there is a positive correlation between increasing neurogenesis and improvements in stroke recovery, methods used to increase PCs and neurogenesis also alter many other forms of plasticity, making it difficult to determine the function of PCs per se. To investigate whether specifically enhancing PC survival is sufficient to improve recovery, the iBax transgenic mouse model was used to remove the pro-apoptotic gene Bax inducibly from nestin-expressing PCs either before or after focal strokes induced by photothrombosis. Increasing PC survival before or after stroke in the iBax mice increased the number of PCs in the peri-infarct region. Interestingly, the majority of the cells that migrated to the peri-infarct region expressed the glial fibrillary acidic protein (GFAP) which is found in astrocytes when Bax was removed prior to stroke, yet when Bax was removed after stroke the majority of the cells expressed doublecortin (DCX) which is expressed in neuroblasts. Irrespective of this significant increase in the different populations of surviving PCs following stroke, there was no change in long-term behavioural deficits on the adhesive removal, horizontal ladder, and cylinder tasks up to 90 days post stroke. Additionally, enhancing PC survival before or after stroke resulted in a significant increase in adult-generated neurons within the dentate gyrus, which was associated with a modest change in spatial learning on the Barnes maze. Together, these experiments suggest strategies that enhance the survival of the PCs by preventing cell death will, by themselves, be insufficient to promote sensorimotor recovery following stroke.

Many young adult university students engage in frequent alcohol bingeing and have high depression scores, both of which are factors that can reduce hippocampal neurogenesis in rodents. Rodents with depleted neurogenesis exhibit selective deficits on high interference memory tasks including visual and spatial pattern separation. We predicted that young adult humans with high bingeing and depression scores would exhibit similarly impaired spatial pattern separation as a result of neurogenesis reductions. The relationships between alcohol bingeing, depression, and spatial pattern separation have, to this point, not been investigated in humans. We developed a novel computerized memory task for assessing spatial pattern separation in humans, loosely based on the “Concentration” memory card game. To further identify how sensitive this pattern separation function is to spatial separation between two stimuli, we developed the spatial separation recognition task (SSRT). We found that young adults with elevated depression and alcohol consumption scores exhibited impaired spatial pattern separation, in spite of intact performance on control tasks, consistent with a selective neurogenesis reduction. Further, this difference in performance seemed to be driven by performance at relatively larger separations. / Thesis / Master of Science (MSc)

Alzheimer’s disease is a progressive neurodegenerative disorder leading to dementia. Interestingly, AD is more prevalent in women than men, where two third of the diagnosed AD population is female. The underlying neuropathology is characterized by extracellular A𝛽 plaques and intracellular tau tangles leading to neuronal cell death. The hippocampus, the main site for learning and memory exhibits the most significant neuronal loss in AD. It is also one of the primary neurogenic niches in the adult brain. A decline of hippocampal neural stem cells is a common feature of AD indicating a defect in neurogenesis. To model AD neuropathology, a triple transgenic model of AD (3xTg) was used. We hypothesize that the defects associated with adult neurogenesis precede the onset of AD hallmarks. Our data showed that neurogenesis defects are present as early as post-natal day 5 of age in the 3xTg model of AD, well before the development of the neuropathology. The early defects were also observed in the TgCRND8 model of AD at 3 months of age. Moreover, no statistically significant difference was detected between male and female mice at 3 months and 9 months of age when investigating NSC populations. This could indicate that sex may not need to be taken in consideration in research design when investigating NSC decline in 3xTg mice. These findings are of clinical relevance, as they may identify early changes that may open opportunities for therapeutic interventions aiming at preventing or delaying neurodegeneration.

The ADNP (Activity dependent neuroprotective protein) gene encodes a transcription factor that is essential for embryonic development and brain formation. Mutations within this gene cause a neurodevelopmental disorder known as Helsmoortel-Van Der Aa syndrome. ADNP is one of the most commonly mutated single genes associated with autism spectrum disorder. However, its role in neurodevelopment is unclear. Our goal in this study is to dissect the role of Adnp in neurogenesis using the mouse retina as a tractable model system. We hypothesized that Adnp might have a crucial role in retinal neurogenesis. We found that Adnp was consistently expressed in all progenitor cells throughout retinal development. Interestingly, Adnp expression was relatively high in differentiated cells and persisted in the adult retina. Adnp was found to regulate retinal size, possibly by controlling cell survival during retinal development. In the retina, Adnp was found to interact with the Chd4 chromodomain helicase protein and might therefore be involved in chromatin remodelling. On the other hand, it also interacted with Pogz – a zinc finger protein associated with heterochromatin and might have a specific role in neural gene regulation. Altogether, these findings indicate that Adnp plays a crucial role in retinal neurogenesis and identify possible neurodevelopmental mechanisms that might depend on Adnp.

