GASP1 modulerait le trafic intracellulaire des RCPG après endocytose provoquée par les ligands. Au cours de ce travail nous nous sommes focalisés sur l’interaction de GASP-1 avec le récepteur beta2-adrénergique (B2AR) et ses conséquences fonctionnelles. Les agonistes du récepteur B2AR sont des bronchodilatateurs puissants utilisés dans le traitement de l’asthme. Avec le Dr N. Frossard, nous avons montré qu'un traitement chronique avec un agoniste B2AR induit le développement d'une hyper-réactivitée bronchique chez les souris sauvages mais pas chez les KO GASP1. Ce phénotype n’est pas relié à une différence dans la dégradation du récepteur B2AR entre les souris sauvages et KO GASP-1 mais est corrélé avec une augmentation du niveau de collagène dans les poumons des souris sauvages. Nos résultats indiquent que GASP1 joue un rôle important dans ce phénomène adaptatif qui serait relié à un remaniement des tissus bronchiques dépendant de cette protéine. / GASP1 have been shown to modulate the postendocytic sorting of different GPCRs.In order to better understand the role of GASP1 in regulating the activity and intracellular traffic king of GPCRs, we have focused our project on the functional consequences of the interaction between GASP1 and beta2-adrenergic receptor (B2AR). B2AR agonists are potent bronchodilators used in the treatment of asthma. With Dr. N. Frossard, we have shown that achronic treatment with a B2AR agonist induces the development of bronchial hyperresponsiveness in wild-type but not in KO GASP1 mice. Furthermore, we have shown that this phenotype is not related to a difference of B2AR receptor degradation between wild type and KO animals but correlates with an increase in collagen levels in the lungs of wild type mice that is not observed in GASP1KO animals. Altogether, our data suggest thatGASP1 is critically involved in these adaptations, which could be related to a GASP1-dependent modification of lung tissues.

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists. Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations. The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22073号 / 農博第2365号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5227(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 保川 清, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that β1 integrins play a protective role in chronic β-adrenergic receptor-stimulated cardiac myocyte apoptosis and heart failure. l-isoproterenol (iso; 400 μg/kg per hour) was infused in a group of wild-type (WT) and β1 integrin heterozygous knockout (hKO) mice. Left ventricular structural and functional remodeling was studied at 7 and 28 days of iso-infusion. Western blot analysis demonstrated reduced β1 integrin levels in the myocardium of hKO-sham. Iso-infusion increased heart weight:body weight ratios in both groups. However, the increase was significantly higher in WT-iso. M-mode echocardiography indicated increased left ventricular end-diastolic diameter, percentage of fractional shortening, and ejection fraction in the WT-iso group. The percentage of fractional shortening and ejection fraction were significantly lower in hKO-iso versus hKO-sham and WT-iso. Peak left ventricular developed pressure and left ventricular end-diastolic pressure measured using Langendorff-perfusion analyses were significantly higher in the WT-iso group (P<0.05 versus WT-sham and hKO-Iso). The number of TUNEL-positive myocytes was significantly higher in hKO-iso hearts 7 and 28 days after iso-infusion. The increase in myocyte cross-sectional area and fibrosis was higher in the WT-iso group. Matrix metalloproteinase-9 protein levels were significantly higher in WT-iso, whereas matrix metalloproteinase-2 levels were increased in hKO-iso hearts. Iso-infusion increased phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in both groups. The increase in c-Jun N-terminal kinase phosphorylation was significantly higher in hKO-iso (P<0.001 versus WT-iso). Thus, β1 integrins play a crucial role in β-adrenergic receptor-stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13126号 / 論医博第2135号 / 新制||医||1024(附属図書館) / (主査)教授 湊谷 謙司, 教授 椛島 健治, 教授 岩井 一宏 / 学位規則第4条第2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24133号 / 医博第4873号 / 新制||医||1059(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 生田 宏一, 教授 濵﨑 洋子, 教授 森信 暁雄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ataxia-telangiectasia mutated kinase (ATM) is a serine/threonine kinase. Mutations in the ATM gene cause a rare autosomal multisystemic disease known as Ataxia-telangiectasia (AT). Individuals with mutations in both copies of the ATM gene suffer from increased susceptibility to ionizing radiation, predisposition to cancer, insulin resistance, immune deficiency, and premature aging. Patients with one mutated allele make-up ~1.4 to 2% of the general population. These individuals are spared from most of the symptoms of the disease. However, they are predisposed to developing cancer or ischemic heart disease, and die 7-8 years earlier than the non-carriers. DNA double-strand breaks activate ATM, and active ATM is known to phosphorylate an extensive array of proteins involved in cell cycle arrest, DNA repair, and apoptosis. The importance of ATM in the regulation of DNA damage response signaling is fairly well-established. This review summarizes the role of ATM in the heart, specifically in cardiac remodeling following β-adrenergic receptor stimulation and myocardial infarction.

Brown (BAT) and white (WAT) adipose tissues are significant contributors to whole-body energy homeostasis. A disturbance in their metabolic function could result in the development of obesity and subsequent metabolic complications. The energy-sensing enzyme of the cell, AMP-activated protein kinase (AMPK), has been vastly studied in skeletal muscle and liver, but its role in BAT and WAT metabolism is elusive. We generated an inducible, adipocyte-specific knockout mouse model for the two AMPK β subunits (iβ1β2AKO) and found that iβ1β2AKO mice were intolerant to cold, and resistant to β3-adrenergic activation of BAT and browning of WAT. These defects in BAT activity were not due to the AMPK-ACC axis, but instead were due to compromised integrity of mitochondria. Mitochondrial morphology, function, and autophagy were all distorted in iβ1β2AKO mice, measured via transmission electron microscopy (TEM), respiration, and immunoblotting, respectively. These findings provide strong evidence that adipocyte AMPK regulates a fine-tuned program that responds to environmental and pharmacological inputs by maintaining mitochondrial integrity through autophagy and subsequent mitochondrial biogenesis in chronic settings. / Thesis / Master of Science (MSc) / Traditionally, there are two types of adipose tissue that appear and function differently. White adipose tissue (WAT) has evolved to store away energy in an efficient manner for later use. In contrast, brown adipose tissue (BAT) is a unique organ in mammals that has evolved over time to maintain body temperature. In essence, BAT has the ability to burn away calories as heat and is a promising therapeutic target to combat obesity and metabolic diseases such as type 2 diabetes. In our study, we have identified a potential factor that not only promotes BAT activity, but also promotes WAT to function more like BAT. By targeting this factor through drugs, there is potential to increase resting metabolic rate and fight the global epidemic of obesity.

Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors.