Nous avons identifié des inhibiteurs pharmacologiques de REST capables d’augmenter l’expression d’un ensemble de gènes cibles de REST (gènes RE1) neuronaux dans des cellules souches neurales (NSC) issues de cellules souches embryonnaires humaines (HESC). De tels composés ont pour intérêt de constituer un nouveau type d’outil pour étudier la fonction de REST dans la prolifération et la différenciation des NSC normales ou pathologiques et pourraient posséder des propriétés thérapeutiques dans les maladies ou une sur-activation de REST participe ou marque la pathologie cellulaire telles que la maladie de Huntington ou certaines tumeurs du cerveau. L’identification des inhibiteurs de REST a été réalisée grâce à la technologie puissante du criblage à haut débit (HTS). Le succès de cette méthode a reposé sur l’élaboration d’un test cellulaire fonctionnel robuste de l’activité de REST dans les NSC. Un système rapporteur de cette activité a été construit autour d’une cassette d’expression de la Luciferase Renilla placée sous le contrôle d’un promoteur constitutif fort. Plusieurs sites RE1 ont été insérés en amont de cette cassette afin de rendre l’expression de la Luciferase dépendante de l’activité de REST. Nous avons ainsi isolé le compose  x5050, un benzimidazole qui entraîne, comme montre par l’étude transcriptomique, la surexpression spécifique des gènes RE1 neuronaux. x5050 ne modifie ni la transcription de REST ni la fixation de REST sur une séquence oligonucléotidique RE1 marquée. En revanche, x5050 entraîne la diminution du niveau de la protéine REST, vraisemblablement en modulant la dégradation de REST par le système ubiquitine-protéasome. / Our goal was to identify pharmacological inhibitors of REST that would be able to increase the expression of a set of neuronal gene targets of REST (RE1 genes) in human neural stem cells (NSCS) derived from human embryonic stem cells (HESC). These compounds would at first provide a new type of tool to better understand REST action on proliferation and differentiation in normal or pathological NSCS and could have therapeutical properties for diseases in which an over-activation of REST is implicated in or influences cellular pathology such as huntington’s disease or some brain tumors. Identification of REST inhibitors was performed using the powerful technology of high throughput screening (HTS). Success of this method was based on the set up of a robust functional cell assay of REST activity in NSCS. A reporter system of this activity has been constructed using an expression cassette of the renilla luciferase placed under control of a strong constitutive promoter. Several RE1 sites have been inserted upstream of this cassette to make the expression of Luciferase dependent on REST activity. We have isolated x5050 compound, a benzimidazole which leads to upregulation of RE1 genes as shown by transcriptomic studies. x5050 modified neither rest transcription nor rest fixation on a labeled nucleotidic RE1 sequence. On the contrary, x5050 treatment induced the decrease in rest protein level, probably by modulating REST degradation by the ubiquitin-proteasome system.

