The subventricular zone (SVZ) of the mammalian brain serves as one of only two sources of adult born neurons. Adult born neural progenitors - known as neuroblasts - acquire the ability to migrate and travel large distances to their destination in the olfactory bulb. Disruption of neuroblast migration is associated with learning and memory deficiencies and, following injury, neuroblasts are re-routed to promote neurodegeneration. While a lot of research has attended to augmenting the production and survival of neuroblasts, the body of evidence for pharmacological targets or compounds that promote migration is comparatively sparse. This thesis set out to identify novel strategies to modulate neuroblast migration for brain repair by studying proteins known to modulate migration and identifying new ones through compound screening. Firstly, an explant migration assay from the mouse SVZ was used to investigate the potential to use growth factors to stimulate neuroblast migration. This confirmed that that insulin-like growth factor 1 (IGF1) and IGF2 regulate neuroblast migration, as previously reported by other research groups. The role of IGF2 is investigated further using a mouse model in which the binding of IGF2 to IGF2R is disrupted, resulting in increased proliferation in the embryonic cortical SVZ, brain overgrowth and perinatal lethality. In the second half of this thesis I try to tackle one of the major bottlenecks limiting the search in for pharmaceutical interventions targeting neuroblast recruitment: the lack of high-fidelity in vitro migration assays. Drawing concepts from existing in vitro migration assays and cerebral organoid models, I developed a novel neuroblast spheroid migration assays that permits the investigation of large numbers of interventions, concurrently, in 3 dimensions. Using the spheroid assay I successfully screened 1012 small molecule kinase inhibitors for their effects on neuroblast migration. Several compounds were identified that significantly increased or decreased neuroblast migration. Two genes: MUSK and PIK3CB were selected from the screen as putative biological targets and genetic knockdown of these genes validated that interruption of their activity increased neuroblast migration. In the future these compounds could be studied further to explore their potential for augmenting the recruitments of new neurons to sites of injury so support neuroregeneration, or for decreasing invasion of brain malignancies.

O maior nicho neurogênico no encéfalo adulto está ao redor dos ventrículos laterais, mas a identificação das células que iniciam tal formação é controversa. Há uma inervação do peptídeo CART que pode abrir perspectivas para o entendimento de seu papel na modulação da neurogênese. Propormos estudar a citoarquitetura ultra-estrutural das células proliferativas na região periventricular e descrever a organização dessa região e sua inervação por axonios imunorreativos ao CART. Utilizamos ratos e camundongos adultos, preparados para análise ultraestrutural e neuroquímica em microscópios eletrônicos de transmissão e varredura de alta-resolução, de luz e laser confocal. O estudo da proliferação e inervação ocorreu com a administração do marcador de fase S BrdU e anticorpos anti-BrdU, anti-CART, anti-DCX, anti-GFAP e anti-GFP. Cada tipo celular do nicho neurogênico apresentou uma densidade própria de ir-BrdU. Identificamos células de revestimento ventricular inervadas por axônios. A maior densidade de inervação ir-CART ocorre ao longo do trajeto dos neurônios em formação. / The major neurogenic niche in adult brains surrounds the lateral ventricles, but the identity of the cell that initiates this process in controversial. There is an innervation made by the CART peptide that may lead to perspectives for understanding its role in modulation of neurogenesis. We propose to study the ultrastructural cytoarchitecture of proliferative cells in this region and its innervation by CART immunoreactive axons. We used adult rats and mice, prepared for ultrastructural and neurochemical analysis by transmission and high-resolution scanning electron, light and laser confocal microscopes. The proliferation and innervation studies occured with the S-phase marker BrdU and anti-Brdu, anti-CART, anti-DCX, anti-GFAP, anti-GFP antibodies. Each sort of cells in neurogenic niche presented a proper density of BrdU staining. We identified the cells lining the ventricle being innervated by axons. The major density of CART innervation occurs along the pathway of neurons in maturation process.

