Epilepsy is a neurological disorder that causes recurrent and unprovoked seizures due to imbalances in synaptic transmission in distinct regions of the brain. In both human patients and animal models of epilepsy, there is a marked increase in brain-derived neurotrophic factor (BDNF), a critical signaling molecule in the brain that contributes to two divergent pathways important to disease pathology: 1) the regulation of type A receptors for the major inhibitory neurotransmitter GABA (GABAARs), and 2) aberrant neurogenesis with ectopic expression of new neurons from progenitor cells that disrupt neural network activity in the hippocampus. The first part of my thesis addresses how neurons regulate levels of α1-containing GABAARs through BDNF signaling at its receptors, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR). I hypothesized and showed that BDNF, working at TrkB, rapidly activates the Janus kinase and signal transducers and activators of transcription (JAK/STAT) pathway in neurons and identified a novel intracellular receptor signaling complex composed of p75NTR and JAK2 that is present in neuronal processes, cell body, and nucleus. Based on this finding, we suggest that an intracellular p75NTR/JAK2 signalsome recruits STAT3, a transcriptional activator of the gene coding for the cAMP inducible early repressor (ICER) that blocks synthesis of α1 subunits reducing synaptic GABAARs in response to status epilepticus. This model is consistent with our collaborative studies that show a JAK2 inhibitor, WP1066, inhibits development of spontaneous seizures in an epilepsy model and my observation that WP1066 degrades JAK2 protein in primary neurons. The second part of my thesis addresses BDNF regulation of the Late SV40 Factor (LSF), a ubiquitous transcription factor that regulates cell cycle progression and survival. I show that BDNF through the mitogen-activated protein kinase pathway selectively phosphorylates LSF at serine 291 (p291LSF) and that p291LSF is present throughout neurogenesis, increases with status epilepticus in the hippocampus, and is highest in structures associated with neurogenesis (such as olfactory bulb and hippocampus when compared to cortex). Taken together, these results suggest LSF may play an important role in neuronal development and potentially in epilepsy, providing an additional target for future therapeutic intervention. / 2016-12-15T00:00:00Z

Hippocampal neurogenesis is linked to the onset, progression and remission of major mood disorder such as anxiety and depression. Neurogenesis is the process by which new neurons are formed in the brain. Mitochondria mediate cellular adaption and provide energy to support growth of new neurons. Chronic stress and mood disorders have been associated with impairments in mitochondrial function and neuronal growth. Individuals experiencing stress and mood disorders reportedly use cannabis as a means to self-medicate. The impacts of cannabis on stress-related effects on hippocampal neurogenesis and mitochondria are vastly unexplored. To investigate these effects we generated an in vitro model of hippocampal neuron stress by treating HT22 cells with corticosterone, the major effector molecule of stress in rodents. We first characterized the impacts of corticosterone on markers of neurogenesis and mitochondrial function in HT22 hippocampal cells. We found that corticosterone decreased gene markers of neurogenesis, mitochondrial biogenesis, content, dynamics and decreased mitochondrial membrane potential. Corticosterone also decreased levels of antioxidant enzymes but did not alter levels of reactive oxygen species (ROS) or elicit lipid peroxidation. We then investigated with potential impacts of cannabis components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on corticosterone-induced stress. Individually, THC and CBD decreased markers of neurogenesis, dysregulated mitochondrial dynamics and decreased mitochondrial membrane potential. Interestingly, both THC and CBD increased a marker of mitochondrial biogenesis. Finally, we co-treated HT22 cells with corticosterone and THC or CBD to interrogate the impacts of THC and CBD on corticosterone-induced alterations. Our results indicated THC and CBD had no effect on corticosterone-related reductions in neurogenesis markers or mitochondrial membrane potential. However, THC demonstrated a rescuing effect on a marker of mitochondrial biogenesis and CBD normalized a marker of mitochondrial fission; both of which were decreased with individual corticosterone treatments. This thesis ultimately identifies some of the pathways THC and CBD may impact stress response in relation to neurogenesis and mitochondria. / Thesis / Master of Science (MSc) / Neurogenesis is a process that describes the production of new nerve cells in the brain. It mainly occurs during early life, but persists in a central brain structure responsible for learning and memory, known as the hippocampus, throughout our lives. This active brain structure relies on the function of certain organelles called mitochondria, which are the primary cellular energy producers and promote nerve cell production. Mood disorders, such as anxiety and depression, may result as a consequence of impaired hippocampal neurogenesis. Evidently, people suffering from anxiety and depression turn to cannabis use for management and treatment of their mood disorders. Considering cannabis has been shown to affect neurogenesis and mitochondrial function, our primary objective was to explore its effects on hippocampal neurogenesis by focusing on mitochondrial function, in the context of stress. We demonstrate that components found in cannabis, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), alter the stress-induced changes in mitochondrial functions related to neurogenesis, suggesting that cannabis may play a role in protecting nerve cells.

