O nosso grupo está explorando como alternativa para a identificação de novas estratégias de tratamento o reposicionamento de drogas. Nesse contexto, nosso laboratório mostrou que memantina, um antagonista de receptores de glutamato NMDA, apresenta uma atividade tripanocida no ciclo de vida do T. cruzi, in vitro. Com base nessas informações, avaliamos o efeito da memantina em animais infectados com a cepa Y. O tratamento apresentou uma redução da parasitemia (40%) no 8º dpi e redução da carga parasitária no tecido cardíaco no 15º dpi. Uma vez que a cepa Y invade preferencialmente macrófagos, avaliamos o efeito de memantina em células RAW 264.7. Memantina diminuiu a produção de NO por células estimuladas com LPS, diminuiu o Ca2+i, e não houve uma redução de EROs. Memantina também interferiu no ciclo intracelular do parasita, reduzindo o número de células infectadas. Nossos resultados sugerem que o tratamento com memantina pode direcionar o sistema imune de modo a modular os receptores de tipo NMDA, através de um efeito direto ou indireto produzido pelo tratamento. / The search for new therapeutic targets has been of extreme importance for the specific treatment of Chagas disease. Our group is exploring as an alternative, a strategy for drug repurposing. In this context, our laboratory showed that memantine, an antagonistic of NMDA glutamate receptors, has a trypanocidal activity along the life cycle of T. cruzi, in vitro. Based on this information, we evaluated the effect of memantine in animals infected with Y strain. The treatment showed a reduction of parasitemia (40%) in 8th dpi and reduced parasitic load in cardiac tissues at 15th dpi. As the Y strain preferably invades macrophages, we evaluated the effect of memantine in RAW 264.7 cells. Memantine decreased NO production by cells stimulated with LPS, decreased Ca2+i, and did not cause a reduction in ROS. Memantine also interfere with the intracellular parasite cycle, reducing the number of infected cells. Our results suggest that treatment with memantine can target the immune system to modulate the NMDA receptor, through direct or indirect effect caused by the treatment.

Doctor of Philosophy / Department of Psychological Sciences / Mary E. Cain / Drug addiction is a chronic cyclical disease characterized by periods of drug use and abstinence. Drug craving increases as a function of abstinence period, such that longer periods of abstinence result in greater feelings of craving. Longer periods of abstinence may render cues to become more powerful motivators of drug seeking behavior because of the greater craving response. Neurobiological evidence suggests that changes in glutamatergic transmission in the nucleus accumbens (NAc) plays a pivotal role in the incubation of craving and drug seeking motivation. Specifically, the upregulation of Ca²⁺ permeable AMPA receptors may increase drug seeking following the presentation of a drug cue. Environmental housing manipulations also change the expression of metabotropic glutamate receptors (mGlur) and psychostimulant self-administration. In the current experiments, Sprague-Dawley rats were reared in enriched (EC) or isolated (IC) conditions from PND 21-51. Then rats were implanted with indwelling jugular catheters and allowed to self-administer amphetamine (0.1 mg/kg/infusion) or saline paired with a cue light for 16 days for 1h. Then rats went through a forced abstinence period of 1 day and were then tested in a cue-induced seeking test. Immediately after the seeking test, half the rats were sacrificed and the NAc was dissected and prepared for western blot analyses. The other half of rats rested for 40 days and were tested again in the cue-induced seeking test. Immediately following the seeking test, rats were sacrificed and their NAc was dissected. Factorial ANOVA results indicate that rearing in the IC environment increased drug seeking when compared to EC rats after 1 day of abstinence and after 40 days of abstinence, but drug seeking did not increase after 40 days. Rats in the saline groups showed an increase in seeking after 40 days of abstinence, providing evidence of increased responding. Saline responding was significantly lower when compared to rats that responded for amphetamine. When rats self-administered saline, generally IC rats had more responding than EC rats. Western blot analyses indicated that expression of AMPA subunits GluA1, and GluA2, as well as metabotropic glutamate receptors 1 and 5 (mGlur1, and mGlur5) were not different across the experimental groups, suggesting another mechanism could be implicated in drug seeking after short and long abstinence periods. These results suggest that early life experience can have long lasting effects into adulthood and increase the vulnerability of drug abuse. Our results provide mixed results of incubated seeking. Positive early life experiences reduce drug seeking motivation after short and long abstinence periods, providing evidence for further research to examine how early life experience changes the reward seeking and subsequent structures in the mesocorticolimbic pathway.