As epilepsias acometem entre 1-2% da população mundial. De um modo geral, de todas as epilepsias quase um terço deste total de pacientes apresenta a síndrome epiléptica conhecida como Epilepsia de Lobo Temporal (ELT), a qual se instala geralmente após um insulto inicial ou em decorrência de outras patologias como, por exemplo, trauma ou tumor, e parece ser decorrente de anormalidades intrínsecas do lobo temporal tais como, amígdala, hipocampo e córtex piriforme. Depois de um período de latência variado, promove o surgimento de crises convulsivas. Dentre os pacientes que apresentam ELT, cerca de 20 a 30% deles apresentam resistência ao tratamento farmacológico. Para melhor estudar os processos plásticos envolvidos no processo de epileptogênese ocorridos após a instalação do insulto inicial que levam ao aparecimento de crises recorrentes espontâneas, ratos Wistar foram eletricamente estimulados na amígdala para indução de Status Epilepticus (SE). Foram feitas histoquímicas e immunohistoquímica para marcar neurônios ativados (c-Fos+), novos neurônios (Doublecortin DCX+) e em degeneração (FluoroJade C - FJC+) após as crises. Após a indução do SE observamos que quanto mais graves as crises, maior o número de áreas ativadas (c-Fos+) e maior número de neurônios em degeneração (FJC+). Além disso, não houve associação direta entre as áreas cerebrais ativadas e grau de neurodegeneração, nem associação entre gravidade do SE e intensidade de neurogênese (DCX). A segunda fase deste projeto, executada na University of Cincinnati, refere-se ao estudo do impacto do SE, induzido por pilocarpina (PILO) sistêmica, sobre a neurogênese hipocampal. Utilizando a injeção de BrdU, para marcar o dia do nascimento de novos neurônios granulares, em camundongos Thy1-GFP foram submetidos ao SE por PILO. Foram analisadas a plasticidade dendrítica de neurônios granulares em fase de maturação (imaturas, 1 semana) e maduras (8 semanas). As células imaturas sofreram drásticas modificações na sua morfologia e na densidade dendrítica. Por outro lado, as células maturas não sofreram alterações morfológicas na árvore dendrítica, mas apresentaram uma intensa redução na densidade dos espinhos dendríticos, mostrando assim que as células imaturas estão mais suceptíveis ao impacto das crises epilépticas. / The epilepsies affect between 1-2% of the world. In general, all epilepsies almost a third of total patients had an epilepsy syndrome known as temporal lobe epilepsy (TLE), which usually settles after the initial insult or due to other pathologies such as, for example, trauma or tumor, and seems to be due to intrinsic abnormalities such as temporal lobe, amygdala, hippocampus and piriform cortex. After latency period varied, promotes the emergence of seizures. Among the patients with TLE, about 20 to 30% of them are resistant to pharmacological treatment. To better study the processes involved in plastic epileptogenesis occurred after the installation of the initial insult leading to the appearance of spontaneous recurrent seizures, rats were electrically stimulated in the amygdala to induce status epilepticus (SE). Histochemical and immunohistochemistry were done to mark neurons activated (c-Fos +), newborn neurons (Doublecortin - DCX+) and degenerating (FluoroJade C - FJC+) after the crisis. After SE induction observed that the more serious crises, the greater the number of activated areas (c-Fos+) and greater number of degenerating neurons (FJC+). In addition, there was no direct association between the brain areas activated and the degree of neurodegeneration, or association between the severity and intensity of the SE of neurogenesis (DCX+). The second phase of this project, performed at the University of Cincinnati, refers to study the impact of SE induced by pilocarpine (Pilo) system on hippocampal neurogenesis. Using the injection of BrdU, to label the daybirth of new granule neurons in Thy1-GFP mice subjected to SE. We analyzed the dendritic plasticity of granule neurons undergoing maturation (immature, 1 week) and mature (8 weeks). The immature cells have undergone drastic changes in their dendritic morphology and density. On the other hand, the mature cells did not undergo morphological changes in dendritic tree but showed a marked decrease in the density of dendritic spines, thus showing that immature cells are more susceptible to the impact of epileptic seizures.

The discovery of stem and progenitor cells capable of ongoing neurogenesis in the adult mammalian brain has raised hope that we will one day be able to harness their intrinsic regenerative capacity following injury. Development of such therapeutic strategies relies on a comprehensive understanding of the underlying regulation of the neurogenic process. To this end, I show, in this thesis, that cultured post-natal hippocampal neural progenitor cells (NPCs) express a specific repertoire of connexins (Cx), a family of channel forming proteins critical for communication prior to the development of functional chemical synapses. I show that this pattern of Cx expression, specifically Cx43 and Cx45, is modulated by interaction with the extracellular matrix component laminin providing evidence of extracellular matrix-cell interaction in the regulation of intrinsic Cx expression and function in postnatal NPCs. In adult brain, I show, for the first time, that Cx45 localizes to all cell types of the neuronal lineage with the exception of the type 3 doublecortin (DCX)-positive NPCs. Using a loss of function approach, I show that this expression is required for the normal proliferation of type 1 nestin and glial fibrillary acidic protein-positive stem like NPCs but not for the differentiation or survival of their progeny in the adult hippocampus. With respect to exogenous pharmacological cues that influence hippocampal neurogenesis, this thesis also demonstrates that chronic treatment with a sub-set of selective serotonin reuptake inhibitor antidepressants, fluoxetine and escitalopram, increases the proliferation but not the survival of adult NPCs in healthy, non-depressed mice. Further, standard post-operative analgesia with the opiate buprenorphine inhibits the proliferation of DCX-positive adult NPCs and increases the survival of their progeny. Finally, over the course of the research for this thesis, it became clear that exposing research animals to even very subtle environmental changes can influence the basal neurogenic process. Ultimately this work further highlights the exquisite sensitivity of the regulation of what is already recognized to be a highly dynamic process and provides important insight into the neurogenic process that can be used to inform future therapeutic development and application.

