Il est maintenant reconnu que la ségrégation latérale des composants de la membrane plasmique, ou compartimentation membranaire, joue un rôle important dans la régulation de la fonction de nombreuses protéines membranaires. Les tétraspanines constituent une famille de protéines intégrales à quatre régions transmembranaires possédant une capacité unique à s’associer entre elles et avec des nombreuses autres molécules de surface pour former un réseau d'interactions moléculaires, le « tetraspanins web ». Il a été proposé que via l’organisation de ce réseau, les tétraspanines joueraient un rôle dans la compartimentation membranaire des protéines auxquelles elles s’associent. Elles jouent également un rôle dans la compartimentation cellulaire en contrôlant le trafic de certaines des protéines associées. Le laboratoire a précédemment démontré que six tétraspanines conservées (Tspan5, 10, 14, 15, 17 et 33) de la sous-Famille des TspanC8 (caractérisées par la présence de huit cystéines dans le grand domaine extracellulaire) interagissent directement avec la métalloprotéase ADAM10 et régulent sa sortie du réticulum endoplasmique. Cette protéase ancrée à la membrane est responsable du clivage protéolytique de l'ectodomaine de diverses molécules de surface et est indispensable pour l'activation de la voie de signalisation NOTCH. Nous démontrons que Tspan5 et Tspan14 sont des régulateurs positifs de la voie de signalisation Notch et que Tspan15 est un régulateur négatif, agissant en amont de la γ-Sécrétase. Cette régulation négative est associée à un changement d’environnement membranaire ainsi qu’à une dynamique différente d’ADAM10 comme le montre un suivi de la protéase à l’échelle de la molécule unique. Par ailleurs, l’analyse par spectrométrie de masse quantitative a montré que la capacité d’ADAM10 à s’associer à des composants connus du réseau de tétraspanines était influencée par son interaction avec ces tétraspanines. Enfin, nous avons identifié une région de la cellule enrichie en CD9 qui contenait également ADAM10 lorsque Tspan5 est exprimée, mais ce n’est pas le cas avec Tspan15. Cette étude démontre que les différentes TspanC8 influencent différemment la capacité d’ADAM10 à cliver certains de ses substrats et illustre la capacité des tétraspanines à réguler l’activité de leurs protéines partenaires en contrôlant leur compartimentation membranaire. / It is now recognized that the lateral segregation of components of the plasma membrane or membrane compartmentalization, play an important role in the regulation of many membrane proteins functions. Tetraspanins are a family of integral membrane proteins with four transmembrane domains with a unique ability to associate with one another and with many other surface molecules to organize a molecular interactions network, the “tetraspanins web”. It was suggested that via the organization of this network, the tetraspanin play a role in membrane compartmentalization of proteins to which they associate. Tetraspanins also play a role in cellular compartmentalization by controlling the trafficking of some associated proteins. In the lab, it has been shown that six conserved tetraspanin (Tspan5, 10, 14, 15, 17 et 33) of the TspanC8 subgroup (characterized by the presence of eight cysteines in the large extracellular domain) interact directly with the metalloproteinase ADAM10 et mediates its exit from the endoplasmic reticulum. This membrane-Anchored metalloprotease mediated ectodomain shedding of various integral molecules and is essential for Notch signaling pathway activation. We demonstrate that both Tspan5 and Tspan14 are positive regulators of Notch signaling pathway, whereas Tspan15 appears as negative regulator, acting in upstream γ-Secretase. This downregulation is associated with a modification of membrane environment as well as different dynamics of ADAM10, as shown by monitoring of protease using single molecule tracking. Furthermore, quantitative mass spectrometry analysis revealed that ADAM10 ability to associate with known components of “tetraspanin web” was influenced by its interaction with these tetraspanins. Finally, we have identified a CD9 enriched cell area which also contained ADAM10 when Tspan5 is expressed but not with Tspan15.This study demonstrates that different TspanC8 influence differently the ADAM10 ability to cleave some of its substrates and illustrate the ability of tetraspanins to regulate the activity of their proteins partners by controlling their membrane compartmentalization.

