2.5 million people sustain a traumatic brain injury (TBI) annually in the United States. Although there is potential for functional recovery following TBI, there is no definitive treatment to improve recovery after TBI. Our lab has shown that TBI enhances an endogenous neurogenic response in the subventricular zone and hippocampus. TBI-induced neural stem cells (NSCs) can integrate into regions such as the hippocampus and olfactory bulb. Although the mechanism behind TBI-enhanced neurogenesis remains unknown, the Notch signaling pathway has been implicated as a regulator in the maintenance and survival of NSCs. This thesis explores the effects of Notch pathway manipulation on functional recovery following TBI. We hypothesize that Notch signaling plays a critical role in recovery after TBI. Activation of this pathway via a Notch agonist (Notch1) will facilitate post-injury recovery while inhibition of this pathway via a Notch antagonist (recombinant Jagged-1 Fc) will deter post-injury recovery. Functional recovery was assessed within 30 days or 60 days post-injury in two different cohorts of animals. The behavior assays conducted in this study included motor, cognitive, and olfactory assessment. In the 30-day phase, Notch pathway manipulation following TBI did not affect functional performance. In the 60-day study, significant group differences were found. While the FPI+Vehicle animals exhibited a functional recovery in Morris water maze, injured animals with Notch inhibition failed to show this cognitive recovery, indicating the involvement of the Notch pathway in cognitive recovery at the chronic stage following TBI. Motor and olfaction were not significantly affected by Notch pathway manipulation.

Les protéines Vestigial-like constituent une famille de cofacteurs de transcription contenant un domaine très conservé, appelé Tondu, qui permet l’interaction avec les facteurs de transcription de la famille TEAD. L’état de l’art des connaissances actuelles sur cette famille, en termes de répertoire, de structure et de fonction des gènes dans les différents groupes d’animaux, a fait l’objet d’une revue. Durant la thèse, a été étudiée la fonction de deux gènes vestigial, vestigial-like 3 et vestigial-like 4, dans le modèle amphibien xénope. Ce choix découle d’une part, des travaux antérieurs de notre laboratoire qui a caractérisé la famille des gènes vestigial chez le xénope et d’autre part des avantages de ce modèle expérimental qui permet les analyses cellulaires et moléculaires. Les approches de gain et perte de fonction indiquent que vestigial-like 3 est plus particulièrement impliqué dans la migration des cellules de la crête neurale. Vestigial-like 4 a un rôle dans la neurogenèse précoce et la formation de la crête neurale. / Vestigial-like proteins belong to a transcription co factors family with a conserved domain, called tondu, which allows their interaction with TEAD family transcription factors. The state of the art on the current knowledge about this family in terms of gene repertory, structure and functions in different animals has given rise to a review. PhD work has focused on vestigial-like 3 and vestigial-like 4 genes functions in the Xenopus amphibian. This choice stemmed from the laboratory previous works that has described vestigial like gene family in Xenopus, and from the Xenopus model advantages that allows cellular and molecular analysis. Gain and loss of function approaches indicate that vestigial-like 3 is especially implicated in neural crest cells migration. Vestigial-like 4 plays a role in early neurogenesis and neural crest formation.

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.

<p>Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA.</p><p>We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times.</p><p>We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells.</p><p>The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.</p>

Horizontal cells are retinal interneurons that modulate the output from photoreceptors. Two horizontal cell (HC) subtypes are commonly identified in the vertebrate retina: axon-bearing and axon-less HCs. In this work, we have identified Isl1 as a novel HC marker and demonstrated that Lim1 and Isl1 distinguish axon-bearing and axon-less HCs, respectively. In the chick retina, axon-less HCs are furthermore split into two different subtypes based on the expression of GABA and TrkA. We have demonstrated that during early chick retinogenesis, HCs expressing either Lim1 or Isl1 are generated consecutively as two equally large sub-groups at different time points. Moreover, these newborn HCs undertake an unexpected bi-directional migration before settling in their final laminar position. Different HC subtypes complete this migration at different times. We investigated the role of activin signaling during HC subtype generation. Activin or its inhibitor follistatin was administrated during the main phase of HC generation and analyzed when HCs had completed migration. Activin caused a significant decrease in both HC subtypes and decreased the proliferation of retinal precursor cells. Follistatin increased the number of late born (Isl1+) HCs, which migrated to the HC-layer during a prolonged migration period. Both treatments affected retinal histology, but only activin influenced the generation of retinal populations other than HCs. These effects were most likely mediated by altered proliferation in certain retinal precursor cells. The data on HC subtype ratios, birth-dates, migration, apoptosis and extrinsic activin modulation favor a scenario where the mature proportions of HC subtypes are generated sequentially from a specific HC-precursor cell lineage early in development and remain stable thereafter. These proportions are not adjusted by apoptosis, but rather by the combined actions of transcription factors and extrinsic signaling. Our studies on HC subtypes and their development promises to facilitate future studies on HC development, evolution and function.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. Fast activation of microglia in response to neuronal damage requires the rapid availability of a large amount of energy to trigger diverse cytotoxic or neuroprotective signals. ATP-dependent potassium (K(ATP)) channels play important roles in many cellular functions by coupling cell metabolism to electrical activity. K(ATP) channels were first detected in cardiac myocytes and later found in beta-cells of the pancreas, skeletal muscle, neurons, smooth muscle, heart, pituitary, and tubular cells of the kidney. Our group and others have also demonstrated its expression in reactive microglia after brain injury. In rat models of stroke, blockade of the sulfonylurea receptor (SUR), with glibenclamide (Gbc) reduced cerebral edema and infarct volume. Furthermore, clinical data suggest the effectiveness of Gbc to treat stroke. Gbc close the K(ATP) channel by interaction with two drug-binding sites on SUR subunits, as well as, the astroglial NC(Ca-ATP) channel, which mediates the Gbc-induced prevention of edema after cerebral ischemia. In these studies however, the function of the K(ATP) channel remained unclear. Therefore, as Gbc may bind to constitute functional K(ATP) channels after ischemic stroke, other possible effects of Gbc might explain the effectiveness of this drug in the treatment of stroke. Giving the fact that, SUR1-regulated channels are exquisitely sensitive to changes in the metabolic state of the cell, and that microglia are sensing the environment, the expression of K(ATP) channels in activated microglia, will couple cell energy to membrane potential. We herein postulate, that the effectiveness of Gbc to treat stoke, at least in part, is caused by the KATP channel closure expressed by activated microglia, which may then be critical in determining, their participation in the pathogenic process. Given the analogy with beta-cells, K(ATP) channel blockade in microglia would response faster and more efficiently to the external signals released after brain injury. If true, blockade of microglial K(ATP) channel with low doses of Gbc during the early stages of stroke might foster neuroprotective microglial activity, could enhance ischemia-induced neurogenesis in the SVZ, and consequently will lead to an improved functional outcome. The work presented in this thesis demonstrates that, Gbc improves functional neurological outcome in stroke, accompanied by neuron preservation in the core of the ischemic brain. In this region, reactive microglia from tMCAO rats upregulate the K(ATP) channel, which makes microglia a target to Gbc actions in the early stages of stroke. Furthermore, Gbc also strengthens the neuroprotective role of microglia in the acute phase after focal cerebral ischemia, enhance long-term neurogenesis and brain repair processes. As such, identify microglial K(ATP) channels as a key target for stroke treatment. Overall, these results provide new therapeutic avenues for the treatment of other neurological disorders that involve microglia.