京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20633号 / 工博第4371号 / 新制||工||1679(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 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Yes / Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. / BBSRC, MRC and the European Research Council

El treball presentat en aquesta tesi és un estudi de la diversitat genètica en un conjunt de gens implicats en funcions neurològiques ("Gens cerebrals"). Hom ha examinat vint-i-dos gens implicats en els sistemes de neurotransmissió dopaminèrgic, serotoninèrgic i glutamatèrgic. L'objectiu de l'estudi té dos vessants: per una banda l'anàlisi de la diversitat genètica en un conjunt de gens implicats en malaltia humana, en aquest cas en malaltia psiquiàtrica, en poblacions humanes mundials, amb la intenció d'assentar les bases per propers esforços de mapatge genètic; i per altra banda analitzar la diversitat genètica en aquest conjunt de gens per tal de descobrir evidències d'esdeveniments històrics, incloent possibles evidències de selecció. / The work presented in this thesis is a study of the genetic variation in a set of genes related to neurological function ('Brain genes'). Twenty two genes are examined, all of which are involved in either the Dopaminergic, Serotonergic or the Glutamatergic systems of neurotransmission.The objective of the study has two aspects: on the one hand the analysis of genetic variation in a set of genes which are implicated in human disease, in this case psychiatric disease, across global human populations, towards the end of providing some new insight for gene mapping efforts, and on the other hand the study of genetic variation in this set of genes may reveal traces of the population history events undergone, including possible evidence for selection.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

No description available.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

La reconnaissance moléculaire est un phénomène omniprésent dans les systèmes vivants et intervient dans de nombreux processus biologiques comme la reconnaissance cellulaire ou encore la transmission de signaux par les neurotransmetteurs. L’élaboration de molécules synthétiques capables de mimer l’action des récepteurs naturels en complexant sélectivement un substrat cible est, à l’heure actuelle, très recherchée pour la détection ou le diagnostic en biologie et médecine. Parmi l’ensemble des récepteurs synthétiques, les hémicryptophanes sont des molécules cages composées d’un cyclotribenzylène connecté à une autre unité moléculaire par trois bras espaceurs. Les travaux de cette thèse reposent sur l’élaboration de nouveaux hémicryptophanes et l’étude de leurs propriétés de complexation vis-à-vis de molécules d’intérêt biologique. Dans un premier temps, la chiralité de ces récepteurs a été utilisée pour étudier leurs propriétés de reconnaissance stéréosélective face à différents sucres et analogues de neurotransmetteurs. De bonnes diastéréosélectivités et énantiosélectivités ont ainsi pu être observées en milieu organique pour les substrats étudiés. En parallèle, plusieurs hémicryptophanes hydrosolubles ont été synthétisés et ont permis de reconnaitre sélectivement des neurotransmetteurs comme la choline dans l’eau. Enfin, une dernière partie de cette thèse à été consacrée à la mise en place d’une voie de synthèse pour rendre ces récepteurs fluorescents, dans le but d’élaborer par la suite des sondes moléculaires capables de détecter et de suivre spatio-temporellement des molécules d’intérêt biologique dans les systèmes vivants par fluorescence. / Molecular recognition is a very important phenomenon for living systems as it occurs in many biological processes like cell-cell recognition or transmission of signals by neurotransmitters. Nowadays, the design of synthetic host molecules able to mimic natural receptors by complexing selectively a target substrate, is much sought-after for detection or diagnostic in biology and medicine. Among all the different synthetic receptors, hemicryptophanes are cage-shape molecules which are composed of a cyclotribenzylene moiety connected to another molecular unit by three spacer arms. This thesis is about the synthesis of new hemicryptophanes and the study of their complexation properties toward biologically important molecules. First, the stereoselective recognition of carbohydrates and neurotransmitter analogues by these chiral receptors was investigated in organic solvents and revealed good enantioselectivities and diastereoselectivities. In parallel, several water-soluble hemicryptophanes were synthesized and showed an aptitude for recognizing selectively ammonium substrates like choline in water. The last part was devoted to the development of a synthetic pathway to make hemicryptophanes fluorescent, in order to design molecular probes able to track biologically important molecules in living systems by fluorescence.