O nosso grupo está explorando como alternativa para a identificação de novas estratégias de tratamento o reposicionamento de drogas. Nesse contexto, nosso laboratório mostrou que memantina, um antagonista de receptores de glutamato NMDA, apresenta uma atividade tripanocida no ciclo de vida do T. cruzi, in vitro. Com base nessas informações, avaliamos o efeito da memantina em animais infectados com a cepa Y. O tratamento apresentou uma redução da parasitemia (40%) no 8º dpi e redução da carga parasitária no tecido cardíaco no 15º dpi. Uma vez que a cepa Y invade preferencialmente macrófagos, avaliamos o efeito de memantina em células RAW 264.7. Memantina diminuiu a produção de NO por células estimuladas com LPS, diminuiu o Ca2+i, e não houve uma redução de EROs. Memantina também interferiu no ciclo intracelular do parasita, reduzindo o número de células infectadas. Nossos resultados sugerem que o tratamento com memantina pode direcionar o sistema imune de modo a modular os receptores de tipo NMDA, através de um efeito direto ou indireto produzido pelo tratamento. / The search for new therapeutic targets has been of extreme importance for the specific treatment of Chagas disease. Our group is exploring as an alternative, a strategy for drug repurposing. In this context, our laboratory showed that memantine, an antagonistic of NMDA glutamate receptors, has a trypanocidal activity along the life cycle of T. cruzi, in vitro. Based on this information, we evaluated the effect of memantine in animals infected with Y strain. The treatment showed a reduction of parasitemia (40%) in 8th dpi and reduced parasitic load in cardiac tissues at 15th dpi. As the Y strain preferably invades macrophages, we evaluated the effect of memantine in RAW 264.7 cells. Memantine decreased NO production by cells stimulated with LPS, decreased Ca2+i, and did not cause a reduction in ROS. Memantine also interfere with the intracellular parasite cycle, reducing the number of infected cells. Our results suggest that treatment with memantine can target the immune system to modulate the NMDA receptor, through direct or indirect effect caused by the treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20583号 / 工博第4363号 / 新制||工||1678(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 白川 昌宏 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

