Ulm, Univ., Diss., 2007.

Thesis (M.Sc.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.112-122

Summary Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death. Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli. To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons. In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells.

La fonction des protéines de signalisation est déterminée par la nature des domaines qui les composent. Une meilleure compréhension des voies de signalisation passe par l'étude de ces domaines et de leur régulation. PTPN4 est une tyrosine phosphatase qui joue un rôle anti-apoptotique. Lors de l'infection par une souche atténuée du virus de la rage, sa fonction est perturbée, conduisant à la mort des cellules. Cette perturbation est due à l'interaction du motif de reconnaissance au domaine PDZ (PBM) de la glycoprotéine virale avec le domaine PDZ de PTPN4. Nous avons montré que ce domaine PDZ a un rôle d'inhibiteur allostérique de l'activité catalytique de la phosphatase de PTPN4. Ceci représente la première description de la régulation d'une phosphatase par un domaine PDZ. Cette inhibition est levée lors de la fixation d'un ligand au domaine PDZ, tel que le PBM de la glycoprotéine virale. Notre étude structurale révèle que la fixation d'un PBM perturbe les interactions transitoires entre les deux domaines et rétablit ainsi les propriétés catalytiques de la phosphatase. Nous avons par ailleurs identifié un ligand endogène de PTPN4, la MAP Kinase p38 qui, à travers son interaction avec PTPN4, participerait à la régulation de l'homéostasie cellulaire. La formation du complexe implique le recrutement du PBM de p38 par le domaine PDZ de PTPN4. Ainsi, en plus d'avoir une fonction de régulation du domaine phosphatase, le domaine PDZ permet également le recrutement de partenaires et la présentation de substrats au site actif de la phosphatase de PTPN4. Cette étude contribue ainsi à améliorer notre connaissance du rôle des domaines PDZ dans les voies de signalisation cellulaires. / The function of signaling proteins is determined by the nature of the domains from which they are made up. A better understanding of cell signaling pathways will result from the study of these domains and their regulation. PTPN4 is a non-receptor tyrosine phosphatase with an anti-apoptotic function. Upon infection with an attenuated rabies virus, its function is hijacked, which subsequently leads to cell death. This phenotype is arises from the interaction of the PDZ binding motif (PBM) of the viral glycoprotein with the PDZ domain of PTPN4. In this study, we show that this PDZ domain is an allosteric inhibitor of the catalytic activity of the PTPN4 phosphatase domain. This is the first description of the regulation of a phosphatase by a PDZ domain. This inhibition is released by the interaction of a ligand to the PDZ domain, such as the viral glycoprotein PBM. Our structural study revealed that the PBM recognition disrupts the transient inter-domain interactions and restores the complete phosphatase catalytic properties. As well, we identified a PTPN4 endogenous ligand, the MAP Kinase p38, which may participate in the regulation of the cellular homeostatic through its interaction with PTPN4. Thus, in addition to its phosphatase regulatory role, the PDZ domain also allows the recruitment of partners and the introduction of substrates to the PTPN4 phosphatase active site. This study contributes to our understanding of the role played by PDZ domains in cell signaling pathways.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21840号 / 農博第2353号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5212(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (R(te)) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (I(SC)) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated I(SC). Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated I(SC). In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated I(SC) and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.

Organophosphates are neurotoxic compounds that inhibit acetylcholinesterase producing excess cholinergic stimulation. This produces various toxic signs including excitotoxic neuronal damage. Oximes can be used as a treatment for organophosphate poisoning by reactivating inhibited acetylcholinesterase. Traditional oximes do not penetrate the blood-brain barrier, limiting protection of the central nervous system. Novel, brain-penetrating oximes have the potential to protect the brain from organophosphate induced damage. Adult male rats were used to examine the ability of model organophosphates to produce neuropathology and the ability of novel oximes to prevent this damage. Additionally, adult male rats were used to examine changes in gene expression of the MAP kinase system resultant of treatment with model organophosphates and novel oximes. Results of these experiments support that the model organophosphates can be used to study neurodegeneration, the novel oximes may prevent neurodegeneration, and both organophosphates and novel oximes affect expression of MAP kinase genes.

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No description available.