Neurological diseases are devastating illnesses that affect over one billion people worldwide. Drosophila melanogaster provides a genetically tractable system in which to study gene function and the mechanisms of pathogenesis of neurological diseases. In this study I have investigated the function of survival motor neuron (SMN), the causative gene in the neuromuscular disease spinal muscular atrophy (SMA), in growth and differentiation in Drosophila. In addition, I have used the fruit fly to investigate a putative modifier of a previously characterised Drosophila model of Parkinson's disease. Spinal muscular atrophy is an autosomal recessive neurological disease that is characterised by motor neuron loss resulting in muscle weakness. The disease is caused by the deletion or mutation of the survival motor neuron (SMN) gene. In Drosophila, SMN was found to be highly expressed in dividing tissues and a reduction in SMN levels resulted in growth defects, stem cell defects and developmental delay. SMN was also shown to regulate chromosome morphology of the endocycling nurse cells of the female germline. Therefore it appears that SMN has a role in growth control and development in Drosophila. Parkinson's disease is a common disorder that results in widespread neurodegeneration with a predilection for dopaminergic neuron loss resulting in movement defects. A defining neuropathological feature of the disease is the presence of alpha-synuclein containing inclusions. Using a Drosophila model of PD, I have shown that specific alpha-synuclein-induced phenotypes in the fly can be suppressed by the overexpression of the E3 ubiquitin ligase, Nedd4.

<p> Activating mutations in fibroblast growth factor receptors (FGFR) -1, -2, -3 cause craniosynostosis, the premature fusion of one or more cranial sutures, in Apert, Crouzon, Pfeiffer, Muenke, and Beare-Stevenson cutis gyrata syndromes. These conditions are also characterized by other skeletal anomalies and variable effects on brain development and function. The variability of central nervous system defects and the occurrence of severe cognitive impairment despite early surgical intervention, particularly in Apert syndrome, led us to hypothesize that the activating FGFR mutations have a direct, adverse effect on brain development. In this study we looked for aberrations in diverse cellular processes affected by Fgf signaling in the developing telencephalon of an Fgfr2+/S252W Apert mouse model to uncover the mechanisms by which these defects occur. We present evidence that in the Apert mouse model, the radial glial cells to intermediate progenitor transition is depressed during mid-to-late neurogenesis. We also observed a significant increase in apoptosis in the anterior forebrain in Apert newborns. Lastly, an increase in the migration of astrocytes to the dorsolateral cortex by post-natal day five was observed. These results suggest that important physiological roles of Fgfr2 signaling in the development of the cerebral cortex are adversely affected by syndromic activating mutations of Fgfr2, which may lead to gross brain abnormalities. </p>

Parkinson's disease (PD) is an incurable neurodegenerative motor disorder caused by the inexorable loss of dopamine neurones from the substantia nigra pars compacta. Cell loss is characterised by the perturbation of multiple physiological processes (including mitochondrial function, autophagy and dopamine homeostasis) and much of this pathophysiology can be reproduced in vitro using the mitochondrial toxin MPP+ (1-methyl-4-phenylpyridinium). It was hypothesised that MPP+ toxicity could be modelled using protein-protein interaction networks (PPIN) in order to better understand the interplay of systems-level processes that result in eventual cell death in MPP+ models and PD. Initially, MPP+ toxicity was characterised in the human, dopamine-producing cell line BE(2)-M17 and it was confirmed that the neurotoxin resulted in time and dose dependent apoptosis. A radio-label pulse-chase assay was developed and demonstrated that MPP+ induced decreased autophagic flux preceded cell death. Autophagic dysfunction was consistent with lysosome deacidification due to cellular ATP depletion. Pertinent PPINs were sampled from publically available data using a seedlist of proteins with validated roles in MPP+ toxicity. These PPINs were subjected to a series of analyses to identify potential therapeutic targets. Two topological methods based on betweenness centrality were used to identify target proteins predicted to be critical for the crosstalk between mitochondrial dysfunction and autophagy in the context of MPP+ toxicity. Combined knockdown of a subset of target proteins potentiated MPP+ toxicity and the combined resulted in cellular rescue. Neither of these effects was observed following single knockdown/overexpression confirming the need for multiple interventions. Cellular rescue occurred via an autophagic mechanism; prominent autophagosomes were formed and it was hypothesised that these structures allowed for the sequestration of damaged proteins. This thesis demonstrates the value of PPINs as a model for Parkinson's disease, from network creation through target identification to phenotypic benefit.

