The aim of this thesis was the functional characterization of the zebrafish parl (Presenilin-Associated Rhomboid-Like) genes which code for mitochondrial proteins involved in cell survival. A mutation in PARL has been described in Parkinson’s disease patients. I investigated the role of mitochondrial PD-related proteins using a zebrafish parla and parlb deficiency model. I found that the knockdown of both parl genes is lethal. Parla plays a larger role in patterning of the DA neurons in the ventral diencephalon than Parlb. The human PARL rescued the double morphant phenotype, suggesting function conservation between zebrafish and humans. I was able to rescue the mortality and DA neuron mispatterning observed in double morphants with synthetic pink1 mRNA. This suggests that parl genes are epistatic to pink1 in zebrafish. To visualize mitochondria specifically in dopaminergic neurons of live zebrafish, I established a transgenic line Tg(dat:tom20 MLS-mCherry) where regulatory elements of the dopamine transporter (dat) were used to drive expression of a Tom20-mCherry fusion protein that is targeted to the mitochondria. I characterised the expression of Tom20-mCherry to the mitochondria of the majority of DA neuron groups. In addition, I observed a decrease in mCherry fluorescence following MPTP exposure of live fish. The PD-related mutation in PARL is located in a cleavage site of the mammalian protein, which is necessary for the production of the beta peptide; however, this site is predicted to be absent in the zebrafish Parls. To establish the cleavage patterns of the zebrafish Parls and compare them to those of human PARL, I examined the cleavage of Parl-Flag constructs in cultured cells. I detected one band for Parla-Flag and two bands representing Parlb-Flag. The parla and parlb deficiency model along with the characterization of the cleavage patterns of Parl and the Tg(dat:tom20 MLS-mCherry) transgenic line are tools which will help elucidate the role of mitochondrial proteins in PD research.

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation.

The specification and development of nervous system diversity is a driving question in the field of Neurobiology. The overarching goals of the projects described in this thesis are to describe tools to aid in the description of nervous system development and to show the use of the described tools to study nervous system development in the nematode Caenorhabditis elegans. The first chapter of this thesis describes a complete map of the male C. elegans nervous system using a tool developed in the lab to uniquely label all neurons in the C. elegans nervous system, NeuroPAL. The second chapter of this thesis largely focuses on a well-studied homeobox gene, unc-86, and its role in fate transformations in dopaminergic and GABAergic neuron types. These two seemingly disparate projects are united in their effort to investigate nervous system development and neuronal fate determination. NeuroPAL is a multicolor transgene that uniquely labels all neurons of the C. elegans hermaphrodite nervous system and here I show it can be used to disambiguate all 93 neurons of the male-specific nervous system. I demonstrate the wide utility of NeuroPAL to visualize and characterize numerous features of the male-specific nervous system, including mapping the expression of gfp-tagged reporter genes and neuron fate analysis. NeuroPAL can be used in combination with any gfp-tagged reporters to unambiguously map the expression of any gene of interest in the male, or hermaphrodite, nervous system. Furthermore, NeuroPAL is used in mutants of several developmental patterning genes to confirm previously described defects in neuronal identity acquisition. Additionally, I show that NeuroPAL can be used to uncover novel neuronal fate losses and identity transformations in these mutants because of the unique labeling of every neuron. Lastly, we show that even though the male-specific neurons are generated throughout all four larval stages, the neurons only terminally differentiate in the fourth and final larval stage, termed ‘just-in-time’ differentiation. In the second part of this thesis, I describe a few examples of mutant analysis of homeobox gene family members and describe their function in the C. elegans nervous system. I focus largely on a couple potential examples of homeotic fate transformations in mutants of the POU homeobox gene, unc-86. In unc-86 mutants, I describe the ectopic expression of multiple GABAergic terminal identity features in one cell in the head of C. elegans. I raise the hypothesis that this cell may be a transformation of a non-GABAergic ring interneuron, RIH, into that of its GABAergic sister cell, AVL, in unc-86 mutants. While ectopic dopaminergic neurons were previously described in unc-86 mutants, I expand the study to show the ectopic expression of all dopaminergic synthesis and packaging genes. I show support that all non-dopaminergic anterior deirid neurons, ADA, AIZ, FLP, and RMG, lose the expression of some of their wild type terminal fate genes and transform to a fate like that of their dopaminergic sister cell, ADE, as assessed by NeuroPAL expression. Taken together, these studies describe tools and methods for studying nervous system development as well as describe many examples of cell fate transformations.