Les tétraspanines sont des protéines à quatre domaines transmembranaires ayant la capacité d’interagir avec des partenaires et de les intégrer au sein d’un réseau dynamique d’interactions nommé tetraspanin web. Parmi elles, les TspanC8 représentent une sous-famille partageant un même partenaire, ADAM10.Cette protéase, essentielle au développement, est responsable du clivage de nombreux substrats dont le récepteur Notch, plusieurs cadhérines et facteurs de croissance. ADAM10 est également impliquée dans la régulation de plusieurs pathologies, telles que la maladie d’Alzheimer ou des cancers.Les précédentes recherches du laboratoire ont montré que les TspanC8 régulent la fonction d’ADAM10 à travers son expression et sa compartimentation membranaire. Les travaux exposés dans cette thèse montrent que plusieurs TspanC8 régulent également l’activité d’ADAM10 à travers une endocytose et une stabilité différentielle et en explorent les mécanismes sous-jascents. Ils démontrent également l’existence d’une compétition entre TspanC8 pour l’association avec ADAM10, et portent une réflexion sur l’utilisation de tags et sur les biais expérimentaux qui peuvent en découler. / Tetraspanins are a family of proteins containing four-transmembrane domains that can interact with partners to include them in a dynamic network of interactions named tetraspanin web. Among them, the TspanC8 tetraspanin subfamily is known to share one partner, the ADAM10 metalloprotease.The ADAM10 protease is essential to development and is responsible for the shedding of a number of substrates, including the Notch receptor ectodomain, several cadherins and growth factors. ADAM10 is also implicated in the regulation of several pathologies including Alzheimer’s disease and carcinogenesis.Previous studies from our laboratory show that TspanC8 family members regulate the function of ADAM10 through its expression and its membrane compartmentalisation.Data presented in this thesis demonstrate that several TspanC8s also regulate ADAM10’s activity through differential stability and endocytosis, and explore the subjascent mechanisms. We also show that TspanC8 family members compete for association with ADAM10; thus, we bring elements of reflexion the use of tags and subsequent experimental bias.

CD23, the natural negative regulator of IgE, has been shown to be involved in asthma progression through its regulation of IgE. To investigate if its sheddase, ADAM10, is also involved in asthma progression, three mouse models were utilized; an IgE/mast cell dependent model, an IgE dependent, mast cell independent model and a mast cell and IgE independent model. Experimental asthma was then induced in mice which were selectively deficient for ADAM10 in B cells (ADAM10-/-) and compared to WT controls. The ADAM-/- mice had decreased signs of asthma, including eosinophilia, AHR and IgE synthesis in the IgE dependent model compared to LM controls, while with the IgE independent model there was no significant difference. Thus, CD23Tg and ADAM10-/- B cell mice have reduced IgE dependent lung inflammation in mouse models compared to WT controls. As a follow up, ADAM10 was inhibited in WT mice by intranasal administration of an ADAM10 inhibitor, compared to carrier (DMSO) treated mice. As with ADAM10-/- mice, inhibition of ADAM10 was only able to control IgE dependent models. These results thus show that ADAM10 is a possible target in controlling IgE dependent allergic disease, possibly as blocking ADAM10 would cause an increase in CD23 membrane expression. To better understand how ADAM10 cleaves CD23 we first sought to confirm previous studies that CD23 is internalized, with the hypothesis that shedding takes place intracellularly, rather than at the cell surface as previously assumed. Indeed, ADAM10 is more highly expressed intracellularly than at the cell surface. At 37 ºC, crosslinking CD23, especially with the anti-stalk mAb 19G5, resulted in extensive CD23 internalization. In addition, the expected increase in soluble CD23 (sCD23) production when 19G5 was added was blocked by the addition of NH4Cl. NH4Cl is known to block the progression of the endosomal pathway. These findings thus confirmed our hypothesis that cleavage of CD23 requires internalization and progression through the endosomal pathway before it is released into the extracellular space. We further demonstrated that ADAM10 is not only involved in cleaving CD23, but also in sorting CD23 into exosomes, as B cells lacking ADAM10 do not incorporate CD23 into exosomes. In addition, we found that exosomes secreted from the cell contain full length CD23, thus showing that they could bind IgE/antigen complex and be involved in the known CD23 dependent enhancement of antigen presentation by the injection of IgE/antigen complexes compared to antigen alone. These results also show that the change in ADAM10 expression specifically in a B cell could be involved in enhancement of IgE dependent inflammation. To determine what signals change ADAM10 expression, ADAM10 promoter studies were initiated. We found that both IL-21 and anti-CD40 increased ADAM10 promoter activity, while IL-4 and IL-13 had no effect. Overall our data show that increasing ADAM10 activity and expression leads to increased inflammation and IgE and is a possible target in controlling IgE dependent diseases.