<p> The underlying goal of this dissertation is to understand how the amygdala, a brain region involved in establishing the emotional significance of sensory input, contributes to the processing of complex sounds. The general hypothesis is that communication calls of big brown bats (<i>Eptesicus fuscus</i>) transmit relevant information about social context that is reflected in the activity of amygdalar neurons. </p><p> The first specific aim analyzed social vocalizations emitted under a variety of behavioral contexts, and related vocalizations to an objective measure of internal physiological state by monitoring the heart rate of vocalizing bats. These experiments revealed a complex acoustic communication system among big brown bats in which acoustic cues and call structure signal the emotional state of a sender. </p><p> The second specific aim characterized the responsiveness of single neurons in the basolateral amygdala to a range of social syllables. Neurons typically respond to the majority of tested syllables, but effectively discriminate among vocalizations by varying the response duration. This novel coding strategy underscores the importance of persistent firing in the general functioning of the amygdala. </p><p> The third specific aim examined the influence of acoustic context by characterizing both the behavioral and neurophysiological responses to natural vocal sequences. Vocal sequences differentially modify the internal affective state of a listening bat, with lower aggression vocalizations evoking the greatest change in heart rate. Amygdalar neurons employ two different coding strategies: low background neurons respond selectively to very few stimuli, whereas high background neurons respond broadly to stimuli but demonstrate variation in response magnitude and timing. Neurons appear to discriminate the valence of stimuli, with aggression sequences evoking robust population-level responses across all sound levels. Further, vocal sequences show improved discrimination among stimuli compared to isolated syllables, and this improved discrimination is expressed in part by the timing of action potentials. </p><p> Taken together, these data support the hypothesis that big brown bat social vocalizations transmit relevant information about the social context that is encoded within the discharge pattern of amygdalar neurons ultimately responsible for coordinating appropriate social behaviors. I further propose that vocalization-evoked amygdalar activity will have significant impact on subsequent sensory processing and plasticity.</p>

The activity of networks subserving memory and learning in the hippocampus is under the control of GABAergic interneurons. In order to test the contribution of distinct cell types, I have recorded extracellularly, labelled, and identified different types of interneuron in area CA3 of the hippocampus, a region implicated in the generation of gamma and theta oscillations, and the initiation of sharp-waves. I present here a detailed analysis of the spike timing of parvalbumin-positive (PV) basket and physiologically identified pyramidal cells in area CA3, relative to various network states recorded in area CA3 and CA1 simultaneously. Additionally, I have shown by detailed analysis that five classes of previously recorded and identified CA1 interneuron fired with cell type specific firing patterns relative to local gamma oscillations. In CA3, PV basket cells fired phase locked to theta and gamma oscillations recorded in CA1 as well as in CA3, and increased their firing rates during CA1 sharp-waves. Pyramidal cells in CA3 were also phase-locked, but fired at phases different from basket cells. During theta oscillations, CA3 pyramidal and PV basket cells were phase locked to both CA1 and CA3 theta equally, suggesting a wide coherence of these oscillations; in contrast, cells fired more strongly phase-locked to gamma oscillations in CA3 than in CA1, suggesting a specific role for CA3 in the generation of this rhythm. In contrast to theta and gamma oscillations, CA3 basket cells were phase-locked to ripples in area CA3 but not in CA1. Overall, my results show that the spike timing of several types of interneuron in CA1, and PV basket cells in CA3, is correlated in a cell- and area-specific manner with the generation of particular states of synchronous activity.

