Rozprawy doktorskie na temat „Drosophila melanogaster dopamine transporter”

Drosophila melanogaster has a long history of being used as an animal model for a wide variety of human diseases, including genetic diseases, neurodegeneration and alcoholism. Despite the fact that Drosophila was first used as an animal model over 100 years ago, it still remains an extremely relevant model today, thanks to its short life cycle, its low cost ease to rear in laboratory conditions and the sophistication of the molecular tools available for genetic manipulation in Drosophila melanogaster. This model also has far less genetic redundancy with respect to mammals, making the study of the role of certain genes far more straightforward, and yet despite this, still possesses an ortholog for 75% of human disease-causing genes. All of these properties contribute to the relevance of this model and were taken advantage of during this project. In the first part of this project, Drosophila melanogaster was used as a model for mitochondrial deoxynucleotide transport. The Drosophila homolog CG18317 of the yeast gene RIM2, which was previously reported to be a pyrimidine dNTP transporter, was characterized. Knock-out (K.O.) flies for gene CG18317, here referred to as drim2, were characterized for mitochondrial function and mtDNA integrity. The two human homologs for this gene, PNC-1 and SLC25A36 were also expressed in this mutant background, in order to investigate the functional homology of these genes and confirm the validity of this model for human mitochondrial dNTP transport. This project also focuses on further characterizing a K.O. fly line for dTTC19, a gene whose human homolog has already been tied to mitochondrial encephalopathy and psychosis in humans. This characterization was also accompanied by the generation of three K.O. lines which express the dTTC19 gene in a mutant background, in order to finally confirm that the entirety of the mutant phenotype is due to the absence of transcription of the dTTC19 gene. Finally, this project attempts to propose a new protocol which will enable researchers to use Drosophila melanogaster as a model for neurological disorders which present with antisocial symptoms. A protocol was developed to investigate social behaviour in Drosophila melanogaster and to demonstrate that subtle changes in either dopamine levels or previous social contact can have dramatic effects on their social interactions. We therefore propose that Drosophila can also be a useful model for the investigation of the genetic factors involved in diseases which present with antisocial behaviour such as autism, obsessive compulsive disorder, depression and so forth. In conclusion, this project takes full advantage of Drosophila melanogaster as an animal model for mitochondrial biology and disease. Furthermore, it proposes yet another way in which Drosophila can be used as a model which has not yet been done.
Drosophila melanogaster ha una lunga storia come animale modello per tante malattie umane, incluse le malattie genetiche, la neurodegenerazione e l’alcolismo. Anche se Drosophila fu inizialmente utilizzata come animale modello più di 100 anni fa, rimane comunque un modello rilevante oggi grazie al suo ciclo vitale breve, il suo basso costo e la sofisticazione degli attrezzi molecolari disponibili per la sua manipolazione genetica. Questo modello ha anche meno ridondanza genetica rispetto ai mammiferi, rendendo lo studio della funzione di questi geni molto più diretto, ma malgrado questo possiede un ortologo per 75% dei geni legati a malattie umane. Tutte queste proprietà contribuiscono alla sua rilevanza come modello e sono state sfruttate durante questo progetto. In primis, Drosophila melanogaster è stata usata come modello per il trasporto mitocondriale di deossinucleotidi. Il gene RIM2 in lievito, che è stato precedentemente caratterizzato come trasportatore mitocondriale di deossinucleotidi pirimidinici, ha un omologo in Drosophila: CG18317, qui chiamato drim2, che è stato caratterizzato in questo progetto. Questo gene è stato rimosso in vivo e la funzione mitocondriale e l’integrità del mtDNA sono state caratterizzate. I due omologhi umani per questo gene, PNC-1 e SLC25A36, sono stati espressi nel mutante, per determinare l’omologia funzionale di questi geni e per confermare la validità di questo mutante come modello per il trasporto mitocondriale umano di deossinucleotidi. Questo progetto si è anche focalizzato su una caratterizzazione più approfondita di una linea mutante per dTTC19, un omologo di un gene umano che è già stato collegato alla encefalopatia mitocondriale e la psicosi. Questa caratterizzazione è stata accompagnata dalla generazione di tre linee mutanti che esprimono dTTC19, per confermare che il fenotipo mutante osservato sia dovuto alla mancanta trascrizione di dTTC19. In fine, questo progetto propone un nuovo protocollo che, nella nostra opinione, permetterà di utilizzare Drosophila melanogaster come modello per disturbi neurologici che presentano con sintomi asociali. Un protocollo è stato sviluppato per studiare il comportamento sociale in Drosophila melanogaster e per dimostrare che piccole differenze nei livelli di dopamina o nel contatto sociale dopo l’eclosione possono avere effetti drammatici sulle interazioni sociali in Drosophila. Proponiamo che Drosophila può essere un modello utile per lo studio dei fattori genici coinvolti nelle malattie che presentano con comportamento asociale come l’autismo, il disturbo ossessivo compulsivo, la depressione eccetera. In conclusione, questo progetto sfrutta interamente Drosophila melanogaster come animale modello per la biologia e le malattie mitocondriali. In più, propone un nuovo modo per utilizzare Drosophila come modello che non è stato finora sfruttato.

Le comportement sexuel des drosophiles est déterminé génétiquement, il fait intervenir différents mécanismes de communication (chimique: phéromones, acoustique: chant et visuel) et des comportements plus simples (activité locomotrice). Il a de plus été démontré que les enzymes finales de la synthèse de la dopamine (DDC: dopa décarboxylas et TH:tyrosine hydroxylase) étaient produites sous la cuticule et dans le SNC. Dans un premier temps, la recherche s'est portée sur d'éventuelles différences des hydrocarbures cuticulaires dues à la présence ou à l'absence de dopamine. Pour cela on utilise des approches pharmacologiques et génétiques: des inhibiteurs d'enzymes de la TH sur des souches sauvages, des souches mutantes (Ddc ts) complémentées avec les composés qui leur font défaut et des souches mutantes au sein des gènes de structure: Ddc et pale. On observe alors une diminution corrélée de la quantité de dopamine et des hydrocarbures femelles spécifiques. Par la suite, nous avons montré que ce défaut, comparable à l'effet de la décapitation est compensé par une application topicale unique chez ces femelles. Dans un deuxième temps, différents aspects du comportement ont été analysé. Nous avons montré que les mouches mâles ayant une quantité de dopamine et de sérotonine réduite ont une plus faible réussite à la copulation, une plus faible activité locomotrice et un chant modifié. La dopamine semble donc impliquée dans la régulation du comportement sexuel chez la drosophile à différents niveau: production phéromonale, production acoustique et activité locomotrice. Enfin, une étude des variations de production phéromonale en fonction de la température réalisée sur deux souches sauvages de type Drosophila melanogaster permet de conclure à une augmentation de la production des hydrocarbures à longues chaînes avec l'augmentation de la température
Sexual behavior in Drosophila is genetically determined. It implies different communication mechanisms (chemical : pheromones; acoustic : song and visual) and simpler behaviors ( like locomotor activity). In addition, it has been demonstrated that terminal enzymes for dopamine synthesis (DDC : dopa decarboxylase and TH : thyrosine hydroxylase) were formed under the cuticle and in the central nervous system. Initially, our research focused on potential differences in cuticular hydrocarbons, linked to the presence or not of dopamine. For that, genetical and pharmalogical approaches were used: TH enzyme inhibitor on wild type strains, mutant strains (Ddc ts) complemented with their lacking compounds and mutant strains for their structural genes: Ddc and pale. We observed a correlation between diminution of dopamine and of female-specific hydrocarbons. Then, we demonstrated that this defect, comparable to the decapitation effect, is compensated for the females by a single topical application of dopamine. We also analysed different behavioral aspects. We demonstrated that male flies having reduced levels of dopamine and serotonine have a lower success rate for copulation, a lower locomotor activity and a modified song. Dopamine seems to play a significant role in sexual behavior regulation at different levels : pheromons production, acoustic production, and locomotor activity. Finally, a study of pheromonal variations as a function of temperature bas been performed. It shows that higher temperatures favor the production of longer chains of hydrocarbons either in males and females in two wild type strains of D. Melanogaster

