The discovery of stem and progenitor cells capable of ongoing neurogenesis in the adult mammalian brain has raised hope that we will one day be able to harness their intrinsic regenerative capacity following injury. Development of such therapeutic strategies relies on a comprehensive understanding of the underlying regulation of the neurogenic process. To this end, I show, in this thesis, that cultured post-natal hippocampal neural progenitor cells (NPCs) express a specific repertoire of connexins (Cx), a family of channel forming proteins critical for communication prior to the development of functional chemical synapses. I show that this pattern of Cx expression, specifically Cx43 and Cx45, is modulated by interaction with the extracellular matrix component laminin providing evidence of extracellular matrix-cell interaction in the regulation of intrinsic Cx expression and function in postnatal NPCs. In adult brain, I show, for the first time, that Cx45 localizes to all cell types of the neuronal lineage with the exception of the type 3 doublecortin (DCX)-positive NPCs. Using a loss of function approach, I show that this expression is required for the normal proliferation of type 1 nestin and glial fibrillary acidic protein-positive stem like NPCs but not for the differentiation or survival of their progeny in the adult hippocampus. With respect to exogenous pharmacological cues that influence hippocampal neurogenesis, this thesis also demonstrates that chronic treatment with a sub-set of selective serotonin reuptake inhibitor antidepressants, fluoxetine and escitalopram, increases the proliferation but not the survival of adult NPCs in healthy, non-depressed mice. Further, standard post-operative analgesia with the opiate buprenorphine inhibits the proliferation of DCX-positive adult NPCs and increases the survival of their progeny. Finally, over the course of the research for this thesis, it became clear that exposing research animals to even very subtle environmental changes can influence the basal neurogenic process. Ultimately this work further highlights the exquisite sensitivity of the regulation of what is already recognized to be a highly dynamic process and provides important insight into the neurogenic process that can be used to inform future therapeutic development and application.

At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

Indiana University-Purdue University Indianapolis (IUPUI) / Glioblastoma is a lethal cancer that arises from support cells in the nervous system and kills around 20,000 people in the United States each year. While much is known about the highly malignant primary glioblastoma, the natural history of lower grade glioma (LGG) is less understood. While the majority of LGGs are initiated by a mutation in isocitrate dehydrogenase, the events leading to their malignant progression into a grade IV tumor are not known. Analysis of primary tumor sample data has revealed that low transcript levels of Angiomotin-like-1 (AmotL1) strongly associate with poor outcomes of patients with these cancers. Follow-up RNA-sequencing of human embryonic astrocytes with AmotL1 silencing revealed the downregulation of many transcripts that encode proteins mediating gap junctions (GJ) between astrocytes, especially connexin-43 (Cx43). Cx43 protein oligomerizes to form functional channels comprising the astrocytic GJ. AmotL1 knockdown through RNA interference decreases Cx43 transcript and protein levels while increasing its distribution to GJs. This suggests increased GJ formation and intercellular communication, as similar localization patterns are observed in differentiated astrocytes. Astrocytes with AmotL1 knockdown also display a pronounced pancake-like morphology, suggesting that the actin cytoskeleton is affected. Imaging reveals that cells with reduced AmotL1 have characteristic losses in both stress fibers and focal actin under the cell body but notable increases in cortical F-actin. Consistent with previous studies, AmotL1 may promote increases in the number and thickness of F-actin fibers. Because actin binding to related angiomotins is inhibited by phosphorylation from the LATs kinases, I define the effects of expressing wildtype AmotL1 versus mutants that mimic or prevent phosphorylation by LATs1/2. Interestingly, expression of AmotL1 S262D in combination with NEDD4-1, a ubiquitin ligase, results in a profound loss of actin stress fibers. Dependence on NEDD4-1 suggests that this phenotype is due to the induced degradation of proteins that promote F-actin, e.g. RhoA. These results directly support a model in which phosphorylated AmotL1 specifically inhibits F-actin formation as opposed to unphosphorylated AmotL1 which is known to promote stress fiber formation. Thus, in addition to regulating polarity and YAP/TAZ transcriptional co-activators, AmotL1 plays major functions in dictating cellular F-actin dynamics. / 2021-01-01