Glial cells maintain homeostasis that is essential to neuronal function. Injury to the nervous system leads to the activation and proliferation of glial cells and pericytes, which helps to wall off the damaged region and restore homeostatic conditions. Sonic hedgehog is a mitogen which is implicated in injury-induced proliferation of glial cells and pericytes. The mitogenic effects of sonic hedgehog require primary cilia, but the few reports on glial or pericyte primary cilia do not agree about their abundance and did not address effects of injury on these cilia. Primary cilia are microtubule-based organelles that arise from the centrosome and are retracted before cells divide. Depending on cell type, proteins concentrated in cilia can transduce several mitotic, chemosensory, or mechanosensory stimuli. The present study investigated effects of stab wound injury on the incidence and length of glial and pericyte primary cilia in the area adjacent to the injury core. Astrocytes, polydendrocytes and pericytes were classified by immunohistochemistry based on cell-type markers. In normal adult mice, Arl13b immunoreactive primary cilia were present in a majority of each cell type examined: astrocytes, 98±2%; polydendrocytes, 87±6%; and pericytes, 79±13% (mean ± SEM). Three days post-injury, cilium incidence decreased by 24% in astrocytes (p< 0.008) and 41% in polydendrocytes (p< 0.002), but there was no significant effect in pericytes. Polydendrocytes labeled with the cell cycle marker Ki67 were less likely to have cilia compared to resting, Ki67- polydendrocytes. Considering post-injury rates of proliferation for astrocytes and polydendrocytes, it appears that resorption of cilia due to cell cycle entry may account for much of the loss of cilia in polydendrocytes but was not sufficient to account for the loss of cilia in astrocytes. Under normal conditions, astrocytes rarely divide, and they maintain non-overlapping territories. However, three days after injury, there was a 7-fold increase in the number of paired mirror-image astrocytes (p< 0.018), which are most likely daughter cells from astrocytes that recently divided. Cilia incidence tended to decrease in these pairs compared to single astrocytes (p< 0.057) in injured mice. This is the first systematic investigation of cilia of astrocytes, polydendrocytes, and pericytes in the brain. Moreover, the examination of effects of brain injury on cilia adds to the understanding of injury-induced proliferation in these cells.

By 2040, the World Health Organization expects neurodegenerative diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease, to surpass cancer as the second most common cause of death worldwide. Currently, only treatments for symptoms of these diseases are available. Thus, research is critical to alleviate this public health burden by elucidating the pathogenic processes and developing novel therapies. While exact mechanisms by which these heterogeneous neuropathological conditions become manifest in patients remain unclear, growing evidence suggests that oxidative stress (OS) makes a significant contribution to neuronal dysfunction and apoptosis in all major neurodegenerative diseases. Recently, the gene oxidation resistance 1 (Oxr1) has emerged as a critical regulator of neuronal survival in response to OS. Oxr1 is expressed throughout the central nervous system, and its highly conserved TLDc domain protects neurons from oxidative damage through an unknown mechanism. This thesis aimed to define mechanisms by which Oxr1 confers neuronal sensitivity to OS, and to determine its role in neurodegenerative diseases. I found that Oxr1 mediates cytoplasmic localization of ALS-associated proteins Fused in Sarcoma (FUS) and transactive response DNA binding protein 43 kDa (TDP-43) through a TLDc domain- and arginine methylation-dependent pathway. Next, I investigated in vivo neuroprotective functions of Oxr1, and demonstrated that neuronal Oxr1 over-expression extends survival and ameliorates behavioural dysfunction and pathology of an ALS mouse model. In particular, neuronal Oxr1 over-expression strikingly delays neuroinflammation during ALS pathogenesis. Finally, I characterised a mouse model that specifically deletes Oxr1 from motor neurons. While loss of Oxr1 in ChAT-positive motor neurons does not cause overt neurodegeneration in the spinal cord, constitutive loss of Oxr1 leads to neuroinflammation in the cerebellum and spinal cord. Taken together, these studies illuminate functions of Oxr1 in the complex antioxidant defence network and present implications for future therapeutic strategies.