The present work focuses on the mechanisms of selection for the control of action in Drosophila melanogaster (D. melanogaster), also known as fruit fly. D. melanogaster has a rich repertoire of innate and learned behaviours and a quite simple brain, composed by roughly 100,000 neurons, which can be studied by means of sophisticated techniques. Therefore, it offers the possibility to study complex behaviour in a brain structure simpler than that of higher organisms. As a consequence the neurobiological underpinning of its behaviour can be understood in an easier manner. The comparison of its behaviour with similar behaviours shown by different and evolutively distant animals can provide important insights about their relationship with different or conserved underlying neural circuits. This thesis was conceptualized to sketch out whether selection for action processes underlying the behaviour of mammals might be shared with lower organisms such as D. melanogaster. Selection for action entails a close interaction between visual and motor systems allowing to select a specific stimulus in the environment to which act upon. This process allows to filter out irrelevant information for action. The first experiment was aimed at investigating whether flies have an action-based attention. Are flies able to inhibit via attentional mechanisms the response to an upcoming stimulus in order to successfully end an ongoing action? In particular, I observed whether flies are prone to interference effects caused by the upcoming appearance of a competitive stimulus (i.e., a distractor). I expected this inhibitory mechanism to be played out on spatial trajectories. In this study, flies were engaged in a walking task aimed at reaching a visual target (i.e., a bright stripe) while an abrupt identical distractor was laterally presented. The second experiment pointed at extending the finding of the first experiment. In particular, angular distances between target and distractor were considered. The aim of this study was to test the hypothesis that the shorter the distance between target and distractor the greater is the level of inhibition. Then, in the third experiment I targeted the hypothetical neural circuit underlying the behavioural effects observed in the previous experiments. Based on the increasing evidence for an intriguing homology between a specific neuropil of flies (Central Complex; CX) and the mammals’ neural structure involved in action selection, the idea was to test flies with a lesioned CX during the behavioural task used in the previous experiments. To do this, I used a technique based on the GAL4-UAS binary system in order to downregulate specific dopamine receptors in a very selected neural circuit, the so-called E-PG neurons. Moreover, I adopted an optogenetic technique for in vivo neural manipulation. I employed flies bearing light-sensitive ion channels in the same selected neural circuit of CX to briefly activate such neurons during the task. This neural circuit forms a donut-shape structure which it has been proposed to be an integrative circuit between visual and motor systems and to perform an attention-like function. Finally, in the fourth experiment I characterized a series of neural circuits of CX from a neurochemical perspective. The hypothesis was that the dopaminergic system, involved in the action selection process of mammals, could also modulate the neurophysiological response within the CX of flies. Specifically, I recorded in vivo the neural response to dopamine application in CX of flies by using a bioluminescence technique based on a genetically encoded calcium indicator. All in all, this work represents an attempt to tackle the mechanisms of selection for the control of action in flies. The interference paradigm I developed establishes a powerful platform to further explore the problem of selection for action in flies which might be useful for clarifying similar processes in higher organisms.
Il presente lavoro si focalizza sui meccanismi di selezione per il controllo dell’azione utilizzati da Drosophila melanogaster (D. melanogaster), anche nota come moscerino della frutta. D. melanogaster ha un ricco repertorio di comportamenti e un cervello semplice, composto di circa 100,000 neuroni, che può essere studiato con tecniche raffinate. Perciò, offre la possibilità di studiare un comportamento in una struttura cerebrale semplice rispetto a quella di organismi più complessi. Le basi neurobiologiche del suo comportamento possono essere così più facilmente comprese. Il confronto con comportamenti simili mostrarti da animali evolutivamente più lontani può fornire importanti intuizioni sui circuiti neurali sottesi. Questa tesi è stata concettualizzata per verificare se i processi di selezione per l’azione dei mammiferi potessero essere condivisi con organismi più bassi come D. melanogaster. La selezione per l’azione implica una stretta interazione tra sistema visivo e motorio che consente di scegliere uno stimolo nell’ambiente per agire su di esso. Questo processo permette di filtrare le informazioni irrilevanti per l’azione. Il primo esperimento era finalizzato a indagare se i moscerini mostrano un’attenzione basata sull’azione. Sono capaci di inibire la risposta a uno stimolo grazie a meccanismi attentivi per terminare un’azione? In particolare ho indagato se i moscerini sono inclini all’effetto d’interferenza causato dalla comparsa di uno stimolo competitivo (cioè, un distrattore). Mi aspettavo che questo meccanismo inibitorio fosse evidente nelle traiettorie spaziali. In questo studio i moscerini erano impegnati a raggiungere un target visivo (cioè, una striscia luminosa) mentre un distrattore compariva lateralmente. Il secondo esperimento mirava a estendere le conclusioni del primo esperimento. In particolare sono state considerate le distanze tra target e distrattore. L’obiettivo di questo studio era di verificare l’ipotesi che più corta è la distanza tra target e distrattore, più elevato è il livello d’inibizione. Nel terzo esperimento ho individuato l’ipotetico circuito neurale responsabile degli effetti comportamentali osservati negli esperimenti precedenti. Basandomi su crescenti evidenze in favore di un’affascinante omologia tra un neuropilo dei moscerini (il Complesso Centrale, CC) e la struttura neurale dei mammiferi implicata nella selezione dell’azione, ho testato il comportamento in moscerini con CC danneggiato. A questo scopo ho usato una tecnica basata sul sistema binario GAL4-UAS per ridurre l’espressione di specifici recettori dopaminergici in un circuito neurale molto selettivo, i cosiddetti neuroni E-PG. Inoltre, ho adottato una tecnica optogenetica per la manipolazione neurale in vivo. Ho utilizzato moscerini che esprimevano canali ionici foto-attivabili nello stesso circuito del CC per eccitare tali neuroni durante il compito. Questo circuito neurale forma una struttura a ciambella che è stata considerata un centro d’integrazione tra il sistema visivo e motorio deputato all’attenzione. Infine, nel quarto esperimento, ho caratterizzato da un punto di vista neurochimico una serie di circuiti neurali del CC. L’ipotesi era che il sistema dopaminergico implicato nel processo di selezione dell’azione nei mammiferi potesse modulare anche nei moscerini la risposta neurofisiologica all’interno del CC. Nello specifico, ho registrato in vivo la risposta neurale ad applicazioni di dopamina nel CC dei moscerini utilizzando una tecnica di bioluminescenza basata su indicatori del calcio geneticamente codificati. Nel complesso questo lavoro rappresenta un tentativo di affrontare i meccanismi di selezione per il controllo dell’azione nei moscerini. Il paradigma d’interferenza che ho sviluppato costituisce una potente piattaforma per esplorare il problema della selezione per l’azione che potrebbe essere utile al fine di chiarire processi simili in organismi più complessi.