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18863号 / 医博第3974号 / 新制||医||1008(附属図書館) / 31814 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 岩田 想, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24493号 / 医博第4935号 / 新制||医||1063(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 浅野 雅秀, 教授 万代 昌紀, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of this thesis project was to examine the function of connexin-36 (Cx36) gap junctions (GJs) in producing alcohol's intoxicating and rewarding effects. GABA neurons are thought to inhibit dopamine (DA) neurotransmission in the mesocorticolimbic system, which originates in the midbrain ventral tegmental area (VTA) and projects to limbic structures such as the nucleus accumbens (NAcc). The mesolimbic DA system is believed to be the neural substrate of alcohol intoxication and addiction (Tepper, Paladini, & Celada, 1998). Alcohol suppresses the firing rate of GABA neurons in the VTA (Gallegos, Criado, Lee, Henriksen, & Steffensen, 1999) and presumably disinhibits DA neurons thereby resulting in enhanced release of DA in the NAcc. Interestingly, VTA GABA neurons appear to form part of a larger syncytium of GABA neurons in the reticular formation that are linked by electrical synapses via Cx36 GJs (Allison, et al., 2006; Stobbs, et al., 2004; Lassen, et al., 2007). Gap junction blockers, including the Cx36-selective antagonist mefloquine, also suppress the excitability and electrical coupling of VTA GABA neurons (Stobbs, et al., 2004). Thus, I hypothesized that Cx36 GJs cause synchrony in VTA GABA neurons which alcohol blocks to cause intoxication and reward. To accomplish these studies I compared the effects of intoxicating doses of ethanol in Cx36 knockout (KO) mice and mefloquine-treated mice and their wild-type (WT) controls with two tests that index ataxia, an open field activity system and the fixed-speed rotarod apparatus, as well as with ethanol self-administration. I found that Cx36 KO and mefloquine-treated mice exhibit significantly more ethanol-induced loss of movement in the open field test, a paradigm which indexes gross motor activity and tremor, but less ataxia than their WT controls in the rotarod paradigm, a paradigm which indexes balance and coordination. Most importantly, both Cx36 KO and mefloquine-treated mice consumed less ethanol than their controls. These findings provide evidence in support of the hypothesis that Cx36 GJs are important targets for ethanol effects in the mesolimbic system.

The coordinated contraction of the heart occurs because of the propagation of action potential between the cardiomyocytes. Gap junctions consisting primarily of connexin43 (Cx43) connect cardiomyocytes at regions of contact between cells known as the intercalated disc to facilitate cellular coupling. Cardiac pathologies frequently manifest with disrupted gap junctional intercellular communication which can generate potentially fatal arrhythmias, thus, it is essential to elucidate mechanisms underlying Cx43 regulation and altered intercellular communication. Phosphorylation of residues in the Cx43 carboxyl terminus can alter the subcellular localization, channel gating, and internalization of Cx43. The channel open probability of gap junctions is regulated, in part, by the phosphorylation of S368. Phosphorylation of S365 and S373 have been reported to exert gatekeeper effects on the phosphorylation of S368 and these phosphorylation events further affect protein interactions with 14-3-3 and zonula occludens-1 (ZO-1). While it is established that pS365 creates a conformational change preventing pS368, it is currently unclear precisely how pS373 regulates pS368. Further, it is unclear if alterations to these residues might impact protein binding and pathological