Pythons are a well-studied model of postprandial physiological plasticity. Consuming a meal has been shown by past work to evoke a suite of physiological changes in pythons and elicit one of the largest documented increases in post-feeding metabolic rates relative to resting values. However, little is known about how this plasticity manifests in the brains of ball pythons, Python regius. Previous work using the cell-birth marker 5-bromo-12’-deoxyuridine (BrdU) has shown that cell proliferation in the python brain increases six days following meal consumption. This study aimed to confirm these findings and build on them in the long term by tracking the survival and maturation of these newly created cells across a two-month period. We investigated whether these cells differentiated into neurons using double-immunofluorescence for BrdU and a reptile-specific neuronal marker (Fox3). We did not find significantly greater rates of cell proliferation in snakes six days after feeding, but we did observe more newly created cells in neurogenic regions in fed snakes two months after the meal. Feeding did not influence neurogenesis, but feeding does appear to have a neuroprotective effect. More newly created cells survived in fed snakes two months later, particularly in the olfactory bulbs and lateral cortex. These findings shed light on the extent of postprandial plasticity and regional differences in the creation of new neural cells in the brains of ball pythons.

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Methylphenidate (MPH) is the most commonly prescribed drug to treat Attention Deficit Hyperactivity Disorder (ADHD). ADHD is now considered a life-long disorder; therefore, patients take MPH from adolescence into adulthood, highlighting the need for research studying chronic MPH use. MPH increases dopamine and norepinephrine within the synaptic cleft; therefore, chronic use of MPH may lead to changes within important dopaminergic pathways. One pathway, the mesolimbic pathway, includes the hippocampus, an area where adult neurogenesis occurs. We investigated the effects of chronic low and high doses of MPH on neurogenesis and examined levels of a few key proteins linked to cell proliferation in the hippocampus. Low dose MPH appears to increase cell proliferation and cell survival in the hippocampus, and these effects are accompanied by increases in vascular endothelial growth factor (VEGF), the receptor for brain-derived neurotrophic factor (TrkB), and beta-catenin. While high dose MPH may initially increase neuronal proliferation, newly-generated neurons are unable to survive long-term, and decreases in VEGF, TrkB, and beta-catenin are observed with chronic high dose MPH. Another major dopaminergic pathway is the nigrostriatal pathway, which is involved in motor control and degenerates with Parkinson’s disease. Chronic use of MPH appears to sensitize dopaminergic neurons within this pathway to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but the cause of this sensitization is unknown. The autooxidation of excess dopamine forms dopamine-quinones that lead to free radical production, but the antioxidant, glutathione, can protect neurons. However, we showed that chronic MPH increases dopamine-quinone formation and causes a subsequent glutathione depletion within the striatum. Therefore, oxidative stress may sensitize dopamine neurons to MPTP. We also assessed the vulnerability of dopaminergic neurons in the nigrostriatal pathway to MPTP after chronic MPH in females. Interestingly, proestrus (high estrogen) females were more sensitive to MPTP than anestrus (low estrogen) females. Similar to males, chronic MPH caused a depletion in glutathione that was further decreased following MPTP exposure. However, chronic MPH did not significantly alter dopaminergic neuronal numbers or quinone formation in females. These studies highlight some of the potential effects of chronic MPH use.

