Parkinson’s disease (PD) is a debilitating movement disorder. The cardinal symptoms of PD are bradykinesia, resting tremors and rigidity. PD is characterized by degeneration of dopaminergic neurons of A9 region, substantia nigra pars compacta (SNpc) and loss of dopaminergic terminals in striatum while the dopaminergic neurons of A10 region, ventral tegmental area (VTA) are relatively protected. Putative mechanisms, such as mitochondrial dysfunction, dysregulation of the ubiquitin proteasome system and increased oxidative stress have been hypothesized to mediate PD pathology. However, precise mechanisms that underlie selective vulnerability of SNpc dopaminergic neurons to degeneration are unknown. The aim of this thesis was to evaluate the pathological mechanisms that may contribute to degeneration of SNpc dopaminergic neurons in PD. Dopaminergic neurons of SNpc are pacemakers and constant calcium entry through L-type calcium channel, Cav1.3 has been reported in these neurons during pacemaking. In addition, these neurons have poor calcium buffering capacity. Together, this leads to dysregulation of calcium homeostasis in the SNpc dopaminergic neurons leading to increased oxidative stress. Gene expression of the full length channel and the variant was investigated in the mouse midbrain and further their presence was verified in mouse SNpc and VTA and also in SNpc and VTA in the MPTP mouse model of PD. Gene expression of Cav1.3 -42 and its variant was also studied in SNpc from autopsy tissue from PD patients and age matched controls. Having studied differential expression of the calcium channels, global changes in gene expression in SNpc from the MPTP mouse model of PD and PD autopsy tissues were next examined. This is the first report of transcriptome profile alterations from SNpc in mouse model and PD tissue performed using RNA-seq. Gene expression profiles were examined from SNpc 1 day post single exposure to MPTP, in which case there is no neuronal death and 14 days after daily MPTP treatment where SNpc has undergone ~50% cell death. Further, RNA- seq was performed to study gene expression alterations in SNpc from human PD patients and age- matched controls. The RNA-seq data was taken through extensive analyses; analysed for differential gene expression, gene-set enrichment analysis, pathway analysis and network analysis. Glutaredoxin 1 (Grx1) is a thiol disulfide oxidoreductase that catalyses the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. Down-regulation of Grx1 has been established to exacerbate neurodegeneration through impairment of cell survival signalling. Previous work from our laboratory has demonstrated that perturbation of protein thiol redox homeostasis through diamide injection into SNpc leads to development of PD pathology and motor deficits. It was therefore investigated if Grx1 down-regulation in vivo, leading to increased glutathionylation and protein thiol oxidation, could result in PD pathology. This work is thus the first study of RNA-seq based transcriptomic profile alterations in SNpc from human PD patients. This work also highlights several differences between mouse model and human PD tissue indicating that the underlying mechanisms of PD pathogenesis differ from mouse to humans in addition to developing a novel model for PD.