A epilepsia é um transtorno do funcionamento cerebral caracterizado por crises epilépticas recorrentes que acomete cerca de 1 a 2% da população mundial. A epilepsia do lobo temporal (ELT) é o subtipo mais prevalente. A refratariedade aos medicamentos é comum e cerca de 40 % destes pacientes apresentam transtornos psiquiátricos. Neste trabalho utilizamos o método de TacMan real time PCR para quantificar o mRNA de subtipos dos receptores de noradrenalina, dopamina, serotonina e substância P em hipocampos cirurgicamente removidos de pacientes com ELT para conhecer o papel destes na ELT com ou sem comorbidade psiquiátrica (depressão ou psicose). Nossa amostra foi de 48 pacientes com ELT sem (Epilepsia - 24) ou com comorbidade psicótica (Psicose - 10) ou depressiva (Depressão - 14) e 8 Controles (necrópsias). O receptor adrenérgico-α2A (AD2A) apresentou diferença entre os grupos (p = 0,0059) com significância para a variável Antiepiléptico (p = 0,0374) e pós-teste significante de maior expressão do mRNA de AD2A no grupo Epilepsia comparado com Controle e com Psicose. A ativação dos receptores α2A no hipocampo pelos antiepilépticos pode explicar nossos achados do grupo Epilepsia comparado ao Controle, corroborando a literatura acerca do AD2A na epilepsia e em relação aos antiepilépticos. O AD2C mostrou diferença entre os grupos (p = 0,0016), sem significância nas variáveis de controle e significante maior expressão do mRNA de AD2C no grupo Epilepsia comparado ao Controle e Psicose. O AD2C é encontrado em áreas que processam informações sensoriais e controlam atividades motoras e emocionais relacionadas, o que pode explicar nossos resultados. Parece ser importante na patologia relacionada à ELT e merece ser estudado. A não diferença entre Epilepsia e Depressão para AD2A e AD2C, parecem confirmar uma relação bi-direcional ou um mecanismo patogênico comum entre epilepsia e depressão, enquanto a menor expressão de AD2A e AD2C nos psicóticos parece indicar diferenças nos mecanismos adrenérgicos ligados a psicose e epilepsia. D2 mostrou diferença entre os grupos (p = 0,0125) com resultado significativo para a variável Subtipo de Diagnóstico Psiquiátrico (p = 0,0239), provavelmente devido a cronicidade da doença e a quantidade de episódios depressivos apresentados pelos sujeitos. Quanto maior a freqüência das crises (p = 0,0381) maior a expressão do D2 no grupo Epilepsia e no Depressão comparados ao Controle. Estes achados sugerem a participação deste receptor na depressão comórbida na ELT; corroboram que o monitoramento dopaminérgico límbico pode ser útil para desenvolver novos antidepressivos e propõem pesquisas futuras sobre D2 em epilépticos. A participação de 5-HT2A na ELT é indicada, pois, sua maior expressão no grupo Epilepsia em relação ao Controle foi significativa (p = 0,0273). Quanto maior a freqüência das crises epilépticas maior a expressão do 5-HT2A (p = 0, 0433). Não encontramos resultados significativos referentes aos receptores D4, 5-HT1A, 5-HT2C e NK1. Nossos resultados mostraram a possibilidade da aplicação do TacMan real time PCR no estudo de receptores de neurotransmissores, sugeriu a importância dos receptores estudados na ELT e comorbidades psiquiátricas, e que outras estruturas límbicas, como a amígdala, sejam focos de investigação. / Epilepsy is a mental functional disorder characterized by recurrent seizures that affect about 1 to 2% of world population. Temporal lobe epilepsy (TLE) is the most prevalent subtype. The refractory to medication is common and about 40% of these patients have psychiatric disorders. This study used the TacMan real time PCR method to quantify noradrenergic, dopaminergic, serotoninergic and substance P receptors subtypes mRNA expression in hippocampus surgically removed from patients with TLE to know their role in TLE with or without psychiatric commorbity (depression or psychosis). Our sample consisted of 48 TLE patients without (Epilepsy - 24) or with psychotic (Psychosis - 10) or depressive (Depression - 14) commorbity and 8 Controls (necropsies). The α2A adrenergic receptor (AD2A) showed difference between groups (p = 0.0059) with significance for Antiepileptic Medication variable (p = 0.0374) and post-hoc test significantly greater AD2A mRNA expression of Epilepsy group compared with Control and Psychosis. The activation of hippocampus α2A