Within the adult mammalian brain, Neural Stem Cell (NSC)s are maintained in distinct neurogenic niches in a mostly quiescent state. Activation of quiescent NSCs first requires re-entry into the cell cycle in order for the pool to proliferate and eventually commit to a neural fate, giving rise to newborn neurons. The canonical Retinoblastoma (Rb)-E2 Promoter Binding Factor (E2f) pathway is not only key in overcoming the Gap 1 Phase (G1)/S-phase restriction, but novelly appears to be involved in adult neurogenesis and NSC activation. I hypothesized that activator transcription factors E2 Promoter Binding Factor 1 (E2f1) and E2 Promoter Binding Factor 3 (E2f3) are crucial for exit from a quiescent state in adult NSCs. The contribution of the activator E2fs in this transition was studied using a Nestin-driven Cre Recombinase-Estrogen Receptor Tamoxifen-2 Ligand Binding Domain (Cre-ERT2) system to induce targeted deletion of E2f1/3 within NSCs in adult mice. We show that loss of E2f1/3 causes significant neurogenic defects, including pro-neural activation and decreased pools of adult NSCs, that preferentially adopt a quiescent profile in the subventricular zone. We employed this model to further isolate subventricular zone-derived NSCs using a Rosa26:Yellow Fluorescent Protein (YFP) reporter and subsequently analysed transcriptional profiles by RNA sequencing. Loss of E2f1/3 shifts NSC transcriptomes towards one overlapping with quiescent neural stem cell signatures (Codega et al., 2014; Basak et al., 2018), further highlighting the requirement of these E2fs for initial activation. A significant portion of these differentially expressed genes are putative E2f targets. Transcriptionally, major pathways involving cell metabolism, cellular signaling, and neural development are perturbed without activator E2f expression. In effect, this combined approach based on in vivo data and bioinformatics analyses offers a method of prospective identification of novel regulators of adult neurogenesis that require the activator E2fs. Preliminary data suggests that AT-Hook Transcription Factor (Akna) is one such target worth pursuing. Cumulatively, this project describes a unique role for E2f1 and E2f3 during NSC exit from quiescence and subsequent activation towards differentiation. As ongoing maintenance of quiescent NSCs is a necessary prerequisite for lifelong neurogenesis, conclusions from this study could determine the therapeutic potential of targeting activator E2fs to combat the niche exhaustion associated with aging, injury, and neurodegenerative diseases.

The mammalian hippocampus is responsible for many crucial brain functions such as learning, memory, and neurogenesis in adults. Its degeneration is a pathology associated with the early stages of Alzheimer’s disease. A variety of genes have been associated with both neuroprotection and neurogenesis in the brain, some of which include progesterone membrane component 1 (Pgrmc1) and kit ligand (KitL). Pgrmc1 is recognized for mediating hormonal functions in both the ovary and neuroendocrine regions such as the anteroventral periventricular nucleus (AVPV), but its functions in the hippocampus are not well known. Both Pgrmc1 and KitL share downstream targets, the most strongly supported being genes in the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway. I hypothesized that Pgrmc1 regulates neural targets through KitL/c-Kit signaling. To investigate this hypothesis I used a variety of in vivo and in vitro techniques. These techniques included mapping both KitL and receptor c-Kit in the adult female rat brain using in situ hybridization. I used Pgrmc1 silencing with siRNA in hippocampal-derived mHe-18 cells and Pgrmc1/2 double conditional knock out mouse brains to study Pgrmc1 regulation of KitL synthesis. To determine common downstream targets of KitL and Pgrmc1 I then treated mHe-18 cells with soluble KitL protein. Finally, to determine whether c-Kit mediated effects of Pgrmc1, I treated cells with both Pgrmc1 siRNA and AG-1296, a c-Kit inhibitor. The results show that Pgrmc1 regulates KitL expression, as well as downstream targets Pias1, 2, 3, and 4. However, AG-1296 did not abrogate Pgrmc1 regulation of the downstream targets, demonstrating regulation independent of KitL signaling. Taken together, these results suggest that while Pgrmc1 alters KitL expression and regulates the same genes as KitL/c-Kit, the mechanism of action likely differs. Considering that these two genes are involved in neurogenesis and neuroprotection, as well as memory and learning, a better understanding of the pathways may help lead the way in treating neurodegenerative diseases in the future.