MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

Le trafic intracellulaire est un processus fondamental qui maintient l'homéostasie cellulaire. Les RAB GTPases sont des régulateurs clés du trafic intracellulaire. RAB6 est la RAB résidente la plus abondante du Golgi. RAB6 est un régulateur clé de l'homéostasie Golgienne. Mon projet de thèse s'est intéressé à l'étude de la fonction de RAB6 dans la sécrétion post-Golgienne. Des études précédentes ont montré que la déplétion de RAB6 inhibe l'arrivée à la membrane plasmique de différents cargos : dans des cellules HeLa, NPY et VSV-G, et TNFα dans les macrophages. Nous avons donc émis l'hypothèse que RAB6 pourrait être un régulateur général de la sécrétion post-Golgienne. A l'aide de cellules MEFs RAB6 KO, nous avons d'abord montré que la sécrétion de toutes les protéines nouvellement synthétisées est inhibée. Pour comprendre les mécanismes entraînant cet effet, nous avons étudié le rôle de RAB6 dans le transport post-Golgien de trois types différents de cargos : GPI-APs (PLAP et CD59), collagen X, une protéine soluble, et une protéine transmembranaire TNFα. Afin de synchroniser le transport de cargos, nous avons utilisé le système RUSH. Ainsi, nous avons montré que RAB6 est présent sur les vésicules post-Golgiennes contenant les 3 types de cargos et que la déplétion de RAB6 affecte leur sécrétion. Les effecteurs de RAB6 sont aussi impliqués: Myosine II dans leur fission du Golgi, KIF5B dans leur transport vers la périphérie cellulaire, ELKS dans leur arrimage à la membrane plasmique. Finalement, nous avons pu montrer que les 3 cargos sont présents dans les mêmes vésicules post-Golgiennes avec RAB6. Ces résultats montrent que RAB6 régule la sécrétion de différents cargos. / Intracellular trafficking is a fundamental process which ensures cell homeostasis. RAB GTPases are key regulators of intracellular trafficking. RAB6 is the most abundant Golgi resident RAB and is a key regulator of Golgi homeostasis. My Ph.D project focused on understanding the function of RAB6 in post-Golgi secretion.Previous reports have shown that RAB6 depletion impairs the arrival at the plasma membrane of different cargoes: in HeLa cells, NPY and VSV-G and TNFα in macrophages. We thus hypothesized that RAB6 could be a general regulator of post-Golgi transport steps. Using MEF cells from RAB6 KO mice, we first showed that the secretion of all newly synthesized proteins is affected. To decipher the mechanisms leading to this inhibition, we have then investigated the role of RAB6 in the post-Golgi transport of three different classes of proteins, GPI-anchored proteins (such as Placental Alkaline phosphatase or PLAP and CD59), collagen X, a soluble protein, and the transmembrane protein TNFα. In order to synchronize transport of newly-synthetized cargoes along the secretory pathway, we used the RUSH system. Here, we show that RAB6 is present on post Golgi vesicles containing the three types of cargo and that RAB6 depletion affects their secretion to the plasma membrane. RAB6 effectors are also implicated: Myosin II for their fission from the Golgi, KIF5B for their transport to the cell periphery, ELKS/RAB2IP2 for their docking with the plasma membrane. Finally, we could show that these three cargoes are present in the same post-Golgi transport carriers with RAB6. Altogether, these results show that RAB6 regulates the secretion of a wide number of cargo proteins.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24843号 / 医科博第151号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齋藤, 潤, 教授 遊佐, 宏介, 教授 松田, 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis (Ph. D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 133 pages. Includes vita. Includes bibliographical references.