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

The recent discovery of adult neural precursor cells (NPCs) in the dentate gyrus and the subventricular zone of the lateral ventricles of most mammals holds much hope for the potential regeneration of damaged brain tissue. However, their use has been limited by their low numbers and relatively quiescent state, particularly in the aging brain. Previous studies from our laboratory have demonstrated a crucial role for the Rb/E2F pathway in the regulation and proliferation of NPCs, and the direct mechanistic involvement of E2F3 in regulating the pluripotency factor, Sox2. More recently, our investigations into the roles of E2F1 and E2F3 in during adult neurogenesis have revealed that loss of both these genes results in a dramatic loss of adult NPCs. Here, we have employed the Emx1-Cre and Nestin-CreERT2 transgenic models, to specifically delete E2F1 and E2F3 in the cerebral cortex and in NPCs in order to investigate the role of both these genes in embryonic neurogenesis. Our results suggest a switch in the requirement for both E2Fs 1 and 3 between embryonic and adult NPCs, demonstrated by a decrease in NPC proliferation and numbers starting only during late embryonic development and persisting through postnatal neurogenesis. These findings suggest that E2Fs 1 and 3 are essential for the maintenance of stem cells and neurogenesis in the adult brain. Moreover, their deletion results in defects in learning and memory. These studies reveal a crucial role for activating E2Fs in the long-term maintenance and proliferation of neural stem cells.

Many animals, including zebrafish (Danio rerio), form social hierarchies as a result of competition for limited resources. Socially subordinate fish experience chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis, leading to prolonged elevation of plasma cortisol, the glucocorticoid end-product of HPI axis activation. Elevated cortisol levels can reduce cellular proliferation and neurogenesis in the brain. Thus, the present study tested the hypothesis that social stress suppresses cellular proliferation in the brain of subordinate zebrafish via a cortisol-mediated mechanism. Cellular proliferation was assessed using the incorporation of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue, as a marker. After 48 and 96 h of social interaction, significantly lower numbers of BrdU-positive cells were present in the forebrain of subordinate male zebrafish compared to dominant or control fish, suggesting a suppression of cellular proliferation in fish experiencing chronic social stress. Treatment of interacting male zebrafish with metyrapone, a cortisol synthesis inhibitor, attenuated the suppression of cellular proliferation in subordinate fish. Subordinate female zebrafish did not experience elevation of plasma cortisol or suppression of cellular proliferation in the forebrain. Collectively, these data provide evidence that cortisol plays a role in regulating cellular proliferation in the forebrain of male zebrafish during social interactions.

The goal of this study is to find an optimal dose of cytostatic Temozolomide (TMZ) for Long-Evans strain of rats. This dose should reduce neurogeneis while having a minimal pernicious health side-effects. Temozolomide is newly used to suppress neurogenesis but similarly to any other cytostatic has an effect on all dividing cells in an organism. This can affect health of an animal. Contrary to the mice, there was no systematic attempt to establish optimal dose. In our experiment rats were divided into four groups - one control group and three treatment groups which received different doses of temozolomide (10, 25, 40 mg/kg of TMZ). To detect level of neurogenesis cells were labeled by bromodeoxyuridine. During the experiment blood element counts were assessed, sensorimotoric tests were conducted, and weight increment was monitored. The results indicate that dose of 10mg/kg is adequate as it reduces neurogenesis by 64% compared to the control group and does not significantly differ from higher doses. In this group weight increment is comparable with the control group, while in the higher doses of TMZ weight increment is significantly lower. Effect of myelosupression is same for all treatment groups.

No description available.

Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK–JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF−/− mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF−/− littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK–JNK–CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.