Like most organisms, Drosophila melanogaster can synchronize its physiological and behavioural processes by possessing internal circadian clock that regulates. Naturally fluctuating timing cues, like light and temperature (also known as Zeitgebers), synchronize these endogenous and self‐sustained clocks with external time. In Drosophila, synchronization of the circadian clock by light has been studied in detail, but much less is known about the molecular mechanisms underlying temperature entrainment. Previous data from our lab shows that Nocte, a Chordotonal organ (Ch organ) located protein, is required for normal temperature entrainment in Drosophila. However, neither the function of Nocte in temperature entrainment nor the molecular underlying mechanisms are clear. To address these issues, a proteomics strategy of combing co‐immunoprecipitation and MS/MS sequencing was applied to isolate potential interactors of Nocte. IR25a was one of the most promising candidates, which was later confirmed by behavioural tests using RNA interference: Reducing IR25a expression in Chorgan resulted in abnormal behaviour during temperature cycles, similar to what had been described for Nocte mutant. To further confirm the interaction between Nocte and IR25a, I showed that IR25a physically interacts with Nocte in vivo. Moreover, using an IR25a‐gal4 line, I was able to show that IR25a is expressed in subsets of chordotonal organs (Ch organ) including Johnston's Organs (JO), where Nocte is also highly expressed. These results, along with the behavioural data mentioned above are consistent with the proteomics results and suggest that Nocte and IR25a physically and functionally interact. IR25a mutants were employed to further investigate the function of IR25a in temperature entrainment. First of all, I found that both central and peripheral clocks in wild type flies can be synchronized to temperature cycles with only two degree differences (12h: 12h, 27 °C: 25 °C). In contrast, synchronization of locomotor activity rhythms in the IR25a null mutants to the same temperature cycles and other TC's with 2°C amplitude was eliminated. Under the same conditions, the oscillations of the core clock proteins TIMLESS (TIM) and PERIOD (PER) that normally occur in fly heads were completely abolished inIR25a null mutants, suggesting that IR25a is required for temperature entrainment of peripheral clocks. In the central brain pacemaker neurons, the oscillations of TIM in dorsal and lateral neurons were also affected by the IR25a mutants. On the contrary, IR25a is not required for light entrainment and temperature compensation, suggesting that IR25a is specifically involved in temperature synchronization. Moreover, temperature entrainment of the IR25a null mutants can be partially restored by applying larger temperature intervals (29°C: 25°C) indicating that IR25amay function as amplitude detector independent of absolute temperature values. Finally, neuronal activity in IR25a+ neurons is crucial for the synchronization of circadian clocks to low amplitude temperature cycles. Re‐constitution of functional olfactory receptors required the assembly of IR25a with IR76a and IR76b. Interestingly, IR76a and IR76b are neither required for temperature entrainment at the behavioural level nor expressed in the Ch organs. To check if other potential IRs interacting with IR25a exist, I screened the expression pattern of most divergent IRs using IR‐gal4/UAS‐GFP flies. IR56a was isolated as a potential partner of IR25a because it is also expressed in the femur chordotonal organs. To investigate the function of IR56a in temperature entrainment, I generated a null mutant of IR56a. Surprisingly, this gene is not required for synchronizing clocks to a temperature cycle (27°C: 25°C) at the behavioural level. However, the behavioural and molecular phenotypes of IR56a mutant under different temperature cycles need to be further characterized.

Aux synapses Fibres Parallèles - Cellules de Purkinje, le récepteur mGluR4 est le seul mGluR du groupe III à moduler la neurotransmission en inhibant les influx calciques qui régulent la libération de glutamate. Dans des systèmes hétérologues, il a été montré que mGluR4 était lié à des protéines G de type Gi/o couplées négativement à l'adénylate cyclase (AC). Afin de rester au plus proche des interactions physiologiques, nous avons débuté notre étude par la définition du réceptosome des récepteurs mGluR4 natifs dans le cervelet de rat. Nous avons identifié 184 partenaires putatifs du récepteur. Afin de confirmer ces interactions, mais aussi de recenser d'autres interacteurs éventuels, nous avons réalisé une approche complémentaire et indépendante de chromatographie d'affinité. Nombre de protéines ont été retrouvées par cette deuxième approche, en particulier des protéines appartenant aux familles de l'exocytose et du trafic cellulaire. Nos résultats suggèrent que le contrôle de la neurotransmission par mGluR4 pourrait s'effectuer, au moins partiellement, par une interaction avec ce type de protéines. D'autre part, nos approches biochimiques n'ont pas mis en évidence de protéines de la voie AC, mais au contraire plusieurs protéines identifiées appartiennent à la voie Phospholipase C/ Protein Kinase C (PLC/PKC). Ces résultats biochimiques corroborent certains résultats fonctionnels du laboratoire et ouvrent de nouvelles pistes quant à la modulation négative de la neurotransmission par les récepteurs mGluR4 natifs dans le cervelet / At Purkinje Cell - Parallel Fiber synapses, mGluR4 receptors are the only glutamatergic metabotropic receptors of group III to modulate glutamatergic transmission by inhibiting calcium presynaptic influx controlling glutamate release. In heterologous systems, mGluR4 has been shown to activate G proteins of type Gi/o that would be negatively linked to adenylate cyclase (AC). In order to conserve most of physiological interactions, we first studied the receptosome of native mGluR4 in rat cerebellum. We identified 184 putative partners of the receptor. Moreover, in order to confirm these interactions, but also to find other partners, we decided to perform an independent and complementary approach of chromatography affinity. Numerous proteins have been found by this method, particularly proteins belonging to exocytosis and cellular trafficking families. Our results suggest that a partial control of neurotransmission could be due to interaction of mGluR4 with these kinds of proteins. On the other hand, biochemical approaches did not reveal interactions of mGluR4 with some proteins belonging to AC pathway, but with proteins of PLC/PKC pathway. These results are consistent with some functional studies of our lab and gave the way for elucidating the native molecular mechanisms of the cerebellar neurotransmission modulation by mGluR4.