cardiac remodeling during stress. Utilizing an ex vivo ischemia model, we find by immunofluorescent confocal microscopy that wildtype Cx43 hearts exhibit significantly decreased Cx43/N-cadherin colocalization during ischemia, while phospho-null mutant hearts retain Cx43/N-cadherin colocalization. Triton X-100 solubility assay indicates S365A/S373A mice have increased junctional Cx43 during ischemia. Additionally, we show that pS368 decay is more rapid in S373A mutants than wildtype suggesting, for the first time, that pS373 may prevent dephosphorylation at Cx43-S368 rather than promote Cx43-pS368. This knowledge could highlight potential therapies for prevention of cardiac remodeling and arrhythmogenesis. / Master of Science / A healthy heartbeat is achieved by the passage of electrical signals from cell-to-cell throughout the heart to cause a coordinated contraction. The muscle cells of the heart, known as cardiomyocytes, pass this electrical impulse through channels connecting neighboring cells, known as gap junctions, which are made from the protein connexin43 (Cx43). Gap junction channels allow for the passage of ions and small molecules from one cell to the next and these ions help initiate contraction in the neighboring cell. To regulate the location and function of Cx43, the protein can be modified by the addition of chemical groups which may alter the shape of the protein, affecting its function or ability to interact with other proteins. The addition of a phosphate group is known as phosphorylation. When the heart is under stress, such as low oxygen (hypoxia) or low blood flow (ischemia), Cx43 is phosphorylated at specific sites along the protein which can alter the function of gap junction channels in a harmful way. If the heart can no longer pass the electrical signal necessary for contraction, this can cause disrupted heart rhythms known as arrhythmias and can lead to death. Phosphorylation at S368 has been shown to decrease gap junction channel function. We know that phosphorylation at S365 changes the shape of the connexin protein and prevents phosphorylation of S368. However, it is unknown how phosphorylation of S373 affects phosphorylation of S368. We show that S373 may prevent phosphorylation of S368, and that mutation of these residues prevents cardiac remodeling. These discoveries may lead to therapeutic targets to prevent arrhythmia and sudden cardiac death.

Kardiovaskuläre Erkrankungen in Deutschland führen die Todesursachenstatistik an (19,1 % 2013) und verursachen die höchsten Krankheitskosten (14,5 % 2008) (Statistisches Bundesamt, 2015a,b). Im Rahmen von ischämischen Ereignissen am Herzen kann es zu Rhythmusstörungen kommen. In der Therapie dieser Störungen werden traditionell klassische Antiarrhythmika mit Wirkort Ionenkanal eingesetzt, welche jedoch stets ein proarrhythmisches Potenzial aufweisen. Im Fokus der Forschung der letzten Jahre stehen deswegen Peptide wie AAP10 (Antiarrhythmisches Peptid 10), welche direkt an den Gap Junctions ansetzen. In Radioligandenbindungsstudien konnte gezeigt werden, dass Desipramin AAP10 von seinem Rezeptor verdrängen kann. In der vorliegenden Arbeit wurde der Einfluss von Desipramin auf die Gap Junction-Leitfähigkeit in adulten humanen atrialen Kardiomyozyten bestimmt (Jozwiak 2012). Die Bestimmung der Leitfähigkeit erfolgte durch die Technik des Double-Cell-Voltage-Clamp. Es konnte gezeigt werden, dass Desipramin die elektrische Kopplung in humanen Kardiomyozyten, welche vorab durch CO2-induzierte Azidose partiell entkoppelt wurden, erhöht. Weiterhin wurde in der Mapping-Analyse mit dem Langendorff-System gezeigt, dass Desipramin in ischämischen Gebieten am ganzen Kaninchenherzen eine Reduktion der Homogenität und eine Steigerung der Dispersion verhindern kann. In anschließend hergestellten Western Blots aus Gewebeproben derselben Kaninchenherzen ließ sich eine verminderte Dephosphorylierung von Connexin 43 in ischämischen Gebieten unter Desipramin nachweisen. Ebenso vermag Desipramin eine Lateralisierung des Connexin 43 entlang der Zellmembran zu verhindern. Die Ergebnisse zeigen, dass Desipramin die Wahrscheinlichkeit für das Auftreten von Herzrhythmusstörungen unter ischämischen Bedingungen signifikant verringern und damit möglicherweise zur Senkung der Morbidität und Mortalität von Herzkreislauferkrankungen beitragen kann.