Polymerase gamma POL G1 mutator mice (POL G) are deficient in the mitochondrial DNA proof-reading capacity leading to an accumulation of mtDNA point mutations, resulting in accelerated aging phenotype and brain atrophy. Endurance exercise training reverses the phenotypic manifestations and rescues much of the progeroid aging phenotype, including brain atrophy. Neurogenesis is mediated by neurotrophins that stimulate cell growth and survival. One of the main neurotrophins is brain-derived neurotrophic factor (BDNF), which is secreted by muscle cells and has been shown to increase with acute exercise in brain and serum. Therefore, we investigated whether continuous delivery of BDNF by in-vivo gene therapy would improve the neurogenesis on the dentate gyrus in POL G mutator mice. Wild-type controls and POL G mutator mice were given intra-peritoneal injections of capsules containing recombinant G8 myoblasts that secreted BDNF, or vehicle (veh), over five months. Cell survival analysis at the level of the dentate gyrus in the brain was measured by BrdU analysis. By nine months of age, BDNF-injected POL G mutator mice did not exhibit improvements in neurogenesis in comparison with POL G controls. Motor assessment through rotarod performance showed no differences between wild type and POL G. CLAMS assessment demonstrated impairment of locomotor activity in POL G mice as expected; and no improvement in the POL G group treated with BDNF. Unexpectedly, wild type animals treated with BDNF exhibited decreased levels of locomotor activity similar to the POL G mutator mice. In conclusion, continuous BDNF administration did not improve neurogenesis at the level of the dentate gyrus in the POL G animal model. It is likely that the prevention of brain atrophy seen with endurance exercise is mediated by additional molecular factors, including BDNF. / Thesis / Master of Health Sciences (MSc) / Polymerase gamma POL G1 mutator mice (POL G) are deficient in the mitochondrial DNA proof-reading capacity leading to an accumulation of mtDNA point mutations, resulting in accelerated aging phenotype and brain atrophy. Endurance exercise training reverses the phenotypic manifestations and rescues much of the progeroid aging phenotype, including brain atrophy. Neurogenesis is mediated by neurotrophins that stimulate cell growth and survival. One of the main neurotrophins is brain-derived neurotrophic factor (BDNF), which is secreted by muscle cells and has been shown to increase with acute exercise in brain and serum. Therefore, we investigated whether continuous delivery of BDNF by in-vivo gene therapy would improve the neurogenesis on the dentate gyrus in POL G mutator mice. Wild-type controls and POL G mutator mice were given intra-peritoneal injections of capsules containing recombinant G8 myoblasts that secreted BDNF, or vehicle (veh), over five months. Cell survival analysis at the level of the dentate gyrus in the brain was measured by BrdU analysis. By nine months of age, BDNF-injected POL G mutator mice did not exhibit improvements in neurogenesis in comparison with POL G controls. Motor assessment through rotarod performance showed no differences between wild type and POL G. CLAMS assessment demonstrated impairment of locomotor activity in POL G mice as expected; and no improvement in the POL G group treated with BDNF. Unexpectedly, wild type animals treated with BDNF exhibited decreased levels of locomotor activity similar to the POL G mutator mice. In conclusion, continuous BDNF administration did not improve neurogenesis at the level of the dentate gyrus in the POL G animal model. It is likely that the prevention of brain atrophy seen with endurance exercise is mediated by additional molecular factors, including BDNF.

Concussion/mild traumatic brain injury (mTBI) is a significant public health concern, particularly for young individuals and athletes. While the vast majority recover quickly and without lasting consequences, some will suffer from potentially long-term neuropsychiatric sequelae. These sequelae have been investigated in mTBI samples consisting largely of motor vehicle-related injuries, but very few have examined these following sport-related concussion (SRC). Further, new evidence indicates that participation in contact sports alone can result in similar problems with cognition and mood/anxiety. This thesis investigates neuropsychiatric sequelae in youth and young adults with concussion and who participate in contact sports. The hippocampus is known to be vulnerable to head injury, and animal models indicate that mTBI impairs hippocampal neurogenesis — the process of the growth, maturation, and integration of adult-born neurons. Hippocampal neurogenesis is well-recognized for its importance to cognition, and has more recently been linked to mood and anxiety. Accumulating evidence indicates that a test involving a component of high memory interference, the mnemonic similarity test (MST), is sensitive to neurogenesis-dependent conditions in humans. To the best of our knowledge, this thesis is the first to investigate whether the MST is sensitive to concussion, contact sport participation, and athletic anxiety. This thesis describes: (1) a critical review of the literature regarding the psychiatric sequelae of concussion, followed by (2) clinical profiles derived from retrospective data which document the new onset of immediate psychiatric problems in youth, and delayed psychiatric problems in youth with persistent concussion symptomology; that (3) anxiety is the most common and persistent psychiatric symptom in youth with concussion; that (4) concussion and contact sport participation negatively affect performance on the MST; that (5) a reprieve from contact sport participation is associated with an improvement in MST performance; that (6) these negative effects on the MST are not seen with other injuries; that (7) athletic anxiety impairs performance on the MST. Taken together, this thesis provides evidence that psychiatric problems can present immediately after concussion in persons without identifiable risk factors, and that concussion, contact sport participation, and athletic anxiety affect performance on a putatively neurogenesis-linked test. These results may implicate impaired hippocampal neurogenesis in postconcussion sequelae. / Thesis / Doctor of Philosophy (PhD)