Les Drosophiles à jeûn peuvent être conditionnées dans le but d’associer une odeur avec du sucre. Un cycle de conditionnement appétitif induit la formation de mémoire à court terme (MCT) et de mémoire à long terme (MLT). Ainsi, nous avons montré que les MCT et MLT sont formées indépendamment l’une de l’autre et impliquent des structures neuronales distinctes au sein des Corps Pédonculés (CPs), le centre de la mémoire olfactive. Nous avons proposé un nouveau modèle de la dynamique des phases de mémoire appétitive où la formation des MCT et MLT s’effectue de manière parallèle. Suite à cette étude, nous avons identifié deux paires de neurones extrinsèques aux CPs impliqués dans la restitution de l’information appétitive à long terme. Enfin, nous nous sommes intéressés aux mécanismes moléculaires et aux réseaux neuronaux impliqués dans la consolidation de la mémoire aversive. Chez la Drosophile, l’appariement d’une odeur à des chocs électriques deux formes de mémoires consolidées, la mémoire résistante à l’anesthésie (MRA) et la MLT (dépendante de la synthèse protéique de novo). Nous avons montré que 3 paires de neurones dopaminergiques aux CPs jouent un rôle «d’interrupteur» contrôlant une bascule entre la MRA et la MLT. Ainsi, bloquer ces trois paires de neurones dopaminergiques durant la période de consolidation induit une augmentation de la MRA et une inhibition de la MLT, alors qu’activer ces neurones après conditionnement entraîne une inhibition de la MRA, et favorise la mise en place de la MLT. En conclusion, nous avons caractérisé fonctionnellement des ensembles neuronaux discrets jouant un rôle dans différentes étapes de l'apprentissage et de la mémorisation olfactifs
When we present an odor associated with sugar to starved flies, they will be attracted by this odor. One cycle of conditioning induces both Short-Term Memory (STM) and Long-Term Memory (LTM). It is accepted that STM and LTM formation is a sequential process but the link between these two memories remains unknown. We adressed this question and clearly demonstrated that STM and LTM can be formed independently and that they involved different neural structures within the Mushroom Bodies (MB), a memory center. We proposed a new model of dynamic of appetitive memory phases where STM and LTM are formed in a parallel way. Then, using the genetically expressed thermosensible toxine allowing a transiently inactivation of neurotransmission, we identified one type of MB efferent neurons involved in appetitive LTM retrieval. Additionally, we were interested to the molecular mechanisms and the neuronal circuits involved in aversive consolidated memories. Pairing an odor with electric shocs induces aversive memory. In drosophila, there are two forms of consolidated memories, the Anesthesia-Resistant Memory (ARM) and LTM (dependent on de novo protein synthesis). We show that three pairs of oscillatory dopaminergic neurons play a essential role of gating between ARM and LTM formation. So, blocking the neurotransmission of these neurons during the consolidation phase leads to a increase of ARM and inhibition of LTM whereas, artificial activation of these neurons after conditioning leads to an inhibition of ARM and favors the implementation LTM. In conclusion, we characterized functionally a restricted population of neurons playing a role in various stage of learning and memory process

The transport of organic cations (OCs) is important for lowering an organism’s susceptibility to the toxic effects of endogenous and exogenous OCs. Endogenous OCs include choline and N¹-methylnicotinamide (NMN) and catecholamines, while exogenous OCs may include xenobiotics, such as drugs, pesticides, and environmental toxins. These potentially toxic compounds must be eliminated from the organism to ensure survival. The midgut and Malpighian tubules of insects have been shown to be involved with the active transport of OCs. Two putative organic cation-like transporters, named orct and orct2, have previously been identified and cloned from adult Drosophila melanogaster. Previous quantitative real-time polymerase chain reaction studies showed mRNA transcripts of orct and orct2 were differentially expressed in the midgut and Malpighian tubules, and expression patterns increased following exposure to prototypical type I OC, tetraethylammonium (TEA). These findings suggest that the recently cloned Drosophila orct and orct2 will function as a transport protein for the OC substrate TEA. In this study, a dual-function expression vector, pXOOM, was used to design a molecular construct for the heterologous expression and functional characterization of Drosophila ORCT in Xenopus laevis oocytes. The kinetic profile of the expressed ORCT for TEA was determined using [¹⁴C]-labeled TEA assays. Analysis of the TEA functional assays revealed that insect ORCT is capable of a saturable, carrier-mediated transport of TEA. Maximal transport capacity (Jmax) and transport affinity for mediated TEA uptake (Kt) were 5 µmol L-¹ per oocyte and 0.33 mmol L-¹, respectively. ORCT-mediated TEA uptake was inhibited in the presence of type I and type II OCs. Quinine and verapamil inhibited TEA influx by 33 and 43%, respectively, whereas cimetidine and vinblastine did not reduce TEA uptake. These experiments coincide with previous physiological research on OC transport across insect renal tissues. The knowledge gained from this thesis may provide the basis for the development of an effective and environmentally benign insecticide, as well as allow a greater understanding of the deleterious effects of environmental pollutants on insect populations.
Irving K. Barber School of Arts and Sciences (Okanagan)
Biology, Department of (Okanagan)
Graduate

Dopamine (DA) governs movement, sleep, reward, and cognition. The presynaptic dopamine transporter (DAT), clears released DA, controlling DA signaling and homeostasis. Genetic DAT ablation causes hyperactivity, sleep reduction, and altered psychostimulant response. DAT surface expression is dynamic; DAT constitutively internalizes and recycles to and from the plasma membrane, and acute PKC activation stimulates DAT endocytosis. Cell line experiments demonstrated that PKC-stimulated DAT endocytosis requires Ack1 inactivation and the GTPase, Rit2. How Rit2 controls PKC-dependent DAT internalization, or whether regulated DAT endocytosis impacts behavior, is unknown. Here, I present data supporting that PKC activation stimulates Rit2/DAT dissociation, mediated by the DAT N-terminus. Further, Ack1 and Rit2 function independently to facilitate PKC-stimulated DAT internalization. Moreover, PKC-stimulated DAT endocytosis was limited to ventral striatum in ex vivo slice preparations, and required Rit2. Our lab previously demonstrated that certain DA-dependent behaviors required DAergic Rit2 in mice, however whether this was due to perturbed PKC-stimulated DAT internalization, or DAT-independent Rit2 function(s) remains untested. To address this, I turned to Drosophila and its Rit2 homolog Ric. I found that Ric and dDAT proteins interact in cell lines, and that constitutively active Ric (RicQ117L) increased dDAT function in cultured cells and ex vivo whole fly brains. However, neither DAergic Ric knockdown nor RicQ117L altered overall locomotion or sleep, suggesting that these fundamental behaviors do not require DAergic Ric. Together, these results expand our understanding of intrinsic mechanisms controlling DAT endocytosis, and their impact on behavior.

La copie de partenaire, ou "mate-copying" est un comportement bien documenté chez de nombreuses espèces, parmi lesquelles des animaux en apparence aussi rudimentaires que Drosophila melanogaster. Chez cette espèce d'insecte, lorsqu'une femelle observe une autre femelle s'accoupler avec un mâle d'un certain phénotype, sa préférence pour les mâles de ce phénotype augmente. Autrement dit, elle copie la préférence de partenaire de la femelle démonstratrice. Ce comportement constitue un modèle d'apprentissage social observationnel que l'on peut exploiter tant au niveau des mécanismes proximaux (par exemple comportementaux et neurobiologiques) que distaux (par exemple pour son influence sur l'évolution). Dans ce travail, ces deux aspects du mate-copying sont abordés. Le premier chapitre de ma thèse étudie la stabilité de cette stratégie de choix de partenaire en fonction de conditions environnementales sociales, particulièrement sur la disponibilité apparente des mâles, et sa stabilité dans le temps (mémoire à long terme). J'ai montré que les femelles adaptent leur sélectivité en fonction de la disponibilité apparente des mâles, mais sans impact sur leur capacité à copier le choix de la femelle démonstratrice. J'ai aussi contribué à montrer que les femelles peuvent former une mémoire sociale à long terme (24h) impliquant la synthèse protéique. Les deuxième et troisième chapitres abordent les mécanismes cognitifs du mate-copying. Ainsi, j'ai montré que les neurotransmetteurs dopamine et sérotonine sont impliqués dans cet apprentissage ; j'ai montré également que le récepteur dopaminergique DAMB (DopAmine Mushroom Bodies) est requis pour cette mémoire sociale à long terme, mais pas à court terme, suggérant l'implication d'un autre récepteur dopaminergique que DAMB dans cet apprentissage social. J'ai enfin élaboré un nouveau protocole de démonstrations basé sur des photographies, qui contribuera à la caractérisation plus efficace des signaux visuels nécessaires, et à moyen terme, des mécanismes neurobiologiques. Enfin, j'ai montré que le mate-copying est un apprentissage basé sur le trait du mâle impliqué dans l'acceptation et non le rejet par la femelle démonstratrice, et impliquant des réseaux neuronaux dopaminergiques en jeu dans l'apprentissage aversif olfactif
Mate-copying has been reported in many Vertebrate and Invertebrate species, including animals as simple in appearance as Drosophila melanogaster. In this species, when a female observes another female mating with a male of a given phenotype, his attraction to other males of this phenotype increases. In other words, she copies the mate preference of the demonstrator female. This behavior constitutes a powerful model of social observational learning in animals, both for proximate mechanisms (for instance behavioral and neurobiological) as well as ultimate mechanisms (notably, as it takes part to sexual evolution). The present work studied these two aspects of mate-copying. The first chapter tested the stability of mate-copying across environmental social conditions, more specifically, apparent availability of males, and across time (long-term memory). I showed that, while sex-ratio affects female choosiness positively, Drosophila females seem to have evolved a mate-copying ability independently of sex-ratio. I also participated in showing that females can form a social long-term memory (24h) involving protein synthesis. Chapters 2 and 3 deal with cognitive mechanisms in mate-copying. I showed that it involves the neurotransmitters dopamine and serotonine, while the dopaminergic receptor DAMB (DopAmine Mushroom Bodies) is required for this social long-term memory, but not for short-term memory, which suggests that another dopaminergic receptor is also involved in this social learning. I designed and tested a new protocol of demonstrations based on photographs, which will ease the study of the visual cues necessary for this behavior, and later the study of the neurobiological mechanisms. Finally, I showed that mate-copying is a learning based on on the trait of the male accepted by the demonstrator female, and not on the rejected one, and I found that, counter-intuitively, dopaminergic networks involved are those for aversive, not appetitive, olfactory learning