Long-term maintenance of adult neural stem cells (NSCs) is an intricate process of activation, expansion, and differentiation while preserving the stem cell pool. Several regulatory mechanisms underlie the delicate balance in the choice between quiescence versus activation for lifelong NSC maintenance and continuous neurogenesis. Perturbations in this dynamic process result in disease manifestation. The quiescence/activation of NSC was shown to be regulated by the Rb/E2F axis through a molecular program mediated by REST (RE1 Silencing Transcription Factor). Loss of Rb family increased NSC activation at the expense of quiescence through activator E2F transcription factors. The activation and neurogenesis of NSCs were impaired by the loss of effector E2Fs, as well as loss of Opa1, the latter indicating that mitochondrial dynamics is important to maintain stem cell state. Single-cell transcriptome analysis from NSC lineages isolated from adult mouse hippocampus revealed that stem cell progenies are uniquely affected in Opa1-KO leading to impairments in NSC activation and differentiation. Unbiased transcriptional profiling suggested a mitochondrial dysfunction in Opa1-KO that results in activation of classic cellular stress response pathway genes (Atf4, Slc7a11 and Chac1). Thus, the regulatory gene network comprising quiescence (Rb) and activation (E2Fs) programs, and mitochondrial metabolism (Opa1) and their interplay ensures the maintenance of the molecular program of NSC, particularly revealing how it enables stem cells survive stress.

Drosophila chordotonal (Ch) organs are internal stretch receptors required for coordination, balance and hearing. The outer dendritic segment of the Ch neuron is a compartmentalised motile cilium, a feature that is exclusive to this neuron subtype. Ch organs are specified early in development by expression of the proneural gene atonal in the proneural cluster and sense organ precursors (SOPs) (Jarman et al., 1993). However little is known about how chordotonal SOP specification is linked to differentiation of Ch organs. fd3F encodes a forkhead transcription factor which has been identified as a potential downstream target of atonal in microarray experiments (Cachero et al., 2011). I have shown that fd3F is exclusively expressed in Ch neurons and their precursors in Drosophila embryos and Ch SOPs in larval imaginal discs. I have also generated an fd3F deletion mutant by imprecise excision of a P element. Mutant adults and larvae exhibit impaired coordination characteristic of Ch neuron defects and a similar phenotype was observed in fd3F RNAi lines. fd3F mutant Ch neurons do not show gross morphological defects, however the tips of the Ch neuron cilia appear swollen when analysed by electron microscopy and there is also some mis-localisation of proteins within the cilia. I have identified several Ch-specific genes that show strongly reduced mRNA expression in fd3F mutant embryos compared with wild type and could therefore be downstream targets of fd3F. These include a number of genes known to be essential for Ch neuron function such as transient receptor potential (TRP) ion channels, dyneins required for motility of the Ch neuron cilium and components of the retrograde transport machinery that may be required for protein localisation within the cilium. In addition several uncharacterised genes were identified as fd3F targets and these genes may therefore also be important for Ch neuron function. I have shown that fd3F directly regulates two of these genes, nanchung and inactive using GFP enhancer constructs and gel retardation assays. I therefore hypothesise that fd3F is an important component of the gene regulatory network that links atonal expression in SOPs to differentiation of Ch organs. In particular fd3F regulates genes required specifically for Ch neuron function and enhances expression of retrograde transport genes that may be required to ensure correct distribution of proteins within the compartmentalised Ch neuron cilium.