Introdução: Até bem pouco tempo atrás a análise mutacional de regiões gênicas não codificantes raramente eram reportadas em estudos genéticos sobre doenças hereditárias. Geralmente, o enfoque desses estudos recaiam sobre as regiões ditas codificantes, também conhecidas como regiões exônicas. Com o progresso da tecnologia em biologia molecular, especialmente após o desenvolvimento do sequenciamento em larga escala, temos observado um avanço jamais visto antes no entendimento de como opera o genoma humano e consequentemente uma mudança de paradigmas. Estudos mais recentes tem abrangido consistentemente a análise mutacional de regiões não codificantes ou regulatórias. No caso da neuropatia de \"Charcot-Marie-Tooth\" (CMT), pouco ainda se sabe sobre os fenótipos resultantes de mutações localizadas em sequencias regulatórias dos genes relacionados a doença. Por exemplo, na CMT ligada ao cromossomo X do tipo 1 (CMTX1), que é causada por mutações no gene GJB1 que codifica para a proteína canal conexina 32, apenas 0.01% das mutações já detectadas no gene estão presentes na região 5\' UTR (Untranslated Region) (KABZINSKA et al., 2011). Objetivos: Em vista disso, o propósito deste estudo foi a analise mutacional da região não codificante 5\' UTR e região promotora P2 do gene GJB1. Mais precisamente, 600 pares de bases upstream a open Reading frame (ORF) do transcrito do gene GJB1 expresso no sistema nervoso. Métodos: Foram investigados 100 pacientes em andamento no ambulatório de neurologia do Hospital das Clinicas da Faculdade de medicina de Ribeirão preto USP (FMRP - USP) com sintomas sugestivo de CMTX do tipo 1 e que não apresentaram mutação alguma na região codificadora do mesmo gene em triagens posteriores. Para tanto foram realizadas as técnicas de PCR (para amplificação do DNA) e posteriormente o sequenciamento do produto amplificado por meio do sequenciamento de Sanger. Resultados: Três variantes foram detectadas em 4 pacientes não relacionados, duas alterações intrônicas (já descritas) e uma nova alteração, encontrada no exon 1B (não codificante) do gene GJB1. Conclusão: Os resultados obtidos em nosso estudo sugerem que mutações na região 5\' UTR do gene GJB1 na população brasileira são incomuns e aparentemente não são responsáveis por casos sugestivos de CMTX1 sem causa genética atribuída. / Introduction: Until recently mutational analysis of non-coding regions of the DNA were rarely reported on genetic studies about inherited disorders. Usually, the approach of those studies relied primarily on the analysis of the coding regions of the DNA, also known as exonic regions. However, with the progress of science in molecular biology, especially after the development of the high throughput sequencing technology, we have observed an advance never seen before regarding of how the human genome operates and consequently a paradigm shift. New studies have consistently covered these regulatory or non-coding regions of the DNA. In the case of Charcot-Marie-Tooth disease (CMT), little is known about the phenotypes resulting from mutations in the regulatory sequences of the genes related to the disease. For example, in the X linked CMT type 1 (CMTX1), caused by alterations in the GJB1 gene that encodes for the Channel like protein connexin32 (CX32), only 0.01% of the mutations ever found in the gene were actually localized in its 5\' untranslated region (5\'UTR) (KABZINSKA et al., 2011). Objectives: Considering it, the purpose of this study was the mutational analysis of the 5\' non-coding region and promoter region P2 of GJB1 gene. More specifically, 600 base pairs (bp) upstream of the open reading frame (ORF) of the transcript expressed in the nervous system (NS). Methods: 100 patients in progress in the Clinical Hospital of the Medical School at University of São Paulo presenting suggestive clinical diagnoses of CMTX1 were screened for mutations in the region of interest, after mutations in the coding region of GJB1 gene were excluded in previous analysis. Therefore, we performed a Polimerase Chain Reaction (to amplify the DNA) followed by sequencing of the amplified material via Sanger sequencing. Results: We detected 3 alterations in 4 unrelated patients, 2 intronic variants (already described) and a novel mutation in the non-coding exon 1B of the GJB1 gene. Conclusion: The obtained results in our study suggest that mutations in the 5\'UTR of the GJB1 gene in the Brazilian population are uncommon and apparently they are not a major cause of CMTX1 in genetic unassigned patients with a suggestive clinical diagnosis of CMTX1.