Evaluating our environment by deciding what is beneficial or harmful, pleasant or punishing is a part of our daily lives. Seeking pleasure and avoiding pain is a common trait all mobile organisms exhibit and understanding how rewarding stimuli are represented in the brain remains a major goal of neuroscience. Studying reward learning in the fruit fly, Drosophila melanogaster has enabled us to better understand the complex neural circuit mechanisms involved in reward processing in the brain. By conditioning flies with sugars of differing nutritional properties, we determined that flies trained with sweet but non-nutritive sugars formed robust short-term memory (STM), but not long-term memory (LTM). However, flies conditioned with a sweet and nutritious sugar or a sweet non-nutritious sugar supplemented with a tasteless nutritious compound, formed robust 24 hour LTM. These findings led us to propose a model of parallel reinforcement pathways for appetitive olfactory conditioning in the fly, in which both sweet taste and nutrient value contribute to appetitive long-term memory. We followed this line of research by examining the neural circuitry in the fly brain that represents these parallel reward pathways. We found that the biogenic amine octopamine (OA) only represents the reinforcing effects of sweet taste. Stimulation of OA neurons could replace sugar in olfactory conditioning to form appetitive STM. Surprisingly, implanting memory with OA was dependent on dopamine (DA) signaling, which although being long associated with reward in mammals, was previously linked with punishment in flies. We found that OA-reinforced memory functions through the α-adrenergic OAMB receptor in a novel subset of rewarding DA neurons that innervate the mushroom body (MB). The rewarding population of DA neurons is required for sweet and nutrient reinforced memory suggesting they may integrate both signals to drive appetitive LTM formation. In addition, OA implanted memory requires concurrent modulation of negatively reinforcing DA neurons through the β-adrenergic OCTβ2R receptor. These data provide a new layered reward model in Drosophila in which OA modulates distinct populations of both positive and negative coding DA neurons. Therefore, the reinforcement system in flies is more similar to that of mammals than previously thought.

A central theme of this dissertation is nervous system plasticity. Activity-dependent plasticity and dopaminergic modulation are two processes by which neural circuits adapt their function to developmental and environmental changes. These processes are involved in basic cognitive functions and can contribute to neurological disorder. An important goal in modern neurobiology is understanding how genotypic variation influences plasticity, and leveraging the quantitative genetics resources in model organisms is a valuable component of this endeavor. To this end I investigated activity-dependent plasticity and dopaminergic modulation in Drosophila melanogaster larvae using neurobiological and genetic approaches. Larval mechanosensory behavior is described in Chapter 2. The behavioral experiments in that chapter provide a system to study mechanisms of plasticity and decision-making, while the electrophysiological characterization shows that sensory-motor output depends on neural activity levels of the circuit. This system is used to investigate activity-dependent plasticity in Chapter 3, i.e., habituation to repetitive tactile stimuli. In Chapter 4, those assays are combined with pharmacological manipulations, genetic manipulations, and other experimental paradigms to investigate dopaminergic modulation. Bioinformatics analyses were used in Chapter 5 to characterize natural genetic variation and the influence of single nucleotide polymorphisms on dopamine-related gene expression. The impact and suggested future directions based on this work are discussed in Chapter 6. Dopamine also modulates cardiomyocytes. Chapter 7 describes biochemical pathways that mediate dopaminergic modulation of heart rate. The final two chapters describe neurobiology research endeavors that are separate from my work on dopamine. Experiments that have helped characterize a role for Serf, a gene that codes for a small protein with previously unknown function, are described in Chapter 8. In the final chapter I describe optogenetic behavioral and electrophysiology preparations that are being integrated into high school classrooms and undergraduate physiology laboratories. Assessment of student motivation and learning outcomes in response to those experiments is also discussed.

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Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum no mundo, afetando cerca de 1% dos adultos com mais de 60 anos. A DP está relacionada com a degeneração de neurônios dopaminérgicos principais componentes da substância negra cerebral, concomitantemente, com a disfunção do complexo I mitocondrial e o estresse oxidativo que desempenham um papel crucial na patogênese desta doença. A rotenona (ROT) é um pesticida natural e muito utilizado para induzir fenótipo de DP em modelos animais, por ser lipofílico pode atravessar facilmente a barreira hematoencefálica causando disfunção do complexo I mitocondrial e possível morte de neurônios dopaminérgicos. Dentre as várias aplicações terapêuticas dos antioxidantes, ressalta-se sua ação neuroprotetora, uma vez que o sistema nervoso central exibe uma maior vulnerabilidade e susceptibilidade aos insultos oxidativos, o γ-orizanol (ORY) é um produto natural composto por uma mistura de ésteres de ácido ferúlico extraídos a partir do óleo de farelo de arroz, e é bem descrito na literatura por possuir propriedades antioxidantes. Assim, o objetivo deste trabalho foi investigar um possível efeito neuroprotetor do γ-orizanol sobre alterações comportamentais e bioquímicas causadas pela exposição crônica de Drosophila melanogaster a rotenona. A mosca da fruta Drosophila melanogaster, é uma espécie alternativa à utilização de modelos mamíferos que vem sendo usada com bastante confiabilidade na reprodução de modelos de disfunção dopaminérgica. As moscas (de ambos os sexos) com idades compreendidas entre 1 a 5 dias de idade foram divididos em quatro grupos de 50 moscas cada um: (1) de controle, (2) ORY 25 μM, (3) ROT 500 μM, (4) ORY 25 μM + ROT 500 μM. As moscas foram concomitantemente expostos a uma dieta contendo ROT e ORY durante 7 dias de acordo com os seus respectivos grupos. Após o tratamento foram feitas as analises comportamentais e bioquimicas. Como resultados, verificamos que o ORY ofereceu proteção contra as alterações locomotoras causadas por ROT, além de prevenir a mortalidade induzida por rotenona, protegeu contra geração de estresse oxidativo e disfunções mitocondriais além de otimizar as defesas antioxidantes celulares. Nossas descobertas apontam uma restauração dos déficits colinérgicos, e nos níveis de dopamina fornecida por pelo tratamento com ORY. Em conclusão, os presentes resultados mostram que ORY é eficaz na redução da toxicidade induzida ROT em Drosophila melanogaster, o que mostrou uma ação neuroprotetora, possivelmente devido à presença dos componentes de antioxidantes tais como o ácido ferúlico.
Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, affecting about 1% of adults over 60 years. The DP is linked to the degeneration of dopaminergic neurons main components of the substantia nigra brain, concurrently with mitochondrial complex I dysfunction and oxidative stress play a crucial role in the pathogenesis of this disease. The rotenone (ROT) is a natural pesticide, and used to induce PD phenotype in animal models, being lipophilic can easily cross the blood-brain barrier dysfunction causing mitochondrial complex I and possible death of dopaminergic neurons. Among the various therapeutic applications of antioxidants, it emphasizes its neuroprotective action, as the central nervous system displays a greater vulnerability and susceptibility to oxidative insults, the γ-oryzanol (ORY) is a natural product composed of a mixture of esters ferulic acid derived from rice bran oil, and is well described in the literature to possess antioxidant properties. The objective of this study was to investigate a possible neuroprotective effect of ORY on behavioral and biochemical changes caused by chronic exposure of Drosophila melanogaster the rotenone. The fly fruit Drosophila melanogaster, is an alternative species to the use of mammalian models that have been used quite reliable reproduction of dopaminergic dysfunction models. The flies (male and female) between the ages of 1 to 5 days of age were divided into four groups of 50 flies each: (1) control, (2) ORY 25 μM, (3) ROT 500 μM ( 4) ORY 25 μM + ROT 500 μM. The flies were simultaneously exposed to a diet containing ROT and ORY for 7 days according to their respective groups. After treatment were made behavioral and biochemical analysis. As a result, we find that the ORY offered protection against locomotor changes caused by ROT, and to prevent the mortality induced by rotenone, protected against the generation of oxidative stress and mitochondrial dysfunction and optimize cellular antioxidant defenses. Our findings point to a restoration of cholinergic deficits, and dopamine levels provided by the treatment ORY. In conclusion, the present results show that ORY is effective in reducing the ROT induced toxicity in Drosophila melanogaster, which showed a neuroprotective effect, possibly due to the presence of antioxidants components such as ferulic acid.

