Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

Recently, the zebrafish has been considered an important animal model that can be used to investigate human diseases and drug development. Parkinson¡¦s disease (PD), an important neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. However, current treatments for PD are limited and mainly improve only the clinical symptoms of the disease. Thus, a neurodegenerative rat model has been widely used for a long while to search for a new treatment for PD. However, the use of rats as an animal model has certain limitations such as breeding, efficiency, and high dosage. Recently, researchers indicated that neurotoxins such as rotenone, 6-hydroxydopamine (6-OHDA), and paraquat can induce Parkinson¡¦s-like symptoms in zebrafish, and this may be a useful PD model because of the complete development of the zebrafish nervous system, low costs, and low dosage. In this study, we treated zebrafish with 6-OHDA and analyzed their locomotor activity to establish an in vivo animal model of PD. Then, we analyzed the mRNA expression of parkin and PINK1 by reverse transcription¡Vpolymerase chain reaction (RT-PCR).Moreover, we observed tyrosine hydroxylase (TH) expression by immunohistochemical (IHC) staining to confirm if this can be used as a PD model. Finally, we found that treatment with 6-OHDA significantly reduced TH expression. We observed a similar declining trend in the case of mammals. Likewise, parkin and PINK1 mRNA expressions were also decreased after treatment with 6-OHDA. In summary, our study provides a feasible in vivo Parkinson¡¦s model, and a small volume of drugs or compounds can be screened using this model.

Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

Stem cells have the ability to reproduce themselves for a long period and differentiate into specific morphological and functional cells. The stem cells are an important material in the developmental biology, genomics, and transgenic methods, as well as in potential clinical applications, gene therapy and tissue engineering. The pluripotent stem cells will be a valuable source in numerous functional degenerated pathologies. Therefore, the objective of this research program was to establish transgenic porcine embryonic stem (pES) cell lines which can express green fluorescent protein (GFP) report gene stably for tracking after transplantation. We also developed a directed differentiation of pES into neural lineages and applied in rat Parkinson¡¦s disease model. Although the establishment of pluripotent ES cell lines from domestic species is much more difficult than that in murine species, our results had successfully isolated and established pES cell lines from pre-implantation blastocysts. Furthermore, we established the novel GFP-expressing pES cell lines (pES/GFP+), which were obtained by electroporation- mediated transfection with exogenous GFP gene. These pES/GFP+ cells exhibited pluripotent markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 as that of human ES cells. The strategy of directed neural differentiation was to culture pES with neurogenic stimulators such as retinoic acid (RA), sonic hedgehog (SHH), and fibroblast growth factor (FGF). Upon directed differentiation toward neural differentiation, these pES-derived cells exhibited typical neuronal morphology and expressed neural lineage-specific markers such as nestin, NFL, MAP2, GFAP, A2B5, TH, ChAT, and GABA. These results showed the pES cells had the potential to differentiate into neural lineages. When pES/GFP+ cells were transplanted into the SD rat¡¦s brain, and their survival and development was determined by the non-invasive In Vivo Imaging System (IVIS 50), and the invasive fibered confocal Cellvizio® Imaging System (Cellvizio®). The results showed that fluorescent signals from pES/GFP+ cells on the injection site of SD rats¡¦ brain could be detected through the experimental period of 3 months. The level of fluorescent signals detected in treatment groups was two folds above that of the control group. Besides, the functional behavior recovery analysis by amphetamine-induced rotation test indicated the PD rat grafted with pES/GFP+ cells and their derived neural progenitors showed no significant recovery of rotation rate in these two treatments because a progressively increased relative rotation through 3 months duration. However, the relative rotation of PD rats grafted with the pES/GFP+-derived mature neurons, showed a stably decrease relative rotation and resulted in a functional recovery from Parkinsonian behavioral defects. Following 3 months completion of behavioral analyses, PD rats were sacrificed for immunohistochemical analysis. In the section of injected site without tumorgenesis and showed the survival and dopaminergic differentiation of grafted pES/GFP+ derived cells when stained with anti-TH and anti-DA. To our knowledge, there have been no reports of establishing GFP-expressing pES cell lines. These novel pES/GFP+ cell lines established in this study might serve as a non-rodent model and could benefit to the studies involving ES cell transplantation, cell replacement therapy, tissue regeneration, and actual approach for pre-clinical research due to their traceable capacity.

Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.

Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23070号 / 医博第4697号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 花川 隆, 教授 溝脇 尚志, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Magister Pharmaceuticae - MPharm / Neurodegenerative diseases (ND) are chronic and progressive in nature, and characterized by the gradual loss of neurons in various regions of the central nervous system (CNS). ND include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and cerebral ischemia/reperfusion (CIR). They have various progressive neurodegenerative pathologies that can result in several severe functional impairments for patients, and ultimately lead to serious health-related issues. According to more recent data, AD accounts for the most common cause of dementia and is believed to contribute to approximately 60–70% of cases. AD is thus seen as the most common form of dementia.

The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.       The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

A doença de Parkinson é uma doença neurodegenerativa, cujo aparecimento pode estar ligado a mutações ou alterações na expressão do gene SNCA, o qual codifica a proteína a-sinucleína. A expressão do gene em Saccharomyces cerevisiae produz fenótipos típicos de células parkinsonianas, como a formação de corpos de Lewy, que levam a um aumento na produção de espécies reativas de oxigênio (ROS) na mitocôndria. Assim, a produção de melatonina, um antioxidante natural, poderia ser um possível tratamento para a expressão do de SNCA. Aqui, mostramos que a produção de a-sinucleína é prejudicial ao crescimento e à respiração de leveduras, fenótipo semelhante ao desafio com o agente oxidante menadiona. A produção ou suplementação farmacológica de melatonina possui efeito protetor, conferindo vantagem em meio seletivo para atividade respiratória, melhorando a função mitocondrial de leveduras parkinsonianas. Nossos resultados apontam para um papel promissor da melatonina na proteção das células contra danos oxidativos da produção de a-sinucleína em leveduras. / Parkinson\'s disease is a progressive neurodegenerative disorder often linked to mutation or expression alteration of the SNCA gene that encodes the a-synuclein (a-syn) protein. SNCA expression in Saccharomyces cerevisiae leads to deposition Lewy bodies that increase mitochondrial reactive oxygen species (ROS) production, one of the hallmarks of the disease. Hence, a possible treatment would be ROS neutralization through natural antioxidants, such as melatonin. Here we show that a-syn production is detrimental to yeast growth and respiration, similar to the phenotype observed during menadione oxidative challenge. Melatonin production or pharmacological supplementation confers antioxidant protection against oxidative agents, improving mitochondrial respiratory parameters from parkinsonian yeast grown with melatonin. Our studies point out to a promising role of melatonin in protecting yeast against a-syn and other species oxidative damage.