The homeobox gene Pitx3 has been implicated as a key regulator for lens development because homozygous mutant aphakia mice, which are hypomorph for Pitx3, fail to develop lenses. One aim of my thesis is to investigate the underlying cellular and molecular mechanism of Pitx3 mediated lens defect by studying knockout mice lacking Pitx3. Chimeric embryos, generated by aggregating the wild type embryos with Pitx3 heterozygous or Pitx3 homozygous mutant ES cells, have been used to analyse lens development. Pitx3 null cells failed to colonise the lens epithelium in Pitx3 null wild type chimeric lens, suggesting that Pitx3 is cell-autonomously required for lens epithelial cells. Further study of Pitx3 null mice revealed an earlier downregulation of the lens epithelial markers PDGFR-alpha and E-cadherin in E11.5 lens epithelium, suggesting the loss of lens epithelial identity in Pitx3 deficient mice. Furthermore, cell cycle inhibitors p27KIP1 and p57KIP2 were ectopically expressed throughout the morphologically normal Pitx3 mutant lens vesicle, suggesting that inactivation of Pitx3 leads to cell cycle exit of epithelial lens cells. In addition, precocious activation of the fibre cell-specific proteins beta- and gamma-crystallins was observed in Pitx3 null lens. Beta-crystallin expression could be observed as early as E10.5 throughout the entire Pitx3 null lens vesicle and gamma-crystallin was detected in the malformed Pitx3 deficient lens at E11.5. RNA in situ hybridisation study revealed that the expression of the transcription factor Foxe3 was lost in Pitx3 null lens at E10.5, suggesting that Pitx3 maintains the lens epithelial cells partly via the regulation of transcription factor Foxe3 during lens development. Accordingly, this study provides the cellular and molecular basis for the lens defect observed in Pitx3 null and Pitx3 hypomorph aphakia mice. Pitx3 is a key transcription factor for the maintenance of lens epithelium and its absence leads to premature activation of fibre cell differentiation programme of lens epithelial cells. In the other part of my PhD, I have further developed the Pitx3-GFP knockin ES cell system with a goal to use this tool for the identification of determinants of midbrain dopaminergic (mDA) neurons, the type of cells lost in Parkinson’s disease (PD) patients. Experimental cell therapy and clinical trials have shown that foetal midbrain tissues, but not tissues from other DA neuron containing regions, can functionally restore the lost mDA neurons when transplanted in Parkinson’s disease patients. Therefore, it is essential to coax mDA properties on stem cell-derived neurons when considering therapeutic development. Within the central nervous system, Pitx3 is expressed exclusively in mDA neurons. Using a Pitx3-GFP knockin mouse line previously generated in the laboratory I have derived heterozygous and homozygous Pitx3-GFP ES cells from mouse blastocysts. In keeping with previous findings in our laboratory, the heterozygous Pitx3-GFP (Pitx3GFP/+) ES cell-derived GFP positive cells of neuronal morphology can be detected after in vitro differentiation using the PA6 coculture system. Furthermore, I have shown that these cells express tyrosine hydroxylase and midbrain markers Engrailed-1 and Nurr-1, demonstrating their midbrain characteristics. I have also generated supertransfectable Pitx3GFP/+ ES cells to offer a rapid and efficient way to express a transgene episomally. The Cre-mediated inducible system of Pitx3-GFP reporter ES cells has also been developed in our laboratory and I have shown that they have high induction efficiency thus allows transgene activation in a temporally controlled manner. The Pitx3 null ES cells showed impaired potential to differentiate into mDA neurons thus they may be used to evaluate candidate Pitx3 downstream target by gain-of-function test. In summary, I have developed a Pitx3-GFP reporter ES cell system to identify mDA regulators functionally by in vitro differentiation.

Aggression and violence pose a significant public health concern to society. Aggression is a highly conserved behavior that shares common biological correlates across species. While aggression developed as an evolutionary adaptation to competition, its untimely and uncontrolled expression is maladaptive and presents itself in a number of neuropsychiatric disorders. A mechanistic hypothesis for pathological aggression links aberrant behavior with heightened dopamine function. However, while dopamine hyper-activity is a neural correlate of aggression, the developmental aspects and circuit level contributions of dopaminergic signaling have not been elucidated. In this dissertation, I aim to address these questions regarding the specifics of dopamine function in a murine model of aggressive behavior. In chapter I, I provide a review of the literature that describes the current state of research on aggression. I describe the background elements that lay the foundation for experimental questions and original data presented in later chapters. I introduce, in detail, published studies that describe the clinical manifestation and epidemiological spread, the dominant categories, the anatomy and physiology, and the pharmacology of aggression, with a particular emphasis on the dopaminergic system. Finally, I describe instances of genetic and environmental risk factors impacting aggression, concluding with studies revealing an important role for interactions among genetics, environmental factors, and age in the development of aggression. In chapter II, I investigate the developmental origins of aggression by examining sensitive periods during which perturbations to the dopaminergic system impact adult aggressive behavior. Previous work in our laboratory has concluded that periadolescent (postnatal days 22-41) elevation in dopamine, via transient dopamine transporter blockade, leads to increased adult aggression and heightened response to amphetamine. I expanded on these findings by temporally refining the opening and closing of this window of sensitivity, specifically to postnatal days 32 to 41, during which increases in dopaminergic tone increase adult aggression and behavioral sensitivity to psychostimulants. The potentiated response to amphetamine indicated to us a state of altered dopaminergic physiology. We next validated this hypothesis and found increased firing rate (in vitro), and increased bursting and population activity (in vivo) at baseline. These data indicate that elevated periadolescent dopamine impacts maturation of the dopamine system, leading to a hyper-active dopaminergic and aggressive predisposition. In conclusion, this chapter introduces a developmental component to the hyper-dopaminergic model of aggression. In chapter III, I report a series of experiments exploring the direct and causal involvement of dopamine in driving aggression. While dopamine hyper-activity is a neural correlate of aggression, the precise brain circuits involved have not been elucidated. Using optogenetics, I established a causal role for the ventral tegmental area (a key source of dopamine) in aggression modulation. I further advanced this finding by demonstrating that the modulatory role of dopamine, is population- and projection-specific. I found that activity of ventral tegmental area, but not substantia nigra, dopamine neurons promotes aggression. Furthermore, controlled stimulation of ventral tegmental area dopaminergic terminals in the lateral septum, but not the nucleus accumbens, mediates increased aggression. I selectively traced connectivity between the lateral septum and the ventral tegmental area using a Cre-driven, population-specific viral vector. I used this virus to show that anatomically distinct clusters of ventral tegmental area dopamine cells send projections to the lateral septum and the nucleus accumbens, thereby dissociating the two target sites both behaviorally and anatomically. Furthermore, I found that while local dopamine release in the lateral septum increases aggression, it has no bearing on reward behaviors thus indicating a stronger association with impulsive, and not motivated, aggression. In conclusion, this chapter offers causal evidence for dopamine’s role in modulating impulsive aggression by identifying a distinct pathway from the ventral tegmental area to the lateral septum that controls aggression. In the work described in chapter IV, my aim was to determine the mechanism underlying ventral tegmental area to lateral septum dopamine-mediated aggression. I first characterized the expression of dopamine receptors in the lateral septum and found that D2 receptors heavily colocalize with the dominant population of neurons in the lateral septum, i.e. GABAergic cells. Moreover, the D2 receptors are perfectly aligned with incoming dopamine afferents. Next we investigated, in acute brain slices, how D2 signaling affects lateral septum function. We revealed that activating D2 receptors hyperpolarizes D2-expressing lateral septum neurons. This effect was abolished with bath application of the D2 receptor antagonist, sulpiride. We validated the functional involvement of post-synaptic D2 signaling in a behavioral test, and found that the aggression induced by direct terminal release of dopamine at the lateral septum is reversed by acutely blocking local D2 receptor signaling. In conclusion, this chapter demonstrates that the ventral tegmental area to lateral septum dopamine pathway, via D2-mediated inhibition of GABAergic lateral septum neurons, is necessary to drive ventral tegmental area-triggered aggression. In chapter V, I engage in a general discussion addressing how the findings from each chapter can be linked to provide a more comprehensive outlook on environmental and genetic risk factors that can modulate ventral tegmental area-triggered aggression. I discuss possible pre- and post-synaptic mechanisms that could impact the functionality of the identified dopaminergic ventral tegmental area to lateral septum pathway. Moreover, in distinguishing this specific dopamine circuit and lateral septum D2 signaling as an underlying correlate of violent pathology, this dissertation aims to evoke deeper understanding of the mechanism of current antipsychotics used to manage aggression. I end this dissertation by proposing new empirical questions, techniques and lines of research that could further develop my findings as well strengthen the links between dominant models of aggression that exist in the field today.

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 76-93). Also available via the World Wide Web.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vii, 79 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 70-78).

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 105-133). Available also in a digital version from Dissertation Abstracts.

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