Circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) regulates daily rhythms in behavior and physiology and is an important part of the mechanisms regulating mammalian homeostasis. SCN are synchronized with a 24hour cycle mainly by light, but they can also be regulated by a variety of nonphotic signals, such as growth factors, opioids, cytokines, or lipopolysaccharide (LPS), which act by inducing the JAK/STAT signaling pathway. STAT family proteins (i.e. signal transducers and activator of transcription) regulate many aspects of cellular physiology, from growth and differentiation to immune response. However, the JAK/STAT signaling pathway has not yet been studied in the SCN and the function of STAT proteins in the SCN has not yet been clarified. In the first part of the thesis, we focused on localization of STAT3 and STAT5 proteins in the rat SCN and determination of rhythm in proteins and mRNA. Our experiments showed the daily rhythm in the levels of STAT3 protein in SCN astrocytes of rat with low but significant amplitude and with maximum in the morning. In addition, we revealed strong but nonrhythmic expression of STAT5A protein in astrocytes and STAT5B protein in nonastrocytic cells of SCN. It was also found that Stat3 mRNA show, similarly to protein, circadian rhythm in...

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13534号 / 論医博第2274号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 伊藤 貴浩, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Epilepsy is a neurological disorder that causes recurrent and unprovoked seizures due to imbalances in synaptic transmission in distinct regions of the brain. In both human patients and animal models of epilepsy, there is a marked increase in brain-derived neurotrophic factor (BDNF), a critical signaling molecule in the brain that contributes to two divergent pathways important to disease pathology: 1) the regulation of type A receptors for the major inhibitory neurotransmitter GABA (GABAARs), and 2) aberrant neurogenesis with ectopic expression of new neurons from progenitor cells that disrupt neural network activity in the hippocampus. The first part of my thesis addresses how neurons regulate levels of α1-containing GABAARs through BDNF signaling at its receptors, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR). I hypothesized and showed that BDNF, working at TrkB, rapidly activates the Janus kinase and signal transducers and activators of transcription (JAK/STAT) pathway in neurons and identified a novel intracellular receptor signaling complex composed of p75NTR and JAK2 that is present in neuronal processes, cell body, and nucleus. Based on this finding, we suggest that an intracellular p75NTR/JAK2 signalsome recruits STAT3, a transcriptional activator of the gene coding for the cAMP inducible early repressor (ICER) that blocks synthesis of α1 subunits reducing synaptic GABAARs in response to status epilepticus. This model is consistent with our collaborative studies that show a JAK2 inhibitor, WP1066, inhibits development of spontaneous seizures in an epilepsy model and my observation that WP1066 degrades JAK2 protein in primary neurons. The second part of my thesis addresses BDNF regulation of the Late SV40 Factor (LSF), a ubiquitous transcription factor that regulates cell cycle progression and survival. I show that BDNF through the mitogen-activated protein kinase pathway selectively phosphorylates LSF at serine 291 (p291LSF) and that p291LSF is present throughout neurogenesis, increases with status epilepticus in the hippocampus, and is highest in structures associated with neurogenesis (such as olfactory bulb and hippocampus when compared to cortex). Taken together, these results suggest LSF may play an important role in neuronal development and potentially in epilepsy, providing an additional target for future therapeutic intervention. / 2016-12-15T00:00:00Z

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The anteroventral periventricular (AVPV) nucleus of the hypothalamus integrates estradiol (E2) and progesterone (P4) feedback signals from the ovaries to stimulate gonadotropin releasing hormone (GnRH) neurons and trigger an ovulatory surge in luteinizing hormone (LH). E2 maintains the daily cyclic LH surge and P4 quickly amplifies the surge and limits it to one day. P4 amplification of the surge and rapid signaling in the AVPV may occur through its non-classical progestin receptors. Previous in vitro studies using a microarray analysis with N42 mouse embryonic hypothalamic neurons suggest that progesterone membrane component 1 (Pgrmc1) regulates genes linked to the janus kinase (Jak)/signal transducer and activator of transcription (Stat) signaling pathway. I hypothesized that P4 alters Jak/Stats through Pgrmc1 regulation of one or more Jak or Stat molecules and then performed a set of in vitro and in vivo studies to test this. I transfected N42 cells with either scramble or Pgrmc1 siRNA followed by treatment with either ethanol vehicle control or 10 nM P4 and measured Jak1, Jak2, Stat3, Stat5a, Stat5b, and Stat6 mRNA levels via quantitative polymerase chain reaction (QPCR). Jak1 and Jak2 mRNAs increased with P4 treatments, and this upregulation required Pgrmc1. Silencing Pgrmc1 in the cells also produced an increase in Jak1 and Jak2 mRNA, suggesting that Pgrmc1 constitutively suppressed jak1 and jak2 in the absence of P4. None of the Stats were significantly regulated by P4 or Pgrmc1 silencing. To determine how Pgrmc1 regulates Jak/Stat in vivo, I took AVPV microdissections from Pgrmc1 and Pgrmc2 double conditional knockout (DCKO) mice and looked at gene expression of jak/stat. Transcript levels of Jak2, but not Jak1, were severely downregulated in the DCKO animals and Stat mRNAs were not significantly changed. Discrepancies from in vitro and in vivo data prompted me to analyze the role of the class II progestin and adipoQ (Paqr) receptors in Jak/Stat signaling. P4 treatments and siRNA experiments in N42 cells showed that Paqr8, but not Paqr7, was required for P4 upregulation of Jak1 and Jak2 mRNAs. Overall, these findings show that Pgrmc1 regulates Jak1 and Jak2 synthesis in a P4-dependent and -independent manner that requires interaction with Paqr8.