Lamina formation in the developing cortex requires precise generation, migration and differentiation of cortical neurons. Cortical projection neurons originate from progenitors of the embryonic dorsal telencephalon. The transcription factor Pax6 is expressed in apical progenitors (APs) throughout corticogenesis in a rostro-lateralhigh to caudo-mediallow gradient. The current studies focus on elucidating the spatial and temporal role of Pax6 in cortical development. I first analysed the cortex of PAX77 transgenic mice that overexpress Pax6 in its normal domains of expression. I show that Pax6 overexpression acts cell-autonomously to reduce the proliferation of late cortical progenitors specifically, resulting in the formation of thinner superficial layers in the PAX77 cortex. Increased levels of Pax6 lengthen the cell cycle of APs and drive the system towards neurogenesis. These effects are specific to late stages of corticogenesis, when superficial layer neurons are normally generated, in cortical regions that express Pax6 at the highest levels. The number of superficial layer neurons is reduced in postnatal PAX77 mice, while radial migration and lamina specification of cortical neurons are not affected by Pax6 overexpression. Then, Pax6 was conditionally inactivated in cortical progenitors at mid- or late-stages of corticogenesis by using a tamoxifen-inducible Emx1-CreER line. I report a novel requirement of Pax6 for continuous suppression of ventral fates and concurrent maintenance of an appropriate dorsal identity in cortical progenitors. Pax6 ablation at either mid- or late-stages of corticogenesis increases the proliferation of late cortical progenitors at all levels across the rostral-caudal axis. In the absence of Pax6 from mid-corticogenesis, late-born neurons are severely under-represented and misspecified in superficial layers of the mutant cortex. Notably, Pax6 inactivation during late corticogenesis also affects superficial laminar fate; although the numbers of late-born cortical neurons are not severely affected in superficial layers of the mutant cortex, substantial numbers of late-born cells fail to migrate to appropriate laminar positions and accumulate in the ventricular zone (VZ) of the postnatal mutant cortex. Collectively, these gain- and loss-of-function studies suggest that disruption of Pax6 levels during different developmental time points leads ultimately to impaired formation of superficial cortical layers but through different cellular and molecular mechanisms.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of; Masters of Science in Medicine (Anatomical Sciences) School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg. 2017. / This research studied adult hippocampal neurogenesis in the dentate gyrus of the hippocampus of the Long-Evans rat. Eighteen male Long-Evans rats were exposed to complex enriched environment, the running wheel environment for exercise as single influencing factor and the standard laboratory environment for 28 days. Thereafter the rats were transcardially perfused with 0.9 normal saline followed by 4% paraformaldehyde. The brains were removed and frozen sagittal sections cut at 50 μm. Brain sections were stained with Cresyl violet for cytoarchitecture. Immunohistochemistry and immunofluorescence techniques were employed for the immature neurons with defined processes using the marker doublecortin (DCX), neuronal proliferation marker Ki-67, the synapse marker, synaptophysin and the dendritic spine marker, synaptobrevin. Giemsa staining was used to identify pyknotic neurons followed by counts for DCX, Ki-67, pyknotic positive cells, and volume density of the dentate gyrus. Results indicated a statistically significant increase in brain weight (p=0.5) for the complex enriched group when compared to the running group and control. The typical cytoarchitecture of the hippocampus in rodents was observed with more densely packed granule cell layer in the dorsal limb of the dentate gyrus compared to the ventral limb especially in the enriched group. The Ki-67 immunopositive cell number between groups showed a variable difference with a three-fold increase each between the standard control and exercise, and between the exercise and enriched but a six-fold increase between the standard control and the complex enriched groups. Comparing the DCX immunopositive results, we observed also that the neuronal numbers, structure, dendritic patterns as well as the neuronal arrangement on the dorsal and ventral limbs of the dentate gyrus varied significantly among groups. The apoptotic cell numberusing pyknotic cells, showed the standard control group to have the highest number of cells compared to the exercise versus the enriched group; noting a five-fold difference between the standard control and exercise, a twenty seven-fold difference between the standard control versus enriched and a twenty one-fold difference between 6 the exercise and complex enriched group. The volumetric analysis showed a 15-fold difference between the standard control and exercise groups, a five-fold difference between the exercise and complex enriched and a nineteen-fold difference between the standard control and complex enriched groups. However, no statistical significant difference was found in the volumetric analysis of the dentate gyrus between the groups. / MT2017

Opioid replacement therapy with methadone or buprenorphine has been recommended for managing opioid dependence during pregnancy. Although opioid replacement therapy decreases harmful consequences from maternal illicit drug seeking behaviors, the effects of methadone and buprenorphine on neurogenesis and myelination in the developing fetus have not been thoroughly reviewed. Methadone and buprenorphine may alter newborn neurobehavioral functions by impairing neurogenesis and changing the developmental pattern of myelination. This review found that therapeutic doses of methadone and buprenorphine disturb both neurogenesis and myelination in rodents. Methadone and buprenorphine may alter newborn neurobehavioral functions by impairing neurogenesis and changing the developmental pattern of myelination. However, further studies are required to bridge the gap in the understanding between changes in neural development and abnormalities in neurobehavioral functions in the newborn.

The adult mammalian brain harbours at least two germinal - or neurogenic - niches in which new neurons are born throughout life. These neurogenic niches comprise the subependymal zone which lines the ventricular system, and the subgranular zone in the hippocampal dentate gyrus. Post-natal hippocampal neurogenesis was in fact first identified experimentally in the 1960s. However perhaps due partly to aforementioned institutionalised belief and partly to a lack of accessible experimental tools, the phenomenon of hippocampal neurogenesis was widely recognised by the scientific community only shortly before the millennium. Consequent study has established that adult hippocampal neurogenesis has been conserved through millions of years of evolution in nearly every mammalian species studied to date. Importantly, post-mortem studies and radioisotope carbon dating techniques suggest that it also occurs in humans. A great deal of this research has focused on understanding the inner workings of the cells that undergo the transformation to become new adult-born neurons. By contrast, relatively little is known about the potential regulatory role of the surrounding extracellular microenvironment. This might be useful to know in light of much evidence that the extracellular matrix is a key regulator of developmental neurogenesis. This thesis describes my study of whether extracellular matrix regulates hippocampal neurogenesis.