receptors by antiepileptic drugs can explain our findings of the Epilepsy group compared with Control, corroborating the literature about the AD2A in epilepsy and for antiepileptic drugs. The AD2C showed differences between groups (p = 0.0016) without significance in the variables of control and significantly greater AD2C mRNA expression of the Epilepsy group compared to Control and Psychosis. The AD2C is found in areas that process sensory information and control motor and emotional related activities, which may explain our results. It seems to be important in the pathology related to TLE and deserves to be studied. No differences between Epilepsy and Depression to AD2A and AD2C seem to confirm a bi-directional relation or a common pathogenic mechanism between epilepsy and depression, while the lowest AD2A and AD2C expression within psychotics seem suggests differences in adrenergic mechanisms linked to psychosis and epilepsy. D2 showed differences between groups (p = 0.0125) with significant results for the variable Subtype of Psychiatric Diagnosis (p = 0.0239), probably due to chronic disease and the number of depressive episodes presented by subjects. The higher the frequency of seizures (p = 0.0381) the higher was the D2 expression within Epilepsy group compared with Control and Depression compared to Control. These findings suggest the involvement of this receptor in TLE commorbid depression; corroborate that limbic dopaminergic monitoring may be useful in developing new antidepressants and propose future research on D2 in epileptics. The participation of 5-HT2A in TLE is indicated, therefore its significant higher expression in the Epilepsy group in relation to Control (p = 0.0273). The higher the frequency of seizures the higher was the 5-HT2A expression (p = 0.0433). We found no significant results for the D4, 5-HT1A, 5-HT2C and NK1 receptors. Our results showed the possibility of TacMan real time PCR method application in TLE neurotransmission receptors study, suggested the importance of the studied receptors in TLE and psychiatric commorbities and that other limbic structures, as the amygdala, should be investigation targets.

A epilepsia é um transtorno do funcionamento cerebral caracterizado por crises epilépticas recorrentes que acomete cerca de 1 a 2% da população mundial. A epilepsia do lobo temporal (ELT) é o subtipo mais prevalente. A refratariedade aos medicamentos é comum e cerca de 40 % destes pacientes apresentam transtornos psiquiátricos. Neste trabalho utilizamos o método de TacMan real time PCR para quantificar o mRNA de subtipos dos receptores de noradrenalina, dopamina, serotonina e substância P em hipocampos cirurgicamente removidos de pacientes com ELT para conhecer o papel destes na ELT com ou sem comorbidade psiquiátrica (depressão ou psicose). Nossa amostra foi de 48 pacientes com ELT sem (Epilepsia - 24) ou com comorbidade psicótica (Psicose - 10) ou depressiva (Depressão - 14) e 8 Controles (necrópsias). O receptor adrenérgico-α2A (AD2A) apresentou diferença entre os grupos (p = 0,0059) com significância para a variável Antiepiléptico (p = 0,0374) e pós-teste significante de maior expressão do mRNA de AD2A no grupo Epilepsia comparado com Controle e com Psicose. A ativação dos receptores α2A no hipocampo pelos antiepilépticos pode explicar nossos achados do grupo Epilepsia comparado ao Controle, corroborando a literatura acerca do AD2A na epilepsia e em relação aos antiepilépticos. O AD2C mostrou diferença entre os grupos (p = 0,0016), sem significância nas variáveis de controle e significante maior expressão do mRNA de AD2C no grupo Epilepsia comparado ao Controle e Psicose. O AD2C é encontrado em áreas que processam informações sensoriais e controlam atividades motoras e emocionais relacionadas, o que pode explicar nossos resultados. Parece ser importante na patologia relacionada à ELT e merece ser estudado. A não diferença entre Epilepsia e Depressão para AD2A e AD2C, parecem confirmar uma relação bi-direcional ou um mecanismo patogênico comum entre epilepsia e depressão, enquanto a menor expressão de AD2A e AD2C nos psicóticos parece indicar diferenças nos mecanismos adrenérgicos ligados a psicose e epilepsia. D2 mostrou diferença entre os grupos (p = 0,0125) com resultado significativo para a variável Subtipo de Diagnóstico Psiquiátrico (p = 