Les cellules immunitaires provenant des deux vagues hématopoïétiques jouent des rôles distincts dans la réponse immunitaire, ce qui pose la question d’une potentielle communication entre les deux vagues d’hématopoïèse. De plus, la réponse immunitaire joue un rôle primordial dans la progression des tumeurs. Les cascades inflammatoires telles que la voie JAK/STAT et la voie Toll régulent l’hématopoïèse et les mutations affectant ces voies sont associées à des défauts hématopoïétiques et au développement de cancer du sang chez l’humain. Les deux voies de signalisation sont conservées au cours de l’évolution. La voie Toll a notamment été découverte chez la drosophile. Comme chez les mammifères, les mutations dans ces cascades produisent chez la larve des tumeurs des cellules du « sang » appelées tumeurs mélanotiques qui sont dues à la prolifération et à la présence d’hémocytes à l’état inflammatoire qui s’agrègent et forment des masses noires mélanisées. Au cours de mon doctorat, j’ai caractérisé l’impact de Gcm, le seul facteur de transcription spécifique de l’hématopoïèse primitive, sur la réponse immunitaire innée et l’activation de l’inflammation. Je me suis concentré sur les voies Toll et JAK/STAT en utilisant le modèle de la drosophile. J’ai pu montrer que Gcm inhibe la formation des tumeurs mélanotiques provoquées par l’activation constitutive de l’une ou l’autre voie. Gcm agit en activant l’expression d’inhibiteurs de chacune des deux voies. De plus, mes données montrent pour la première fois l’interaction entre les vagues d’hématopoïèses primitive et définitive, une interaction qui est nécessaire pour monter une réponse inflammatoire efficace. Dans ce système, Gcm inhibe la sécrétion de cytokines pro-inflammatoire Upd2 et Upd3 des hémocytes embryonnaires. Mes résultats indiquent également que Gcm a un impact sur l’expression de gènes mitochondriaux dans un fond génétique qui conduit au développement de tumeurs mélanotiques et à un état inflammatoire. Enfin, j’ai transposé mes résultats à un système mammifère en montrant que chez la souris, Gcm induit l’expression d’inhibiteur de la voie JAK/STAT dans une lignée cellulaire leucémique humaine. Pour conclure, mes données mettent en évidence l’importance de la communication entre les deux vagues d’hématopoïèse dans le système immunitaire et montrent qu’une voie de régulation développementale régule la capacité du système à répondre à l’inflammation. / Immune cells originating from different hematopoietic waves play role in mounting an efficient immune response, which raises the aspect of communication between distinct waves. In addition, immune responses have pivotal roles in modulating tumor progression. Inflammatory cascades, such as the JAK/STAT and Toll pathways are also known to regulate hematopoiesis and mutations in either of them are associated with hematopoietic defects and blood cancers in humans. Both pathways are highly conserved in evolution and interestingly, the Toll cascade was initially discovered in Drosophila. Like in mammals, mutations within these cascades produce the so called “melanotic tumors” in Drosophila larvae, which are due to blood cell proliferation and to the presence of hemocytes in an inflammatory state that aggregate and form black melanized masses. During my PhD, I proposed to decipher the impact of Gcm, the only known transcription factor specific to embryonic hematopoiesis on innate immune response and inflammation, by focusing on the JAK/STAT and Toll signaling cascades in vivo using the simple Drosophila model. I was able to show that Gcm inhibits melanotic tumors formation induced by the over-activation of both the JAK/STAT and Toll cascades. This is mediated by inducing the expression of JAK/STAT and Toll cascades inhibitors. In addition, my data describes for the first time the interaction occurring between the primitive and definitive hematopoietic waves and necessary to trigger an appropriate inflammatory response, where Gcm inhibits the secretion of the proinflammatory cytokines Upd2 and Upd3 from embryonic hemocytes. Moreover, I show that Gcm impacts the molecular landscape of mitochondrial genes in genetic backgrounds that lead to melanotic tumors and to an inflammatory state. Interestingly, I transpose my findings to vertebrates by showing that a GCM murine gene induces the expression of JAK/STAT inhibitors in a human leukemia cell line. In conclusion, my data highlights the importance of hematopoietic wave communication in the immune response and show that a developmental pathway regulates the competence to respond to inflammation.