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site (Site 1) in the ECD-mGluR1α using a recently developed computational algorithm. This predicted site (D318, E325, D322 and the bound L-Glu) is situated in the hinge region in the ECD-mGluR1α adjacent to the reported Glu-binding site. Mutagenesis studies indicated that binding of L-Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. In addition, the Ca2+ effects on drugs targeting mGluR1α were investigated. Ca2+ enhances L-Quis response of the receptor by increasing L-Quis binding to ECD-mGluR1α and promotes the potency of Ro 67-4853, a positive allosteric modulator of mGluR1α. Increasing Ca2+ concentration, the inhibitory effects of a competitive antagonist ((s)-MCPG) and a non-competitive negative allosteric modulator (CPCCOEt), were eliminated. Furthermore, we also identified another potential Ca2+ binding pocket (Site 2) consists of S165, D208, Y236 and D318, which completely overlapped with L-Glu. Thapsigargin (TG) induced ER Ca2+ depletion reduced surface expression of mGluR1α, and D208I and Y236I also decreased the receptor trafficking to plasma membrane suggesting the role of Ca2+ binding in protein folding and trafficking in the ER. Further, to measure ER Ca2+, a series of genetically encoded biosensors were designed by placing a Ca2+ binding pocket at the chromophore sensitive region of red florescent protein mCherry. The designed sensors are able to bind Ca2+ and monitor Ca2+ concentration change both in vitro and in cells. The findings in this dissertation open up new avenues for developing allosteric modulators of mGluR function that target related human diseases.

Yes / Brain function depends on co-ordinated transmission of signals from both excitatory and inhibitory neurotransmitters acting upon target neurons. NMDA, AMPA and mGluR receptors are the major subclasses of glutamate receptors that are involved in excitatory transmission at synapses, mechanisms of activity dependent synaptic plasticity, brain development and many neurological diseases. In addition to canonical role of regulating presynaptic release and activating postsynaptic potassium channels, GABAB receptors also regulate glutamate receptors. There is increasing evidence that metabotropic GABAB receptors are now known to play an important role in modulating the excitability of circuits throughout the brain by directly influencing different types of postsynaptic glutamate receptors. Specifically, GABAB receptors affect the expression, activity and signaling of glutamate receptors under physiological and pathological conditions. Conversely, NMDA receptor activity differentially regulates GABAB receptor subunit expression, signaling and function. In this review I will describe how GABAB receptor activity influence glutamate receptor function and vice versa. Such a modulation has widespread implications for the control of neurotransmission, calcium-dependent neuronal function, pain pathways and in various psychiatric and neurodegenerative diseases.

No / Most inhibitory signals are mediated via γ-aminobutyric acid (GABA) receptors whereas glutamate receptors mediate most excitatory signals (Trends Neurosci 14:515–519, 1991; Annu Rev Neurosci 17:31–108, 1994). Many factors influence the regulation of excitatory and inhibitory synaptic inputs on a given neuron. One important factor is the subtype of neurotransmitter receptor present not only at the correct location to receive the appropriate signals but also their abundance at synapses (Pharmacol Rev 51: 7–61, 1999; Cold Spring Harb Perspect Biol 3, 2011). GABAB receptors are G-protein-coupled receptors and different subunits dimerise to form a functional receptor. GABAB receptor subunits are widely expressed in the brain and by assembling different isoform combinations and accessory proteins they produce variety of physiological and pharmacological profiles in mediating both inhibitory and excitatory neurotransmission. This chapter will describe the understanding of the molecular mechanisms underlying GABAB receptor regulation of glutamate and GABAA receptors and how they modulate excitatory and inhibitory neurotransmission.