Lors d’une infection microbienne, la défense de l’hôte comprend deux facettes complémentaires. Premièrement, le système immunitaire cible les pathogènes dans le but de les éliminer, une attaque correspondant à la résistance. Dans un second temps, l’organisme doit réparer les dégâts causés par le pathogène ou par la réponse immunitaire de l’hôte, un mécanisme appelé résilience. J’ai étudié les effets d’une infection intestinale par la bactérie Serratia marcescens chez la drosophile. Nous avons mis en évidence un processus de purge dans l’intestin, lors duquel les enterocytes -les cellules principales de l’intestin- se vident partiellement de leur contenu. L’épithélium intestinal devient alors très fin mais se régénère rapidement, protégeant ainsi la mouche des effets délétères de l’infection. J’ai identifié un transporteur d’acides aminés, CG1139, qui est nécessaire à la régénération de l’intestin. CG1139 est requis pour la mobilisation de certaines réserves métaboliques de la drosophile et pour le transport rétrograde de ces dernières vers l’intestin
Upon microbial infections, host defenses comprise two complementary facets. First, immune effectors target and kill the invading pathogen, an attack referred to as resistance. Second, the infected host must repair the damages inflicted by microbes or by the immune response itself, a mechanism called resilience. I have studied the effects of an intestinal infection with the bacterium Serratia marcescens in Drosophila. We have discovered a purge mechanism in the intestine, where enterocytes -the main cell type in the gut- extrude some of their internal contents. The intestinal epithelium thus becomes very thin but rapidly recovers its shape, thereby protecting the fly against the deleterious effects of infection. I have identified an amino acid transporter, CG1139, which is required for the intestinal recovery. CG1139 is necessary to mobilize the fly’s internal metabolic reserves and to transport some these metabolic stores back to the gut, in a retrograde manner

Les acides aminés ont de nombreuses fonctions dans l’organisme en plus de leur rôle comme constituants élémentaires des protéines. Ils peuvent par exemple servir de neurotransmetteur ou de signal pour l’activation de voies de signalisation intracellulaires. Leur passage à travers la membrane plasmique est facilité par des transporteurs de la famille des protéines SLC. Les transporteurs hétérodimériques d’acides aminés HAT appartiennent aux SLC. Les HAT sont constitués d’une chaîne légère SLC7 assurant la spécificité de transport et d’une chaîne lourde SLC3 impliquée dans l’adressage du complexe protéique à la membrane. Ma thèse a porté sur l’étude du rôle d’un homologue de SLC7 chez la drosophile, Minidiscs (Mnd), dans le fonctionnement du système nerveux. Mnd appartiendrait aux transporteurs du système L, principalement connus pour leur rôle dans la prolifération cellulaire. Mes travaux de thèse ont permis de mettre en évidence la localisation de Mnd dans le cerveau de drosophile dans certains neurones (corps pédonculés, neurones dopaminergiques) et dans certaines cellules gliales (glie corticale). La présence de Mnd dans le cerveau semble intervenir dans la modulation de certains comportements, tels que le réflexe de géotaxie négative. Ces travaux ont aussi montré que, comme les HAT de mammifères, Mnd s’associe de façon covalente à un partenaire protéique. Les expériences de transport semblent par ailleurs confirmer l’appartenance de Mnd au système L.Ces résultats suggèrent que Mnd est probablement impliqué dans la régulation de l’activité neuronale et donc dans le fonctionnement du système nerveux, ce qui n’avait encore jamais été décrit pour un transporteur du système L
Amino acids have many functions in the body in addition to their role as basic constituents of proteins. They can for example serve as a neurotransmitter or signal for the activation of intracellular pathways. Carriers of the SLC protein family facilitate their path through the plasma membrane. The heterodimeric amino acid transporters HAT belong to SLC proteins. HAT are composed of a light chain SLC7 ensuring the specificity of transport and a heavy chain SLC3 involved in the addressing of the protein complex to the plasma membrane. My thesis focused on studying the role of a SLC7 homologue in drosophila, Minidiscs (Mnd), in the functioning of the nervous system. Mnd might belong to system L carriers, mainly known for their role in cell proliferation. My thesis work led to highlight the location of Mnd in the drosophila brain in some neurons (mushroom bodies, dopaminergic neurons) and some glial cells (cortical glia). The presence of Mnd in the brain seems to be involved in the modulation of some behaviors such as negative geotaxis reflex. This work also showed that, as for mammal HAT, Mnd is associated covalently to a protein partner. Transport experiments seem also to confirm the belonging of Mnd to the system L. These results suggest that Mnd is probably involved in the regulation of neuronal activity and thus in the functioning of the nervous system, which had never been described for a system L carrier