0,0239), provavelmente devido a cronicidade da doença e a quantidade de episódios depressivos apresentados pelos sujeitos. Quanto maior a freqüência das crises (p = 0,0381) maior a expressão do D2 no grupo Epilepsia e no Depressão comparados ao Controle. Estes achados sugerem a participação deste receptor na depressão comórbida na ELT; corroboram que o monitoramento dopaminérgico límbico pode ser útil para desenvolver novos antidepressivos e propõem pesquisas futuras sobre D2 em epilépticos. A participação de 5-HT2A na ELT é indicada, pois, sua maior expressão no grupo Epilepsia em relação ao Controle foi significativa (p = 0,0273). Quanto maior a freqüência das crises epilépticas maior a expressão do 5-HT2A (p = 0, 0433). Não encontramos resultados significativos referentes aos receptores D4, 5-HT1A, 5-HT2C e NK1. Nossos resultados mostraram a possibilidade da aplicação do TacMan real time PCR no estudo de receptores de neurotransmissores, sugeriu a importância dos receptores estudados na ELT e comorbidades psiquiátricas, e que outras estruturas límbicas, como a amígdala, sejam focos de investigação. / Epilepsy is a mental functional disorder characterized by recurrent seizures that affect about 1 to 2% of world population. Temporal lobe epilepsy (TLE) is the most prevalent subtype. The refractory to medication is common and about 40% of these patients have psychiatric disorders. This study used the TacMan real time PCR method to quantify noradrenergic, dopaminergic, serotoninergic and substance P receptors subtypes mRNA expression in hippocampus surgically removed from patients with TLE to know their role in TLE with or without psychiatric commorbity (depression or psychosis). Our sample consisted of 48 TLE patients without (Epilepsy - 24) or with psychotic (Psychosis - 10) or depressive (Depression - 14) commorbity and 8 Controls (necropsies). The α2A adrenergic receptor (AD2A) showed difference between groups (p = 0.0059) with significance for Antiepileptic Medication variable (p = 0.0374) and post-hoc test significantly greater AD2A mRNA expression of Epilepsy group compared with Control and Psychosis. The activation of hippocampus α2A receptors by antiepileptic drugs can explain our findings of the Epilepsy group compared with Control, corroborating the literature about the AD2A in epilepsy and for antiepileptic drugs. The AD2C showed differences between groups (p = 0.0016) without significance in the variables of control and significantly greater AD2C mRNA expression of the Epilepsy group compared to Control and Psychosis. The AD2C is found in areas that process sensory information and control motor and emotional related activities, which may explain our results. It seems to be important in the pathology related to TLE and deserves to be studied. No differences between Epilepsy and Depression to AD2A and AD2C seem to confirm a bi-directional relation or a common pathogenic mechanism between epilepsy and depression, while the lowest AD2A and AD2C expression within psychotics seem suggests differences in adrenergic mechanisms linked to psychosis and epilepsy. D2 showed differences between groups (p = 0.0125) with significant results for the variable Subtype of Psychiatric Diagnosis (p = 0.0239), probably due to chronic disease and the number of depressive episodes presented by subjects. The higher the frequency of seizures (p = 0.0381) the higher was the D2 expression within Epilepsy group compared with Control and Depression compared to Control. These findings suggest the involvement of this receptor in TLE commorbid depression; corroborate that limbic dopaminergic monitoring may be useful in developing new antidepressants and propose future research on D2 in epileptics. The participation of 5-HT2A in TLE is indicated, therefore its significant higher expression in the Epilepsy group in relation to Control (p = 0.0273). The higher the frequency of seizures the higher was the 5-HT2A expression (p = 0.0433). We found no significant results for the D4, 5-HT1A, 5-HT2C and NK1 receptors. Our results showed the possibility of TacMan real time PCR method application in TLE neurotransmission receptors study, suggested the importance of the studied receptors in TLE and psychiatric commorbities and that other limbic structures, as the amygdala, should be investigation targets.

No description available.