Hintergrund: Connexine (Cx) spielen eine wichtige Rolle bei Wachstum und Differenzierung. Die Angiogenese ist an vielen physiologischen und pathologischen Prozessen beteiligt. Jedoch ist noch unklar, ob Connexine einen Einfluss auf die Angiogenese haben. Fragestellung; In dieser Arbeit sollte die Rolle endothelialer Connexine an der Angiogenese untersucht werden. Methoden: Venöse Zellen der menschlichen Nabelschnur (HUVEC) wurden kultiviert bis sie subkonfluent waren. Vor der 3D Kultur im Matrigel wurden die Zellen entweder mit Nicotin, dem Gap Junction Inhibitor Palmitoleinsäure (PA), einem siRNA-Knockdown von entweder Cx37, Cx40 oder Cx43 oder mit Isoprenalin behandelt. Die Zellen wurden dann auf ein in vitro Angiogenese-Assay (3D-Kultur, Matrigel) gegeben und nach 18h wurden unterschiedliche Angiogeneseparameter erhoben, um die Komplexität der Angiogenese zu beurteilen. Änderungen der Expression der Cx mRNA- und Proteinexpression sowie der Kommunikation über Gap Junctions wurden mittels PCR, Immunfluoreszenzmikroskopie, Western Blot und Lucifer Yellow Dye Transfer untersucht. Bei Gewebeproben der A. mammaria von Rauchern und Nichtrauchern wurden mittels Immunofluoreszenzmikroskopie die Expression der Cx beurteilt. Ergebnisse: Die Behandlung mit spezifischer siRNA führte zu einer signifikanten Abnahme der Expression des jeweiligen Connexins in den HUVECs. Sowohl der Knockdown als auch die Behandlung mit PA verringerte die Kommunikation über Gap Junctions signifikant und reduzierte die Anzahl der Abzweigungen im Angiogenese-Assay. Der Knockdown des Cx43 und Cx40 sowie die Behandlung mit PA reduzierten ebenfalls die Komplexität des Musters im Matrigel-Assay. Nicotin führte zu einer Reduktion der Cx43- und Cx37-Proteinexpression, wohingegen Cx40 durch transskriptionelle Gegenregulation konstant gehalten wurde, sowie zu einer Abnahme der Länge der Kapillar-ähnlichen-Strukturen, der Anzahl der Abzweigungen und des Musters im Matrigelassay, während die Anzahl der Zellen zunahm. Die mRNA-Expression der Connexine war hingegen erhöht. In Gewebeproben von Rauchern konnte analog eine verminderte Expression von Cx43 und Cx37, aber nicht von Cx40, in der Intima gezeigt werden. Isoprenalin erhöhte die Proteinexpression der endothelialen Connexine und verbesserte sowohl die interzelluläre Kommunikation als auch das Muster im Matrigel-Assay. Ebenso waren die Kapillar-ähnlichen Strukturen im Vergleich zur Kontrolle länger. Schlussfolgerung: Aus den erhobenen Ergebnissen lässt sich der Schluss ziehen, dass Connexine bei der Angiogenese involviert sind, vor allem beim Vorgang der Verzweigung. Dies kann teilweise die Veränderung erklären, die eine Nicotinbehandlung auf die Angiogenese hat.