Le but de cette thèse était de comprendre le rôle des protéines de la famille ELAV/Hu dans le développement, le maintien et la fonction des neurones par l’analyse phénotypique de mutants de dérégulation d'elav/Hu sur deux organismes modèles. Chez le xénope, nous avons montré que 3 homologues de la famille elav/Hu (elrB, elrC et elrD) sont exprimés de façon différentielle au cours du développement du système nerveux, suggérant un rôle dans différentes phases de la maturation des neurones et la définition de différents sous-domaines neuraux. La dérégulation de l’expression d'elrB dans les embryons de xénope réduit l'expression de marqueurs neuraux précoces, stoppe la prolifération cellulaire et induit l’apoptose, suggérant un rôle dans la régulation post-transcriptionnelle de gènes impliqués dans le contrôle du cycle cellulaire et/ou l'apoptose. Chez la drosophile, l’expression ubiquitaire d’elav obtenue grâce au système UAS/Gal4 produit un phénotype de dépigmentation de la cuticule et des soies similaire à celui de mutants de la voie de biosynthèse de la dopamine (DA ; neuromodulateur et intermédiaire de synthèse de la cuticule chez la drosophile). Ce phénotype de dépigmentation suggère qu'elav régulerait l'obtention de la tyrosine hydroxylase (TH) neurale qui contrôle la 1ère étape de la biosynthèse de DA. Ce travail suggère que les protéines ELAV/Hu participent au contrôle du cycle cellulaire et/ou de l'apoptose en plus de leur rôle dans la différenciation des neurones. L'identification de la TH comme cible potentielle d'ELAV suggère un rôle dans le contrôle de la synthèse de la DA, établissant ainsi un lien entre la présence d'ELAV et la fonction du système nerveux
The goal of this thesis was to understand the role of ELAV/Hu family proteins in the development, maintenance and function of neurons, using phenotypic analyses of deregulation mutants in two models. In Xenopus, we showed that 3 elav/Hu family homologs (elrB, elrC, and elrD) are differentially expressed during nervous system development, suggesting a role in different phases of neuron maturation and the definition of different neural sub-domains. Deregulating elrB expression in Xenopus embryos reduced early neuronal markers expression, stopped cell proliferation, and induced apoptosis, suggesting a role in post-transcriptional regulation of genes controlling cell cycle and/or apoptosis. In Drosophila, ubiquitous expression of elav, obtained with the UAS/Gal4 system, produced a phenotype of cuticle and bristles depigmentation. This phenotype, similar to that of mutants of dopamine (DA; neuromodulator and cuticle biosynthesis intermediate in Drosophila) biosynthesis. This depigmentation phenotype suggests that elav could regulate production of neural tyrosine hydroxylase (TH), which control the first step of DA biosynthesis. This work suggests that ELAV/Hu proteins participate in the control of cell cycle and/or apoptosis in addition to their role in neuron differentiation. Identification of TH as a potential target of ELAV suggests a role in controlling the biosynthesis of DA, thus establishing a link between ELAV presence and its function in the nervous system

In der vorliegenden Arbeit wurde der Einfluss von Dopamin, Serotonin und GABA auf das Schlafverhalten von Drosophila melanogaster genauer untersucht. Mit Hilfe von Mutanten in Wiederaufnahmetransportern für Dopamin und Serotonin konnte gezeigt werden, dass Dopamin und Serotonin entgegengesetzte Wirkungen auf die Schlafmenge der Fliegen haben. Dopamin hat eine schlafhemmende, Serotonin eine schlaffördernde Wirkung. Die Nutzung eines neuronal dopamindefizienten Fliegenstammes erweitert diese Erkenntnisse. Die Nutzung von RNAi zur Hinunterregulierung der Rezeptoren für Dopamin brachte keine weiteren Erkenntnisse, da sie zu keinem messbaren Effekt führen. Jedoch ergab eine parallel dazu durchgeführte Hinunterregulierung des GABABR2 Rezeptors, dass dieser maßgeblich für die Aufrechterhaltung des Schlafes in der zweiten Hälfte der Nacht verantwortlich ist. Es konnte gezeigt werden, dass für diese Aufgabe vor allem ihre Expression in den l-LNv Neuronen relevant ist. Dabei ist für die GABABR2 Rezeptoren kein Effekt, für Dopamin und Serotonin nur in geringen Ausmaß ein Effekt auf die Innere Uhr in Form von gering veränderter Periode zu beobachten. Durch eine Kombination der Transportermutanten für Dopamin und Serotonin mit dem intakten, als auch mutierten WHITE Transporter zeigte sich eine interessante Interaktion dieser drei Transporter bei der Regulation der Gesamtschlafmenge, wobei die white Mutation zu einer Reduzierung der Gesamtschlafmenge führt. UPLC Messungen der Stämme ergaben, dass der Effekt von white vermutlich auf dessen Einfluss auf den beta-Alanyldopamingehalt der Fliegen basiert. beta-Alanyldopamin wird bei dem Transport von Dopamin über die Gliazellen durch das Enzym EBONY gebildet, dessen Mutation in der Kombination mit intaktem WHITE und mutiertem Dopamintransporter zu einer drastischen Reduktion des Schlafes während der Nacht führt. Im Rahmen der Untersuchung konnte zudem gezeigt werden, dass entgegen des bisherigen Wissens aus Zellkulturstudien in Drosophila melanogaster kein beta-Alanylserotonin gebildet wird. Möglicherweise wird nur Dopamin, nicht jedoch Serotonin über die Gliazellen recycelt. Dies ist ein interessanter Unterschied, der sowohl eine zeitliche, als auch lokale Feinregulation der Gegenspieler Dopamin und Serotonin ermöglicht. Die Untersuchung der Dimerpartner BROWN und SCARLET zeigte, dass lediglich BROWN zu einer Reduktion des Schlafes führt. Ein Effekt, der auch in einer Fliegenlinie mit spontaner white Mutation beobachtet werden konnte. Die genaue Funktion dieses Heterodimertransporters und seine neuronale Lokalisation wurden im Rahmen dieser Arbeit noch nicht geklärt. Dennoch liegt eine Funktion als Dopamin- oder beta-Alanyldopamintransporter in Gliazellen auf Grund der ermittelten Ergebnisse nahe. Zusätzlich konnte zum ersten Mal in Drosophila melanogaster eine Funktion der Amintransporter bei der Anpassung der Inneren Uhr an extreme kurze bzw. lange Photoperioden gezeigt werden. Eine anatomische Lokalisierung des WHITE Transporters im Gehirn von Drosophila melanogaster, die weitere Charakterisierung der Rolle des WHITE/BROWN Dimers und die Zuordnung bestimmter dopaminerger und serotonerger Neurone bei der Modulation der Aktivitätsmaxima stellen spannende Fragen für zukünftige Arbeiten dar
The main focus in the present work, was the observation of the influence of dopamine, serotonin and GABA on the sleep behaviour of Drosophila melanogaster. By utilizing mutants for the dopamine transporter as well as the serotonin transporter, it was possible to show, that dopamine and serotonin have opposing effects on the total sleep amount of flies. Dopamine has a sleep inhibiting, serotonin a sleep promoting function. A neuronal dopamine deficient stock complemented those findings. Usage of RNAi to downregulate dopamine receptors did not enhance the information, since no measurable effect could be detected. But in parallel performed experiments with RNAi mediated knockdown of GABABR2 receptors could show its role in the maintenance of sleep during the second half of the night. I could show that especially the expression in the l-LNv is needed for that. In case of the GABABR2 receptors no effect on the period was observed, for dopamine and serotonin only a minor effect on the clock in form of a mild period change accompanied those drastic sleep phenotypes. Combining the amine transporter mutants with functional as well as mutated white led to some interesting observations regarding the interaction of those transporters in regulating total sleep, in which white reduces the total sleep amount. Following up those experiments with UPLC measurements, it was shown that presumably WHITE causes its effect due to its relevance for the amount of beta-alanyldopamine in adult flies. When dopamine is transported into the glia cells, beta-alanyldopamine is synthesized by the enzyme EBONY. The ebony mutant revealed a drastic sleep phenotype when combined with an intact WHITE transporter and a mutated dopamine transporter. This leads to a dramatic decrease of sleep during the night phase. When doing the UPLC measurements it was furthermore revealed, that unexpectedly regarding the knowledge from cell culture experiments, beta-alanylserotonin cannot be detected. Presumably, only dopamine, but not serotonin is recycled by the glia cells. This interesting difference gives space for a temporal as well as for a local fine regulation of the dopamine and serotonin signals. Investigating the dimer partners of WHITE, BROWN and SCARLET, I found that BROWN just as a spontaneous white mutation that I observed, led to a decrease of total sleep. The function of this heterodimer and its neuronal localisation in the brain remains unknown. Regarding the data presented in this work, it is likely that this dimer transports either dopamine or beta-alanyldopamine in glia cells. Furthermore, I could observe that dopamine and serotonin change the ability of the circadian clock to adapt to different photoperiods, a so far unstudied phenotype. 96 An anatomical approach to localize the WHITE transporter in the brain of Drosophila melanogaster and a further characterization of the function of the WHITE/BROWN dimer, with regard to sleep and eventually the mapping of serotonergic and dopaminergic neurons, which modulate the activity peak responses, are questions for future work