ADAM10, ADAM17, and SPAG6 contribute significantly to humoral immunity and secondary lymphoid tissue architecture. ADAM10 and ADAM17 are two closely related zinc-metalloproteinases. Through cleavage of their ligands CD23 and TNF, respectively, they greatly influence IgE production and secondary lymphoid tissue architecture maintenance. Th1 prone WT strains initially exhibit increased ADAM17 and TNF yet reduced ADAM10 relative to Th2 prone WT strains. In the absence of B cell ADAM10, a compensatory increase in ADAM17 and TNF cleavage is noted only in Th1 prone C57Bl/6, not Th2 prone Balb/c. B cell TNF homeostasis is important for maintaining secondary lymphoid tissue architecture. We show for the first time that excessive B cell TNF production in C57-ADAM10B-/- lymph nodes contributes to loss of B/T segregation, increased HEV number and size, fibrosis, loss of FDC networks, and impaired germinal center formation. Furthermore, B cell ADAM10, which enhances IgE production through CD23 cleavage, is shown to be a marker of Th2 susceptibility. B cell ADAM10 is elevated in Th2 prone mouse strains and allergic patients compared to Th1 prone controls and as B cell ADAM10 level increases, so does IgE production. Lastly, the B cell profile of allergic patients is determined to be B cell ADAM10highADAM17lowTNFlow. Furthermore, the mechanism underlying reduced class-switched antibody production in C57-ADAM10B-/- mice is explored. C57-ADAM10B-/- B cells exhibit a B10, or IL-10 producing, phenotype, which is linked to reduced antibody production. Furthermore, increased Tregs noted in C57-ADAM10B-/- mice contributed to reduced class switched IgE production and disease parameters following a house dust mite airway inflammation challenge. SPAG6, a component of the central apparatus of the “9+2” axoneme, plays a central role in flagellar stability and motility. Immune cells lack cilia, but the immunological synapse is a surrogate cilium as it utilizes the same machinery as ciliogenesis including the nucleation of microtubules at the centrosome. We demonstrate that Spag6 localizes in the centrosome and is critical for centrosome polarization at and actin clearance away from the synapse between CTL and target cells. Furthermore, improper synapse formation and function likely explains reduced CTL function and class-switched antibody production in Spag6KO mice.

CD23 has long been appreciated to be a natural, negative regulator of IgE synthesis. This understanding is due in part to animal models in which CD23 deficient or CD23 transgenic animals display exacerbated or reduced IgE levels respectively. Interestingly, CD23 is susceptible to proteolytic cleavage from the cell surface. When this occurs, CD23 loses its regulatory capability and the solubilized form can lead to pro-inflammatory events through its cytokinergic activity on macrophages. Thus, targeting this specific cleavage would be beneficial to the control of allergic disease by stabilizing CD23 at the cell surface. Inhibitor studies performed by our group as well as others indicate that the enzyme responsible for CD23 ectodomain shedding is a hydroxamate-sensitive metalloproteinase. Through collaboration with the Blobel group, we analyzed various ADAM KO mouse embryonic fibroblasts (MEFs) and found no involvement of ADAMs 8,9,12,15,17,19, and 33 in CD23 shedding, however we did find a role for ADAM10. Using ADAM10 KO MEFs and ADAM10 specific inhibitors, we discovered that ADAM10 is indeed the CD23 cleaving enzyme or “sheddase”. Thus, developing strategies that would target ADAM10 could have an effect on sCD23 release and IgE production. In the CNS, signaling through the kainate receptor (KAR) by glutamate causes an increase in ADAM10 expression. Human B cells were found to express a GluK2 containing kainate receptor and its activation increased ADAM10 expression which is in agreement with KAR activation in the CNS. Although glutamate is considered a neurotransmitter, it signals in the periphery and elevated levels are associated with certain immune disorders. A significant corresponding increase in sCD23 release is observed as well. Remarkably, this activation induced a strong increase in B cell proliferation, IgG, and IgE production and these events can be reversed through the use of NS102, a specific KAR antagonist. Thus, we report for the first time the unique presence on B cells of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation and Ig production. This enhancement and control thereof has implications for allergic and autoimmune diseases. Lastly, the CD23-ADAM10 axis was examined in a non-allergic disease state, B cell chronic lymphocytic leukemia (BCLL). BCLL is characterized by a large accumulation of CD23+ cells and high levels of soluble CD23 in the sera. After further analysis, we show that ADAM10 is indeed over-expressed in BCLL and could account for the high levels seen in this patient population. Furthermore, specifically targeting ADAM10 resulted in reduced soluble CD23 release, reduced proliferation, and enhanced apoptosis induction. Taken together the novel finding that ADAM10 is involved in CD23 shedding allows for targeted therapeutic intervention of both atopic and non-atopic disease states.