Adhesion G-protein coupled receptors (GPCRs) are the second largest class of GPCRs, yet their functions and ligands remain predominantly unidentified. Polymorphisms in the gene encoding the adhesion GPCR latrophilin 3 (ADGRL3) have been associated with an increased risk for attention deficit hyperactivity disorder (ADHD) and substance use disorder (SUD) in various linkage and association studies. Disrupting the function of ADGRL3 homologs across mammalian and invertebrate model systems leads to changes in various dopaminergic phenotypes such as hyperactivity, sleep impairment, and changes in sensitivity to psychostimulants, suggesting that ADGRL3 contributes to behavior by modulating dopamine signaling. Here, I use behavioral and imaging studies to delineate an important role for Cirl, the Drosophila homolog of ADGRL3, in a recently characterized dopaminergic sleep circuit. Sleep impairment is a common symptom in both SUD and ADHD, and sleep studies are well established in Drosophila. Our work shows that fruit flies that carry a null mutation for Cirl are hyperactive and display a deficit in sleep that is enhanced by adult thermogenetic activation of dopamine neurons. Though Cirl displays high expression within dopamine neurons, conditional knockout of Cirl in dopamine neurons does not recapitulate sleep deficits seen in Cirl null flies, and specific rescue of Cirl in a knockout background does not ameliorate them. Intriguingly, activating dopamine neurons in Cirl null flies throughout development rescued the sleep deficits, indicating that this dopaminergic intervention induces lasting changes that can ameliorate lack of Cirl function. Imaging studies reveal that Cirl shows high expression in the central complex, which is involved in sleep and receives dense dopaminergic input. I demonstrate that Cirl functions within different populations of the central complex downstream of dopaminergic innervation to differentially affect night and daytime sleep through both dopaminergic and non-dopaminergic mechanisms. This work delineates a novel role for an adhesion GPCR in modulating sleep behavior, and further characterizes ADGRL3 as a potential therapeutic target for disorders characterized by dysregulation of dopaminergic neurotransmission.

Parkinson’s disease (PD) is characterized by the degeneration of midbrain dopaminergic neurons. Genetic studies have revealed causative and risk loci associated with a proportion of PD cases, such as PRKN/PARK2, encoding parkin and when mutated causes a rare familial form of autosomal recessive PD. Cell-based studies have linked parkin to mitochondrial turnover by autophagy, but to date, manipulating this gene in rodents has not robustly recapitulated core features of PD. Reconciling these results is essential to determine parkin’s role in mitochondrial biology, brain physiology, and PD pathogenesis. Here, we find that global, inducible deletion of Prkn/Park2 (parkin iKO) in the adult mouse leads to age-dependent motor impairments that are responsive to levodopa treatment. We report that these behavioral defects are associated with progressive pathology in dopaminergic neurons, regional gliosis and lipid oxidation changes, culminating in the selective degeneration of nigrostriatal dopaminergic neurons. We also present a new, in vivo mitophagy reporter system to investigate the relationship of parkin’s described roles in mitochondrial homeostasis to the observed phenotypes. These results give critical insight into parkin’s contribution to dopaminergic neuron stability in the mammalian brain, and provide two distinct and novel organismal tools to investigate mitochondrial homeostasis and PD pathogenesis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24086号 / 医博第4862号 / 新制||医||1059(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 井上 治久, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Reinforcement learning occurs when organisms adapt the propensities of given behaviours on the basis of associations with reward and punishment. Currently, reinforcement learning models have been validated in minimalist environments in which only 1-2 environmental stimuli are present as possible predictors of reward. The exception to this is two studies in which the responses of the dopamine system to configurations of multiple stimuli were investigated, however, in both cases the stimuli were presented simultaneously rather than in a sequence. Therefore, we set out to understand how current models of reinforcement learning would respond under more complex conditions in which sequences of events are predictors of reward. In the two experimental chapters of this thesis, we attempted to understand whether midbrain dopaminergic neurons would respond to occasion setters (Chapter 3), and to the overexpectation effect (Chapter 4). In addition, we ran simulations of the behavioural paradigms using temporal difference models of reinforcement learning (Chapter 2) and compared the predictions of the model with the behavioural and neurophysiological data. In Chapter 3, by performing single-neuron recording from VTA and SNpc dopaminergic cells, we demonstrated that our population of neurons were most responsive to the latest predictor of reward, the conditioned stimulus (CS) and not the earliest, the occasion setter (the OS). This is in stark contrast with the predictions of the model (Chapter 2), where the greatest response is seen at the OS onset. We also showed at a neural level that there was only a weak enhancement of the response to the discriminative stimulus (SD) when this was preceded by the OS. On the other hand, at a behavioural level, bar pressing was greatest when the SD was preceded by the OS, demonstrating that rats could use the information provided by the OS, but that dopamine was not controlling the conditioned response. In Chapter 4, our population of dopaminergic neurons showed that they would preferentially respond to only one of the two conditioned stimuli (CSA, CSB) in the overexpectation paradigm. The predictions of the model (Chapter 2) suggested that when the two stimuli would be presented in compound, there would be an inhibitory response if the reward magnitude was kept constant and an excitatory response if the reward magnitude was doubled. The lack of neural firing to one of the two conditioned stimuli, however, does not make for easy interpretation of the data. Perhaps, one of the conditioned stimuli acted as if it were overshadowing the other, resulting in no response to the second CS. Interestingly, at a behavioural level, we did not see increased licking frequency to the compound stimuli presentation, a result that is somewhat at odds with the previous literature. Overall, the results of our experimental chapters suggest that the role that midbrain dopaminergic neurons play in reinforcement learning is more complex than that envisaged by previous investigations.