博士
國立清華大學
生物資訊與結構生物研究所
101
Melatonin is a major hormonal mediator of light-induced photoperiodic changes in circadian biological events and is found in bacteria, protozoa, macroalgae, plants, fungi, invertebrates, and vertebrates. Despite of the structural characterization of melatonin, there has been increasing interest in this “molecular pacemaker” hormone. The physiological roles of melatonin have been widely reported on sleep, mood, immune response, cardiovascular fitness and aging. Research on melatonin biosynthesis could help improve our knowledge of circadian rhythm. The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an arylalkylamine N-acetyltransferase, which is involved in melatonin formation, sclerotization, and neurotransmitter inactivation and has been found in the head, the eyes, the optic lobe and the brain of Drosophila melanogaster. Moreover, Dat belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes the transfer of the acetyl group in acetyl coenzyme A (AcCoA, cofactor) to various arylalkylamines (substrate). In order to unravel the detail molecular mechanism for Dat activity, we worked on the structural and functional studies of Dat. We have determined high-resolution crystal structure of D. melanogaster Dat in apo form, binary complex form (AcCoA-bound), and ternary complex form (acetylarylalkylamine/CoA-bound). A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the binary complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism where nucleophilic attack and leaving group protonation occur in a coordinated manner dependent on catalytic triad. The molecular basis of substrate recognition and the kinetic mechanism by which Dat interacts with substrate and cofactor are unclear. Here, two-substrate kinetic analysis and dead end analog inhibition studies with the tryptophol and palmitoyl CoA indicated that Dat utilizes an ordered sequential mechanism requiring binding of acetyl-CoA first. Furthermore, we presented the first crystal structure of ternary complex in this AANAT family. Detailed analyses of ternary complexes of Dat revealed a hydrophobic substrate-binding pocket near the acetylation active site. The shape and size of the pocket dictate substrate selectivity and specificity. We have mapped two key aromatic residues in the protein-substrate interface essential for substrate binding and selection between phenylalkylamines (PAAs), indoalkylamines (IAAs), and arylamines substrates. The Dat has higher activity with the PAAs than with the IAAs. It appears that the aromatic ring and alkyl chain length on arylalkylamine molecule greatly define the Dat extended substrate specificity profile. By analyzing ternary complex structure as well as site-directed mutagenesis, we demonstrated that Phe43 significantly influence the substrate binding and the activity of Dat, while Tyr64 was an important determinant of substrate preference. Kinetic studies confirmed that the Y64W mutation is sufficient to increase the activity of the enzyme toward IAAs being the preferred substrate for the Y64W mutant, which indicated that this residue modulates the substrate preference of Dat between PAAs and IAAs. These results confirmed that these residues are critical for aromatic interaction between Dat and substrate. This work provides a structural foundation for the detailed understanding of the structural and biological properties of arylalkylamine N-acetyltransferases and of GCN5-related N-acetyltransferase superfamily proteins in general.

碩士
國立清華大學
生物資訊與結構生物研究所
102
Drosophila melanogaster dopamine N-acetyltransferase (Dat, EC 2.3.1.87) belongs to the arylalkylamine N-acetyltransferase (AANAT) family, which catalyzes the synthesis of the hormone precursor (melatonin). We have solved the structures of Dat in apo form, binary complex (Dat / acetyl coenzyme A) and ternary complex form (Dat / acetylarylalkylamine / CoA) and proposed the catalytic mechanism previously. According to the binding study by isothermal titration calorimetry (ITC), the cofactor (Acetyl-CoA) needed to bind to the Dat prior to substrate, which would hinder the substrate entry to its binding site. Therefore, we speculate that an entry tunnel for substrate may exist to facilitate the substrate binding to the active site. In this study, we replaced two residues with tryptophan, M121 and D142, located inside the tunnel to see the effects of tunnel hindrance. Our DTNB-based enzyme activity measurements and enzyme kinetic studies showed that mutant M121W decreased the enzyme activity and the substrate binding comparing to wild type Dat. Among the four substrates (Dopamine, serotonin, phenylethylamine, tryptamine) tested, only the efficiency of dopamine remains. This result confirms that M121W and D142W may hinder the substrate entry, resulting in decreased binding efficiency of the binary complex. Our studies not only confirm the existence of a substrate tunnel, but also show the tunnel size may contribute to the substrate specificity.

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this thesis is described the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction fragment-length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In the course of screening for ine cDNAs, other genes of interest were isolated from the ine genetic region and were further characterized. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na$\sp+$/Cl$\sp-$-dependent neurotransmitter transporter family. Members of this family catalyze the rapid re-uptake of neurotransmitters released into the synapse, and thereby play key roles in controlling neuronal function. The conclusion is that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective re-uptake of the substrate neurotransmitter ine transporter, and thus over stimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology.
Typescript. Includes bibliographical references (leaves 46-56). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99354

The aim of this work was to characterise the influence of adenosine on imaginal disc cell line Cl8+ of Drosophila. I prepared stable cell lines with the overexpression or RNA interference of genes coding adenosine receptor AdoR (CG9753) and adenosine transporter DmENT2 (CG11045) in D. melanogaster. These cell lines were subsequently used to test their response to extracellular adenosine signal by the measurement of cell viability and level of second messengers cAMP and Ca2+ in Cl8+ cells.

碩士
國立清華大學
生物資訊與結構生物研究所
104
Dopamine N-acetyltransferase (Dat) found in Drosophila melanogaster belongs to arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87) family, which is a member of GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes arylalkylamine N-acetylation which transfers acetyl group of acetyl-CoA (Ac-CoA) to arylalkylamine to generate N-acetyl-arylalkylamine and CoA. AANAT had been reported the ordered bi bi sequential mechanism by enzyme inhibition analysis as well. In our previous study, we had also determined Dat is ordered bi bi sequential mechanism using isothermal titration calorimetry (ITC) and enzyme inhibition kinetics. Dat has to bind cofactor (Ac-CoA) first and then followed by substrate (Arylalkylamine). Nevertheless, the underlying structural mechanism still remains ambiguous. Furthermore, we had found the electron density map of products, N-acetyl-arylalkylamine and CoA, on substrate and cofactor binding site in ternary structure by soaking. It seemed products cannot auto-release. Thus, we interested in how Dat conducts enzymatic recycling and the catalytic process. In this study, we solved 1.20 Å resolution ternary structure (Dat/N-acetyl-arylalkylamine/CoA) of Dat by co-crystallization. Comparing apo form, binary form (tDat/Ac-CoA complex) and ternary form (tDat/CoA/Ac-PEA complex) of tDat, we found conformation of apo form Dat was different from binary and ternary form among them; structures of binary and ternary form were similar with each other. Then, we found the conformational change after Ac-CoA binding with tDat. The conformational change of substrate binding site may decide whether tDat can binding substrate or not. Thus, we elucidated ordered bi bi sequential mechanism of Dat by x-ray structural analysis. Additionally, the overall ternary structure of co-crystallization was similar with soaking except for an additional Ac-PEA outside the protein surface. The phenomenon implied may exist some factors to facilitate product release and enzyme recycle of Dat. Using isothermal titration calorimetry (ITC) and x-ray co-crystallization to carry out the competitive experiments, we found Ac-CoA showed dominantly competitive relation with respect to CoA. The dominantly competitive relation between Ac-CoA and CoA may resulted in enzymatic recycle of Dat. Finally, we based on catalytic triad to generate three variants, E47D, E47Q and E47N, for approaching transition state. All of them lost their substrate binding affinity and led to dramatic catalytic activity decrease. According to our results, we suggested Ac-CoA priorly binds to Dat providing a conformation change which facilitates substrate binding and forms ternary form. Ac-CoA may serve as driving force in catalyzation process of Dat. Our results implied Ac-CoA drives CoA and N-acetyl-arylalkylamine in Dat (ternary form) away, and occupies the cofactor binding site of Dat. Then Dat returns to state of binary form and accomplishes enzymatic recycling.