The amyloid-β precursor protein (APP) has been widely studied due to its role in Alzheimer´s disease (AD). When APP is sequentially cleaved by β- and γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is toxic to neurons. However, the main processing pathway for APP involves initial cleavage at the α-site, within the Aβ region, instead generating a neuroprotective soluble fragment, sAPPα. APP is a member of a protein family, also including the proteins APLP1 and APLP2, which are processed in a similar way as APP. In addition, K/O studies in mice have shown that the three proteins have overlapping functions where APLP2 play a key physiological role. The aim of this thesis was to study mechanisms underlying the α-secretase processing of APP and APLP2. We have used the human neuroblastoma cell-line SH-SY5Y as a model system and stimulated α-secretase processing with insulin-like growth factor-1 (IGF-1) or retinoic acid (RA). Our results show that the stimulated α-site cleavage of APP and APLP2 is regulated by different signaling pathways and that the cleavage is mediated by different enzymes. APP was shown to be cleaved by ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a PKC-dependent manner. We further show that protein levels and maturation of ADAM10 and TACE is increased in response to RA, mediated by a PI3K- or PKC-dependent signaling pathway, respectively. Another focus of our research has been O-GlcNAcylation, a dynamic post-translational modification regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase (OGA). We show that decreased OGA activity stimulates sAPPα secretion, without affecting APLP2 processing. We further show that ADAM10 is O-GlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved in a similar way as APLP2 during IGF-1 stimulation by substituting the E1 domain in APP with the E1 domain in APLP2. Together our results show distinct α-site processing mechanisms of APP and APLP2. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript.</p>

Made available in DSpace on 2016-06-02T19:48:19Z (GMT). No. of bitstreams: 1 4177.pdf: 5761817 bytes, checksum: cb8d207716a1afe86708253767f3f7ee (MD5) Previous issue date: 2012-02-24 / Financiadora de Estudos e Projetos / Alzheimer's disease (AD) is the most common cause of dementia in people over 65 years. Platelet studies with ADAM10 have shown to decrease its expression in AD patients. The association between cognitive testing and molecular biomarkers such as levels of platelet ADAM10 protein can be important tools for the accurate and early diagnosis of AD. The aim of this research was to investigate the relationship between the Mini-Mental State Examination - MMSE and the Clinical Dementia Rating - CDR with the ADAM10 expression in two groups of elderly. 30 subjects with AD were compared with 25 matched controls by sex, age and education. All ethical considerations were observed. Individual evaluations were carried out and applied the CDR and MMSE, and then performed the collection of biological material of the elderly. The techniques SDS-PAGE and Western blotting were used to quantify the ADAM10 content in platelets. After collecting the data, they were analyzed using statistical methods of comparison, correlation, association, logistic regression and diagnostic accuracy. The results show that the ratio ADAM10/_-actin was reduced in elderly patients with AD and that this reduction is intensified with the progression of the disease. MMSE and CDR have significant correlations with the values of ratio ADAM10/_-actin, this being the only statistically significant variable (p = 0.01) to increase the probability of occurrence of AD. The cutoff value < 0.4212 in ROC curve captures 70% sensitivity and specificity of 80.77% for the presence of AD according to the ratio ADAM10/_-actin. Therefore, the ratio ADAM10/_-actin seems to be a relevant biomarker for AD. The results bring important contributions to an accurate diagnosis of Alzheimer's disease from the perspective of ADAM10 as a biomarker for this disease. The results are preliminary but encouraging. / A doença de Alzheimer (DA) é a causa mais comum de demência em pessoas com mais de 65 anos de idade. Estudos plaquetários com a ADAM10 têm demonstrado diminuição na sua expressão em pacientes com DA. A associação entre testes de avaliação cognitiva e biomarcadores moleculares, tais como os níveis plaquetários da proteína ADAM10 podem contribuir para o diagnóstico preciso e precoce da DA. O objetivo desta pesquisa foi verificar a relação entre o Mini-Exame do Estado Mental MEEM e o Clinical Dementia Rating CDR com a expressão da ADAM10 em dois grupos de idosos. 30 sujeitos com DA foram comparados com 25 controles, pareados segundo sexo, idade e escolaridade. Todos os cuidados éticos foram observados. Foram realizadas avaliações individuais e aplicados o CDR e MEEM e em seguida realizada a coleta do material biológico dos idosos. As técnicas SDSPAGE e Western Blotting foram utilizadas para detecção da ADAM10. Após a coleta dos dados, estes foram analisados por meio de métodos estatísticos de comparação, correlação, associação, regressão logística e acurácia diagnóstica. Os resultados mostram que a razão ADAM10/_-actina apresenta-se diminuída em idosos com DA e que esta redução se intensifica com o avanço da doença. O MEEM e o CDR apresentam correlações significativas com os valores da razão ADAM10/_-actina, sendo esta a única variável estatisticamente significativa (p = 0,01) para o aumento da probabilidade de ocorrência da DA. O ponto de corte < 0,4212 da curva ROC capta sensibilidade de 70% e especificidade de 80,77%, para a presença de DA segundo a razão ADAM10/_-actina. Portanto, a razão ADAM10/_-actina parece ser um relevante biomarcador para DA. Os resultados trazem contribuições importantes para um diagnóstico preciso da doença de Alzheimer na perspectiva da ADAM10 como um biomarcador para esta doença. Os resultados são preliminares, porém animadores.