Ce manuscrit détaille la stratégie utilisée par nos laboratoires pour identifier de nouvelles molécules neuroprotectrices pour le traitement curatif de la maladie de Parkinson (MP). La MP est une maladie neurodégénérative caractérisée par des symptômes moteurs invalidants qui résultent de la dégénérescence des neurones dopaminergiques (DA) des noyaux gris centraux. Précédemment, nos laboratoires ont synthétisé et identifié au cours d’un criblage 3 molécules chefs de file qui possèdent un effet neurotrophique sur les neurones DA embryonnaires. Des études préliminaires d’ADMEtox nous ont permis de sélectionner la molécule SF41, un dérivé 6-aminoquinoxaline, pour une première évaluation de l’effet neuroprotecteur in vivo de nos molécules. En effet, cette molécule est bien tolérée chez l’animal et, administrée par voie orale, elle est capable de traverser la BHE. SF41 a montré un faible effet protecteur vis-à-vis des fibres DA dans un modèle animal de la MP. Dans le but d’augmenter l’activité neurotrophique de cette molécule, une 50ène de dérivés de seconde génération ont été synthètisés et criblés in vitro dans un modèle de mort spontanée des neurones DA. Ce criblage nous a permis d’identifier 5 molécules lead plus puisssantes et efficaces que SF41. Ces molécules, qui possédent les mêmes propriétés physico-chimiques que SF41, pourraient également atteindre le système nerveux central et ainsi conduire à un effet neuroprotecteur marqué dans un modèle animal de la MP. De plus, ces molécules possèdent un profil pharmacologique intéressant car elles sont capables d’empêcher la mise en place de mécanismes qui peuvent potentiellement contribuer à la mort des neurones DA dans la MP (stress oxydant, stress médié par les astrocytes, dyshoméostasie calcique, stress médié par la diminution en facteur trophique…). Une étude préliminaire avec la molécule PAQ, l’une de ces 5 molécules, a permis d’obtenir un effet neuroprotecteur dans un modèle in vivo de la MP qui semble supérieur à celui de la molécule SF41. Ces résultats encourageants nous donnent bon espoir d’obtenir la preuve de concept de l’activité neuroprotectrice de nos dérivés 6-aminoquinoxaline. / This manuscript describes the strategy used by our laboratories to identify new neuroprotective molecules for the therapy of Parkinson disease (PD). PD is a neurodegenerative disease characterized by disabling motor symptoms resulting from the degeneration of dopaminergic (DA) neurons of the basal ganglia. Previously, our laboratories have synthesized and identified in a screening 3 lead compound which exhibited a neurotrophic effect on embryonic midbrain DA neurons. Preliminary ADMEtox studies allowed us to select the molecule SF41, a 6-aminoquinoxaline derivative, for a first in vivo evaluation of the neuroprotective effect of our molecules in an animal model of PD. Indeed, SF41 is well tolerated in animals and is able of crossing the BBB after oral treatment. SF41 showed a weak protective effect on DA fibers in an animal model of PD.In order to increase the neurotrophic activity of this molecule, around fifty second generation derivatives were synthesized and screened in vitro in a model of spontaneous death of DA neurons. This screening allowed us to identify five lead compounds more powerful and effective than SF41. These molecules, which possess the same physico-chemical properties that SF41, could also reach the central nervous system and lead to a marked neuroprotective effect in an animal model of PD. In addition, these molecules have an interesting pharmacological profile because they are able to prevent the establishment of mechanisms that can potentially contribute to the death of DA neurons in PD (oxidative stress, stress mediated by astrocytes, calcium dyshomeostasis, stress mediated by trophic factor deprivation...).A preliminary study with the molecule PAQ, one of these five molecules, yielded a neuroprotective effect in animal model of PD that seems higher than with SF41. These encouraging results give us hope to achieve proof of concept of the neuroprotective activity of our 6-aminoquinoxaline derivatives.