A fundamental characteristic of neural stem cells (NSCs) is their ability to divide symmetrically, producing daughter cells of different fates. This requires the regulation of proliferation verses differentiation, which could occur in two different ways; at a cellular or environmental level. Although there is some evidence for each model, little is known about how this regulation occurs. One candidate is the integrin family. Integrins are known to regulate key aspects of stem cell behaviour. The integrin subunits α6 and β1, which heterodimerise into a laminin receptor, are highly expressed within the NSCs of the embryonic ventricular zone and loss of integrin β1 (itgβ1) function within the ventricular zone of the embryonic mouse results in NSC detachment and apoptosis. The aim of this thesis is to investigate the role of itgβ1 in the regulation of proliferation verses differentiation in the NSC of the chick embryonic neuroepithelium. To test the function of itgβ1 within this system, we are using transfection of a constitutively active (CA*), wild type (WT) and extracellular portion only (EC only) integrin-β1, via electroporation of the chick embryonic CNS, resulting in a patchwork of expression within the NSCs of the midbrain neuroepithelium. This system allows both expressing cells and their non-expressing neighbours to be studied within the same environment. We predicted that if integrins are acting to regulate NSC behaviour via an intrinsic mechanism, only the cells expressing the CA* integrin will alter their behaviour. If the second model is correct and integrins act via an extrinsic mechanism, we predicted that the neighbouring non-expressing cells will also alter their behaviour. We observed a significant increase in the number of neurons generated upon expression of CA*β1. This increase in neurons was a non-cell autonomous effect; the neurons were GFP and human itgβ1 negative, supporting the second model of extrinsic signals and cell-cell interactions in the regulation of proliferation and differentiation. The increase in neurogenesis was only observed in the midbrain upon CA*β1 expression for 48hrs. A significant increase in mitotic cells was observed 12hrs after electroporation, the earliest time point and by E4 (48hrs) a significant proportion of mitotic cells were abnormally located by 48hrs, resulting in basal mitoses. Investigation of signalling between cells was carried out using microarray analysis of the two populations of cells, CA*β1 positive and negative. One candidate from the microarray results was the bHLH transcription factor Tal2. Tal2 has previously been shown to be specifically expressed within the midbrain neuroepithelium at the time of electroporation and to play a role in the regulation of neurogenesis. In summary, this thesis has showed an important role of itgβ1 in the regulation of proliferation and differentiation of NSCs within the chick embryonic neuroepithelium in a non-cell autonomous manner.

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No description available.

Regenerative medicine offers tremendous potential for the treatment of irreversible damage to the brain. Activation of quiescent adult neural stem cells by clinical means to regenerate tissue can improve pathological outcomes of patients afflicted by brain trauma. Control of the cell- cycle is important in activating quiescent neural stem cells for the purpose of enhancing adult neurogenesis. Here, we uncover the role of cell-cycle regulatory transcription factors E2F1 and E2F3 in adult neural stem cell activation and characterize it. We hypothesize that the Retinoblastoma-E2F pathway is crucial for neural stem cell activation. We characterized the requirement of E2Fs1/3 for adult neural stem cells activation through a combination of multiple knockout timepoints in mice and novel markers used to identify distinct neural stem cell sub- populations. The results show a marked reduction in the neurogenic capacity of the adult brain, with common markers of proliferation and different progenitor-cell lineages decreased. Additionally, the ability of quiescent neural stem cells to transition to an active state is reduced. A whole genome-analysis of RNA isolated from E2Fs1/3-knockout adult neural stem cells has shown a shift from an active identity-state to a quiescent one. In the future, E2Fs1/3 could emerge as key regulators of quiescent stem cell activation, and thus could be potential targets for therapeutic control in order to enhance neurogenesis in patients with brain pathology.

The body and central nervous system of the Nile crocodile (Crocodylus niloticus) is known to grow continuously, even past sexual maturity. Previous studies on crocodilians show a gradual decrease in the rate of growth of the brain as individuals mature; however, the data on brain growth are limited and there are no data on growth rates of the spinal cord and retina. The current thesis provides a description of the growth rates of the brain, spinal cord and eye (ocular volume) of 70 juvenile and sub-adult Nile crocodiles ranging between 92 g and 90 kg in mass. Body growth in the crocodiles is accompanied by growth of all central nervous system structures (brain, spinal cord and eye), although growth occurs at different rates, with the spinal cord being the fastest growing structure, followed by the eye and the brain. The neurogenic regions (areas to which new neurons are continuously added) of the brain were revealed using doublecortin immunohistochemistry. Labelled cells were observed in all the regions of the telencephalon (including the cortical mantle, dorsal ventricular ridge, striatum, septal nuclei and the olfactory bulbs) as well as the molecular and granular layers of the cerebellar cortex, but not in the diencephalon or brainstem.

A Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Master of Science in Medicine. School of Anatomical Sciences, Faculty of Health Sciences University of the Witwatersrand, Johannesburg / This study investigated the presence and pattern of adult neurogenesis in the Subventricular zone (SVZ) of the lateral ventricle, the dentate gyrus (DG) of hippocampus and potential neurogenic sites in three prosimian primates. While two nocturnal species, the Galagoides demidoff phasma (Galago) and the Perodictus potto (Potto) were caught in the wild, the Lemur catta (Lemur) was a zoo kept diurnal animal. Two brain specimens from each species, perfusion-fixed with 4% paraformaldehyde were cut at 50 μm thick frozen sections in sagittal and coronal planes. Using doublecortin (DCX) and Ki-67 antibodies, immature neurons and proliferating cells were identified respectively in the SVZ and DG and in potential sites such as the striatum, corpus callosum, amygdala, and piriform cortex in all the three species. DCX positive cells were observed in the cerebellum of the Lemur and the Galago but not in the Potto. There were no Ki-67 proliferating cells observed in the cerebellum and the neocortex of all the three species. Interspecies analysis indicated that the estimated rate of Ki-67 proliferating cells in Potto was 1.9 times higher than that of the Lemur and 4.8 times higher than that of the Galago. There was no statistical significant variation in the number of estimated Ki-67 cells within the three species but a significant difference (P ≤ 0.05) when comparing Potto with the Lemur and Galago. There was no significant difference (P ≥ 0.05) in the number of Ki-67 cells between the Lemur and the Galago. Variations do exist in the cell proliferation pattern among these three prosimian primates.

Adult neurogenesis is a conserved feature of the central nervous system across the animal kingdom. This process takes place in restricted neurogenic niches of the brain, where active populations of adult stem/progenitor cells are capable of producing newborn neurons. The niche is tightly controlled by intrinsic signals within the microenvironment and from stimuli arising from the external world, which together determine the cellular behaviour of the niche and neuronal output. Currently, our understanding of the biological properties of adult neurogenesis rests mainly on two niches of the vertebrate forebrain. To broaden our view of the diversity of this trait comparative models and new niches must be explored. Here, I have taken advantage of the robust neurogenic capacity of the adult zebrafish brain to examine differences in forebrain and sensory neurogenic niches in regards to cytoarchitectural organization, neurogenic plasticity, and regulation. Five principle findings emerge: (1) up to six morphologically distinct cell types compose forebrain and sensory niches, and are devoid of ependymal cells; (2) heterogeniety in the phenotype of the stem/progenitor cell exists across niches; some having radial glial characteristics; (3) active populations of proliferating stem/progenitor cells reside within primary sensory structures of the adult brain, forming a “sensory neurogenic niche”; different from other models of adult neurogenesis; (4) changes in the social environment induce neurogenic plasticity in sensory niches more readily than integrative niches of the forebrain, and occur independently of cortisol levels; (5) modality-specific stimulation influences stages of adult neurogenesis exclusively in corresponding primary sensory niches as a result of sensory-dependent neurogenic plasticity. Additionally, I have shown that Fibroblast Growth Factor signalling may not be involved in maintaining cell proliferation in sensory niches. These studies showcase the diverse properties of forebrain and sensory neurogenic niches and provide a new perspective concerning the functional role of adult neurogenesis.