Le facteur neurotrophique dérivé du cerveau (BDNF, Brain-Derived Neurotrophic Factor) est une protéine qui joue un rôle essentiel dans la survie et la différenciation des neurones, ainsi que dans l'induction et l'expression de la plasticité synaptique (Deinhardt and Chao, 2014; Lu et al., 2005). Le BDNF est très exprimé à l'âge adulte et la réduction de son expression est impliquée dans de nombreuses maladies neurodégénératives et troubles psychologiques (Anastasia and Hempstead, 2014). Son action sur la plasticité synaptique est majeure pour la mise en place de fonctions cognitives et pour la mise en place de la mémoire (Bekinschtein et al., 2008; Egan et al., 2003).Le BDNF existant sous deux formes ayant des fonctions opposées, on peut parler d'effet Yin et Yang du BDNF (Lu et al., 2005). En effet, le BDNF est synthétisé en tant que molécule précurseur, le proBDNF, qui a des effets négatifs " Yin ". Par sa liaison au récepteur p75NTR, le proBDNF favorise l'apoptose, la rétraction dendritique et la dépression à long-terme. Au contraire, la forme clivée, le BDNF mature (mBDNF), se lie préférentiellement au récepteur TrkB qui active les voies de signalisation de survie cellulaire, de différentiation et de potentialisation à long terme. On parle alors d'effets positifs ou " Yang ". Le clivage de BDNF joue donc un rôle capital dans la régulation de sa balance fonctionnelle vers l'une ou l'autre voie. Il est généralement admis que le proBDNF est clivé en mBDNF par la furine dans le Golgi ou par la pro-protéine convertase 1/3 dans les vésicules de sécrétion (Mowla et al., 2001; Seidah et al., 1996). Le proBDNF restant n'ayant pas été clivé dans la cellule peut être sécrété et être rapidement clivé par la plasmine ou les métalloprotéases de matrice (MMP7) dans l'espace extracellulaire (Lee et al., 2001). Cependant, si les enzymes de conversion du BDNF sont bien connues, les mécanismes de régulation du clivage ne sont pas encore compris.Nous montrons ici un nouveau mécanisme de régulation de la maturation de BDNF via une phosphorylation qui impacte directement la balance fonctionnelle. En effet, nous montrons que la phosphorylation du résidu S130, localisé à l'interface entre le pro-domaine et le domaine mature, diminue l'efficacité du clivage du BDNF par la furine, régulant ainsi l'équilibre entre les formes immature et mature. Cette phosphorylation au site S130 est catalysée par les ectokinases FJX1 et FAM69B qui sont localisées dans l'appareil de Golgi avec BDNF. De plus, grâce à l'utilisation de souris transgéniques knock-in phospho-mutantes, nous montrons que la phosphorylation de BDNF, en favorisant la forme proBDNF, inhibe la potentialisation à long-terme et diminue la dynamique de plasticité des épines dendritiques après stimulation neuronale. Ces résultats suggèrent ainsi une nouvelle voie de régulation de la balance fonctionnelle de BDNF et suggèrent un rôle critique de la phosphorylation S130 dans les processus d'apprentissage et de la mémoire.En parallèle, la recherche de kinases potentielles de BDNF nous a amenés à identifier une exoPKA, localisée dans le Golgi et qui interagit directement avec BDNF. Cette exoPKA atypique phosphoryle BDNF au niveau de la sérine S130 et régule sa maturation et donc l'équilibre entre les formes pro et matures de BDNF. Cette exoPKA est différente de la PKA cytosolique ce qui suggère l'existence d'un nouveau mécanisme de régulation de la plasticité par PKA via BDNF. Enfin, nous sommes en train de tester le rôle de l'exoPKA sur la régulation négative de la plasticité synaptique via la régulation du clivage de BDNF. Ces résultats permettront de déterminer l'existence d'une dichotomie d'action de PKA sur la plasticité en fonction de la forme activée, cytosolique ou golgienne/sécrétée. / Brain-Derived Neurotrophic Factor (BDNF) is a protein that plays an essential role in the survival and differentiation of neurons, as well as in the induction and expression of synaptic plasticity (Deinhardt and Chao, 2014; Lu et al., 2005). BDNF is highly expressed during adulthood and the reduction of its expression is implicated in many neurodegenerative diseases and psychological disorders (Anastasia and Hempstead, 2014). Its action on synaptic plasticity is critical for the establishment of cognitive functions and for the establishment of memory (Bekinschtein et al., 2008; Egan et al., 2003).BDNF exists as two forms with opposite functions, or Yin and Yang effects (Lu et al., 2005). Indeed, BDNF is synthesized as a precursor molecule, proBDNF, which has negative effects "Yin". By binding to the p75NTR receptor, proBDNF promotes apoptosis, dendritic retraction and long-term depression. In contrast, the cleaved form, mature BDNF (mBDNF), binds preferentially to the TrkB receptor that activates the signaling pathways promoting cell survival, differentiation, and long-term potentiation. Thus, mBDNF has positive effects or "Yang". The cleavage of BDNF therefore plays a key role in regulating its functional balance towards one or the other pathway. It is believed that proBDNF is cleaved into mBDNF by furin in the Golgi network or by pro-protein convertase 1/3 in secretory vesicles (Mowla et al., 2001; Seidah et al., 1996). The remaining proBDNF that has not been cleaved in the cell can be secreted and rapidly cleaved by plasmin or matrix metalloproteases (MMP7) into the extracellular space (Lee et al., 2001).Having diametrically opposite roles, together, mature BDNF and proBDNF allow fine regulation of neuronal survival and differentiation and of their activity-dependent synaptic plasticity (Lu et al., 2014; Yang et al., 2014). Since BDNF has critical roles in neuronal functions, it is not surprising that its expression and action are extremely regulated in time and space. However, even if the enzymes processing BDNF are well known, the mechanisms regulating BDNF cleavage are not yet understood.Here we show a new mechanism for the regulation of BDNF maturation via phosphorylation which directly impacts the functional balance. Indeed, we show that phosphorylation of the S130 residue, located at the interface between the pro- and the mature domain, decreases the efficiency of BDNF cleavage by furin, thus regulating the balance between immature and mature forms. This phosphorylation at the S130 site is catalyzed by ectokinases FJX1 and FAM69B which are localized in the Golgi apparatus with BDNF. Furthermore, by using phospho-mutant knock-in transgenic mice, we show that the phosphorylation of BDNF, by promoting the proBDNF form, inhibits the long-term potentiation and decreases the plasticity dynamics of the dendritic spines after neuronal stimulation. These results suggest a novel regulatory pathway for BDNF functional balance and suggest a critical role for S130 phosphorylation in learning and memory processes.In parallel, the search for potential kinases of BDNF led us to identify an exoPKA, located in the Golgi and interacting directly with BDNF. This atypical exoPKA phosphorylates BDNF at serine S130 and regulates its maturation and therefore the balance between the pro- and mature forms of BDNF. This exoPKA is different from the cytosolic PKA which suggests the existence of a new mechanism of regulation of plasticity by PKA via BDNF. Finally, we are testing the role of exoPKA on the downregulation of synaptic plasticity via the regulation of BDNF cleavage. These results will allow to determine the existence of a dichotomous action of PKA on plasticity whether cytosolic or Golgi/secreted PKA is activated.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19806号 / 人博第777号 / 新制||人||187(附属図書館) / 27||人博||777(吉田南総合図書館) / 32842 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24525号 / 医博第4967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 渡邊 直樹, 教授 中川 一路, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Objective: The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned. Methods: Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media. Results: A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation. Conclusions: Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C.Cardiovascular Research 1994;28:72-77.

Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway

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付記する学位プログラム名: デザイン学大学院連携プログラム / 京都大学 / 新制・課程博士 / 博士(情報学) / 甲第24031号 / 情博第787号 / 新制||情||134(附属図書館) / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 阿久津 達也, 教授 山本 章博, 教授 鹿島 久嗣 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

The dystrophin-associated protein complex (DAPC) provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, serving as a mechanosensor and signal transducer across the sarcolemma. Pharmacological stimulation of AMP-activated protein kinase (AMPK) induces the expression of DAPC components in skeletal muscle, whereas physiological reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast glycolytic extensor digitorum longus (EDL) and slow oxidative soleus (SOL) muscles from wild-type (WT) mice, as well as from littermates deficient in both isoforms of the AMPK-β subunit in skeletal muscle (MKO) were analyzed. DAPC mRNA levels, as well as protein expression and localization were similar between genotypes, with the exception of nNOS, which displayed a compensatory sarcolemmal enrichment in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC, such as PGC-1α and KSRP, were also not affected by the loss of AMPK. However, MyoD and myogenin expression was significantly diminished in MKO muscles, which is consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO SOL muscles, despite an adaptive accumulation of PGC-1α at the sarcolemmal compartment. Collectively the evidence indicates that AMPK is sufficient, but not essential for the maintenance of DAPC expression in skeletal muscle. However, AMPK is required for preserving extrasynaptic utrophin levels in slow, oxidative muscles, which underscores the role of AMPK in the gene expression of this disease modifying protein. / Thesis / Master of Science (MSc) / The dystrophin-associated protein complex (DAPC) connects the interior and exterior of muscle cells. Activation of AMP-activated protein kinase (AMPK) increases the expression of the DAPC in skeletal muscle. We sought to determine whether AMPK was necessary for DAPC expression in skeletal muscle. Fast and slow muscles from normal mice, as well as from those deficient in skeletal muscle AMPK (MKO) were analyzed. We found DAPC levels and localization were similar between both groups, with the exception of nNOS, which was enriched at the muscle membrane in MKO muscles. Regulators of the DAPC were also not affected by the loss of AMPK. However, genes important for the production of muscle were significantly diminished in MKO muscles. Furthermore, we observed decrements in utrophin at the muscle membrane selectively in slow MKO muscles. Our work indicates that AMPK is not essential for the DAPC expression in skeletal muscle, however it is required for preserving utrophin levels in slow, oxidative muscles.