No description available.

Der Transkriptionsfaktor Pitx3 wird nur in postmitotischen mDA Neuronen des Mittelhirns exprimiert und zeigt eine vollständige Koexpression mit TH im VTA und der SN. Bei einem Verlust an Pitx3-Expression (aphakia-Maus) zeigen die mDA Neurone des VTA nahezu keine Veränderung, während sich jedoch die Neuronen der SN nicht entwickeln. Die Gründe hierfür sind bisher unklar.  In der vorliegenden Arbeit sollte analysiert werden, welche Rolle Pitx3 bei der Neurogenese der mDA spielt, indem Pitx3 im murinen Mittelhirn spezifisch überexprimiert wurde. Dies sind die ersten Ergebnisse einer in vivo Pitx3-Überexpressionsstudie in der Maus und die Ergebnisse könne wie folgt zusammengefast werden:  • Die Expression von TH-, DAT- und Nurr1-positiven Zellen im medialen Bereich des dopaminergen Areals, aus dem das VTA hervorgeht, ist bei allen Mutanten (Shh-Cre+/Pitx3OE, Foxa2-Cre+/Pitx, Wnt1-Cre+/Pitx3OE und En1-Cre+/Pitx3OE) signifikant erhöht. Dabei war dieser Phänotyp besonders bei den  Wnt1-Cre+/Pitx3OE Mutanten ausgebildet.  • An E12.5 ist die Expression von Lmx1a und Lmx1b  bei allen Mutanten in der VZ signifikant erhöht.  • Bei den Wnt1-Cre+/Pitx3OE Mutanten zeigte sich an E12.5 eine starke ektopische Wnt1-Expression in den Bereichen der VZ und SVZ. Ebenso konnte eine gesteigerte Proliferation (BrdU, Ki67) bei gleichzeitiger leichter Reduktion der Neurogenese (Ngn2) beobachtet werden. In den Bereichen der ektopischen Wnt1-Expression konnte weiterhin eine Inhibierung der Shh-Expression an E12.5 festgestellt werden.  • An E12.5 konnte bei den Wnt1-Cre+/Lmx1aOE und Wnt1-Cre+/Lmx1bOE Mutanten eine ektopische Wnt1-Expression in den Bereichen festgestellt werden, in denen Lmx1a/b konditional überexprimiert wurde. Hierbei zeigte sich eine starke Reduktion der TH- und Pitx3-Expression, die aber wahrscheinlich durch eine Störung des IsO hervorgerufen sein könnte.  Im Allgemeinen kann also gesagt werden, dass Pitx3 nicht nur an der Differenzierung, sondern auch an Prozessen der Koordination von Proliferation und Spezifikation – im Sinne von Aufrechterhaltung des mDA Bereiches bzw. der Abgrenzung alternativer neuronaler Zellschicksale  beteiligt sein könnte. Dabei beeinflusst Pitx3 wahrscheinlich indirekt den kanonischen Wnt-Signalweg, indem es eine Rückkopplungsschleife mit Lmx1a und/oder Lmx1b, oder einem Zwischenfaktor bildet.