La protéine précurseur du peptide amyloïde (APP) est un élément central dans l'apparition et le développement de la maladie d'Alzheimer. L'APP possède deux voies de maturations distinctes. La maturation dite amyloïdogénique, via la béta-sécrétase, a principalement lieu au niveau des endosomes et entraine la production de peptides béta-amyloïdes neurotoxiques au-delà d'un certain seuil. La voie non-amyloïdogénique, via l'alpha-sécrétase, a quant à elle lieu au niveau de la membrane plasmique et entraîne la libération de fragments solubles sAPP alpha; neuroprotecteurs. Dans un premier temps, nos travaux ont mis en évidence que le récepteur de la sérotonine de type 4 (5-HT4R), l'alpha-sécrétase ADAM10 et l'APP forment un complexe protéique et que le récepteur 5-HT4 est capable de moduler à la fois l'adressage membranaire de l'alpha-sécrétase ADAM10 et de l'APP mais aussi leur maturation. Par cette modulation, le récepteur 5-HT4 peut favoriser de façon constitutive la libération de sAPP alpha. Dans un second temps, nous avons mis en place une approche protéomique, basée sur la recherche de protéines interagissant avec ce complexe et pouvant influencer le trafic cellulaire de l'ADAM10 ou de l'APP et favoriser la voie non-amyloïdogénique. Ces travaux nous permettent d'émettre des hypothèses originales quant aux mécanismes communs de régulation, à la fois de l'endocytose et du trafic du récepteur 5-HT4, de l'ADAM10 et de l'APP. Ces mécanismes mettent en cause le complexe AP-2 et un autre complexe protéique impliqué dans le trafic intracellulaire du précurseur de la protéine amyloïde. / The amyloid precursor protein (APP) is the key element in the appearance and the development of Alzheimer's disease (AD). Basically, APP can be processed following two maturation pathways. The fist one, named the amyloidogenic pathway, involves a secretase, occurs mostly in the endosomes and leads to the release ofbeta-amyloïd peptides that are neurotoxics if overproduced. The second one, named the non-amyloidogenic pathway, involves analpha-secretase, occurs mostly at the plasma membrane and leads to the release of soluble APP alpha fragments that are neuroprotective. First, our work demonstrated that a serotonin receptor, the 5-HT4 receptor (5-HT4R), is able to form a protein complex with the alpha-secretase ADAM10 and APP and that this receptor could modulate ADAM10 and APP trafficking and maturation. By this way, the 5-HT4 receptor is able to increase ADAM10 and APP cell surface localization and to constitutively promote sAPP alpha release. In a second time, using an unbiased proteomic approach, we focused on the quest of protein partners able to interact with this complex and to promote the non-amyloïdogenic pathway. This work allowed us to propose original hypothesis about shared mechanisms able to regulate 5-HT4R, APP and ADAM10 endocytosis and trafficking. These mechanisms involve the AP-2 complex and another protein complex involved in the cellular trafficking of the amyloid precursor protein.

In this study we show, to our knowledge, the first description of the role ADAM10 plays on mast cells. ADAM10 is abundantly expressed on mast cells both in vitro and in vivo. Its expression is inhibited by IL-10, a suppressive cytokine. siRNA depletion of ADAM10 on bone marrow-derived mast cells (BMMC) caused decreased IL-6 production following IgE cross-linking and also impaired BMMC stem cell factor (SCF)-induced migration through collagen IV. Mast cells and T helper cells (Th cells) in the peritoneum were reduced in ADAM10 KO mice. In addition, ADAM10 KO BMMC produced significantly less of all cytokines measured following IgE cross-linking, including IL-6, TNF-α, IL-13, and MCP-1, compared to wild type BMMC. Collectively these data show that mast cell ADAM10 can be regulated by a T regulatory cell cytokine, IL-10, and describes key ways in which ADAM10 loss affects prototypical mast cell functions and distribution.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18905号 / 医科博第61号 / 新制||医科||4(附属図書館) / 31856 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 武田 俊一, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM