The striatum is the major input structure of the basal ganglia, and is composed of two major populations of spiny projection neurons (MSNs), which give rise to the socalled direct and indirect pathways, and several types of interneuron. Dopaminergic inputs to striatum are critical for its proper function. Indeed, loss of dopaminergic neurons in Parkinsonism leads to motor disturbances, grossly disturbs striatal activity, and is associated with the emergence of excessively-synchronized network oscillations at beta frequencies (15-30 Hz) throughout the basal ganglia. How the distinct structural, neurochemical and other properties of striatal neurons are reflected in their firing rates and patterns in vivo is poorly defined, as are their possible cell-type-selective contributions to the aberrant oscillations arising in the Parkinsonian brain. To address these issues, I first used multi-electrode arrays to record the spontaneous firing of ensembles of neurons in dorsal striatum in both anaesthetised dopamine-intact and Parkinsonian (6-hydroxydopamine-lesioned) rats during two well-defined brain states, slow-wave activity (SWA) and spontaneous activation. The chronic loss of dopamine led to an overall increase in the average firing rates of striatal neurons, irrespective of brain state. However, many neurons in the Parkinsonian striatum still exhibited the low firing rates and irregular firing patterns typical of neurons in the dopamine-intact striatum. During SWA in Parkinsonian rats, the firing of striatal neurons was more strongly synchronized at low frequencies, in time with cortical slow (~1 Hz) oscillations. During spontaneous cortical activation in Parkinsonian rats, more striatal neurons engaged in synchronized firing in time with cortical beta oscillations. Under the same experimental conditions, I then recorded the spontaneous firing of individual striatal neurons and juxtacellularly labelled the same neurons to verify their cell types, and locations; indirect pathway and direct pathway MSNs were distinguished by the expression (and lack of expression respectively), of the neuropeptide precursor preproenkephalin (PPE). After chronic dopamine loss, and on average, only indirect pathway (PPE+) MSNs significantly increased their firing rates during both brain states, and engaged in widespread, synchronized firing in the beta-frequency range. This did not hold true for all PPE+ MSNs; the Parkinsonian striatum contained many MSNs that were virtually quiescent, which were just as likely to belong to the indirect pathway as the direct pathway. Direct pathway (PPE-) MSNs increased their firing only during SWA after chronic dopamine loss and rarely engaged in aberrant beta oscillations. Taken together, these data suggest that (1) the firing patterns, as well as the firing rates of many striatal neurons are grossly disturbed by chronic loss of dopamine and (2) that the pathological synchronization of the rhythmic firing of a subpopulation of indirect pathway MSNs could contribute to the propagation of aberrant beta-frequency oscillations to downstream basal ganglia nuclei in Parkinsonism.

In the case of Parkinson’s disease, a common neurodegenerative disorder, the loss of motor function results from the selective degeneration of dopaminergic neurons (DNs) in the brain. Current treatments focus on pharmacological approaches that lose effectiveness over time and produce unwanted side effects. A more complete concept of rehabilitation to improve on current treatments requires the production of DNs to replace those that have been lost. Although pluripotent stem cells (PSCs) are a promising candidate for the source of these replacement neurons, current protocols for the terminal differentiation of DNs require a complicated cocktail of factors. Recently, a naturally occurring steroid called guggulsterone has been shown to be an effective terminal differentiator of DNs and can simplify the method for the production of such neurons. I therefore investigated the potential of long-term guggulsterone release from drug delivery particles in order to provide a proof of concept for producing DNs in a more economical and effective way. Throughout my study I was able to successfully encapsulate guggulsterone in Poly-ε-caprolactone (PCL)-based microspheres and I showed that the drug was capable of being released over 44 days in vitro. These guggulsterone-releasing microspheres were also successfully incorporated in human induced pluripotent stem cell (hiPSC)-derived neural aggregates (NAs), providing the foundation to continue investigating their effectiveness in producing functional and mature DNs. Together, these data suggest that guggulsterone delivery from microspheres may be a promising approach for improving the production of implantable DNs from hiPSCs. / Graduate

Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive neurodegeneration of nigro-striatal dopamine neurons resulting in an array of pathological symptoms characteristic of the disease. Current treatment relies on targeting symptomatic aspects of the disease but currently Parkinson’s disease is incurable. Targeting the regeneration of DA neurons in PD patients could offer an alternative therapeutic approach that could stall and perhaps even revert the progression of the disease and improve the quality of life for patients. Here, I describe the generation of a transgenic zebrafish line for the non-invasive, conditional and specific ablation of dopaminergic neurons in both larval and adult zebrafish. Understanding the endogenous regenerative ability of the zebrafish may in the future contribute to the development of novel therapeutic approaches targeting DA neuron regeneration in humans. The Tg(dat:CFP-NTR) line efficiently labels and ablates most clusters of DA neurons in both the larval and the adult zebrafish brain. Neuronal ablation is followed by a locomotor and tail bend phenotype as well as by an increase in exploratory behavior. Using double transgenic larvae, we showed through live imaging that loss of DA neurons induces an increase in nestin expression; in addition we show an increase in the number of proliferating cells and an up regulation of genes involved in neurogenesis and tissue repair. Adult zebrafish were able to fully recover their DA neuronal population in the olfactory bulb within 45 days post ablation. Overall the Tg(dat:CFP-NTR) zebrafish offers a novel tool for the study of the molecular and cellular mechanisms driving the regeneration of DA neurons in the zebrafish brain and will be a useful tool for the field of regenerative medicine.

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.

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).

No description available.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Restricted until October 2005. Bibliography: leaves 71-94.