Programmed cell death (PCD) is an essential biological process in animal development and tissue homeostasis that is necessary to ensure the physiological well-being of the organism. During PCD, phagocytes facilitate the selective removal of excess, damaged, and potentially deleterious cells, in a multi-step engulfment process. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signal transduction pathways that act redundantly to regulate engulfment: the CED-1/-6/-7 and CED-2/-5/-12 pathways. Of these cell death (CED) proteins, the ABC transporter CED-7 is the only protein reported to be required in both the engulfing cell and the dying cell. However, its function in the cell death process remains the most enigmatic and the ced-7 ortholog previously has not been identified in Drosophila. Homology searches revealed a family of putative ced-7 orthologs that encode transporters of the ABCA family in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1 in PCD, we analyzed their engulfment function in oogenesis, during which 15 germ cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. It has been shown that genetically knocking down individual engulfment genes results in inefficient clearance of the germ cells, which then persist in late-stage egg chambers. Only two of the putative ced-7/ABCA1 genes are expressed significantly in the ovary, CG31731 and CG1718, and we have characterized these genes using transposon insertions, deficiencies, and RNAi knockdowns. Our genetic analysis reveals that CG31731 is necessary for germ cell clearance in the Drosophila ovary. Immunostaining shows that genetically knocking down CG31731 results in uncleared germ cells which persist in late-stage egg chambers. Altogether, our findings suggest that CED-7/ABCA1/CG31731 play evolutionarily conserved roles during engulfment.

碩士
國立清華大學
生物資訊與結構生物研究所
104
Dopamine is a neurotransmitter and associated with many physiological mechanisms, for example, cognition, circadian rhythm, aging, memory and learning. Dopamine N-acetyltransferase (Dat), an arylalkylamine N-acetyltransferase (AANAT), is identified in Drosophila melanogaster involves in the catabolism of monoamines and sclerotization. Dat transfers acetyl group from acetyl coenzyme A (AcCoA) to arylalkylamine and produces N-acetylarylalkylamine. Previous study in our laboratory revealed that Dat obeyed an ordered sequential mechanism: AcCoA binding first, substrates binding afterward, and then acetyl group transferring. In order to understand the detail of this catalysis process, isothermal titration calorimetry (ITC) was used to study the thermodynamics changes and binding affinities in reactions. To confirm the ordered sequential mechanism, sequential addition of cofactors and substrates into 15N labeled Dat protein was performed and monitored by nuclear magnetic resonance (NMR), and then the chemical shift changes of 15N heteronuclear single quantum coherence (HSQC) spectra were analyzed. ITC results were consistent with NMR results and proved the existence of ordered sequential mechanism. Recently, ternary structure which was co-crystalized by Dat, AcCoA, and substrate was resolved by our laboratory. However, this ternary structure was consisted of Dat, CoA, and acetyl-substrate. This indicated that final products did not release from Dat after catalysis. To further investigate how the products leave Dat and let the next reaction occur. Titration of AcCoA into Dat containing products was monitored by ITC, and the formation of new products was confirmed by DTNB assay. The results showed that AcCoA replaced CoA in Dat, and further let acetyl-substrate leave. Then a new Dat-AcCoA complex was ready to bind a new substrate and transfer acetyl group to it. To explore residues affecting cofactor or substrate binding, Ligplot+ was used to analyze the binary form of Dat structure (PDB code: 3TE4) and ternary form (unpublished). In Ligplot+ analysis report, R153 and K192 are related with AcCoA/CoA binding, while M121 may participate in substrate binding. In CAVER software analysis, M121 and D142 located in the narrowest of the substrate binding tunnel of Dat. Four residues were replaced to alanine, and ITC and enzyme activity assay was used to check their roles. Unexpectedly, M121A totally lost the AcCoA binding ability in ITC test, and remained only 13% of activity in functional assay. Considering the notable changes in secondary structure of M121A and far distance between M121 and cofactor, the loss of cofactor binding ability of M121A should be caused from the change in structure rather than the interaction with cofactor. R153 was important residue for AcCoA binding because no cofactor binding was detected in ITC test of R153A. K192A showed less binding affinity to AcCoA/CoA in ITC test, but remained its catalytic ability. D142A showed no significant effects on binding with cofactor or substrate, and on catalysis. In conclusion, Dat obeyed an ordered sequential mechanism: AcCoA binding first, substrates binding afterward, and then acetyl group transferring. To start next reaction, AcCoA replaced CoA in Dat, and further let acetyl-substrate leave. Then a new Dat-AcCoA complex was ready to bind a new substrate and transfer acetyl group to it. Besides, M121 affected on protein structure and R153 was important residue participating in cofactors binding to Dat.

Dissertação de Mestradoapresentada no ISPA – Instituto Universitário para obtenção de grau de Mestre na especialidade de Neurociências Cognitivas e Comportamentais.
O presente estudo descreve o papel de neurónios específicos de dopamina na regulação da aprendizagem social, em contexto sexual, em Drosophila melanogaster. A dopamina (DA) é um neuro-modulador que controla a formação de memórias associativas, com valência positiva (memórias apetitivas) ou negativa (memórias aversivas) durante o processo de aprendizagem associal, e a nossa pergunta é se o mesmo mecanismo também ocorre na aprendizagem social. Como paradigma de aprendizagem social, utilizámos o mate-choice copying (MCC), que é uma forma de aprendizagem social por observação das interações sexuais entre conspecíficos, e que se sabe ser regulado pela dopamina. Como controlo, utilizámos o teste de independent mate choice (IMC), que avalia a preferência inata das fêmeas por machos com fenótipos distintos, neste caso fenótipos artificiais de cor azul e rosa. Replicámos estes dois paradigmas comportamentais num grupo controlo (uma linha heterozigótica, a Canton-Special, ou CS), e em duas linhas transgénicas com neurónios dopaminérgicos desativados: a linha PAM para os neurónios apetitivos, e a linha PPL1 para os neurónios aversivos. Os resultados mostram que a linha CS faz MCC, a linha PAM não faz e a PPL1 parece fazer, mas este último resultado não é conclusivo, provavelmente porque o tamanho da amostra é pequeno. Sabendo que as memórias apetitivas são importantes nos processos motivacionais, incluindo a motivação para o acasalamento, a formação de memórias apetitivas durante o MCC é certamente um mecanismo adaptativo. Por essa razão, poderá haver um menor contributo das memórias aversivas, contudo, o nosso estudo deixa essa hipótese ainda em aberto.
The present study describes the role of dopamine-specific neurons in regulating social learning in a sexual context, in Drosophila melanogaster. Dopamine (AD) is a neuro-modulator that controls the formation associative memories, with positive (appetitive) or negative (aversive) valence during the asocial learning process, and our question is whether the same mechanism also occurs in social learning. As paradigm of social learning, we used mate-choice copying (MCC), which is a form of social learning by observing conspecific sexual interactions, and that is known to be regulated by dopamine. As a control, we used the independent mate choice (IMC) test, which assesses female innate preference for males with distinct phenotypes, in this case the artificial blue and pink phenotypes. We replicated these two behavioural paradigms in a control group (a heterozygous line, Canton-Special, or CS), and in two transgenic lines with dopaminergic deactivated neurons: the PAM line for appetitive neurons and the PPL1 line for aversive neurons. The results show that the CS line does MCC, the PAM line does not, and the PPL1 line seems to do, but the latter result is not conclusive, probably because the sample size is small. Knowing that appetitive memories are important in motivational processes, including mating motivation, the formation of appetitive memories during MCC is certainly an adaptive mechanism. For this reason, there may be a minor contribut