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Expansion De Triplets CTG Et Arrêt Prolifératif Précoce Des Myoblastes DM1 Erwan Gasnier

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Expansion De Triplets CTG Et Arrêt Prolifératif Précoce Des Myoblastes DM1 Erwan Gasnier Expansion de triplets CTG et arrêt prolifératif précoce des myoblastes DM1 Erwan Gasnier To cite this version: Erwan Gasnier. Expansion de triplets CTG et arrêt prolifératif précoce des myoblastes DM1. Biochimie, Biologie Moléculaire. Université Pierre et Marie Curie - Paris VI, 2012. Français. NNT : 2012PAO66195. tel-00829312 HAL Id: tel-00829312 https://tel.archives-ouvertes.fr/tel-00829312 Submitted on 3 Jun 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THESE DE DOCTORAT DE L’UNIVERSITE PIERRE et MARIE CURIE École doctorale Complexité du Vivant (ED515) Présentée par Mr Erwan GASNIER Pour obtenir le grade de Docteur de l’université PARIS VI Spécialité Biologie Moléculaire et Cellulaire Sujet de thèse : EXPANSION DE TRIPLETS CTG ET ARRET PROLIFERATIF PRECOCE DES MYOBLASTES DM1 Thèse dirigée par : Dr FURLING Denis Soutenue le 13 Avril 2012 Devant le jury composé de : Pr FRIGUET Bertrand Président/Examinateur Dr BASSEZ Guillaume Examinateur Dr SERGEANT Nicolas Rapporteur Dr AGBULU Onnik Rapporteur Dr FURLING Denis Directeur de thèse EXPANSION DE TRIPLETS CTG ET ARRET PROLIFERATIF PRECOCE DES MYOBLASTES DM1 Thèse réalisée au sein du laboratoire : Thérapie des maladies du muscle strié - Institut de Myologie UPMC Univ. Paris 6, Um76 / Inserm, U974 / CNRS, Umr7215 G.H. Pitié-Salpêtrière - Bâtiment Babinski 47-83, bld de l'hôpital - 75651 Paris cedex 13, France Expansion de triplets CTG et arrêt prolifératif précoce des myoblastes DM1 La dystrophie myotonique de type I est la pathologie neuromusculaire la plus répandue chez l’adulte. Elle est caractérisée par une atteinte multisystémique plus ou moins prononcée en fonction de l’extension des répétitions CTG, mutation à l’origine de l’atteinte. Le muscle squelettique est particulièrement touché avec un phénomène de myotonie ainsi qu’une atrophie. Les myoblastes, à l’origine de la formation des muscles et de leur régénération potentielle, présentent, chez les patients DM1, une capacité proliférative limitée par rapport à des myoblastes issus d’individus sains. C’est l’activation précoce de la voie p16 qui est à l’origine de cette sénescence prématurée des cellules DM1. Les mécanismes conduisant à ce phénotype sont néanmoins inconnus. Au cours de cette étude, nous avons tenté de décrypter une partie de ces mécanismes et notamment les liens potentiels entre les expansions CTG, la sensibilité au stress oxydatif et l’activation précoce de la voie p16 conduisant à la sénescence des myoblastes DM1. Mots clés : myoblastes, expansions, dystrophie, stress oxydatif, p16, capacité proliférative. CTG expansion and early proliferative arrest of DM1 myoblasts Myotonic Dystrophy type I is the most common neuromuscular pathology at adult age. DM1 is characterized by a multisystemic but variable phenotype, correlated with the size of the CTG expansion. Skeletal muscle tissue is particularly affected showing myotonic symptoms and atrophy. Myoblasts, responsible for skeletal muscle growth and repair, present in DM1 patients a reduced proliferative capacity compared to cells isolated from non-affected individuals. During a previous study, p16INK4A was identified as the trigger of this abnormal proliferative arrest in DM1 myoblasts, but mechanisms activating this pathway are still unknown. In this study, we attempted to decrypt these mechanisms, particularly the potential links between CTG expansions, oxidative stress sensitivity and early activation of the p16 pathway leading to myoblasts senescence. Key words: myoblasts, expansions, dystrophy, oxidative stress, p16, proliferative capacity 1 2 Remerciements : ABACDEFBACB BACACBACACCBBBECBBBEABADABAD AAECBBCBBACBBACB CAC C BB!!!!EE F B"A AA CACA CCC #ECCCBFCACCBACCBAAA BCCBCBCBADBCACEB ABB A B BABAEEEEA AA CBBC A B $$ %ABBBCCC&A%ABBBCCC&AEACEEACEEACEA$CCA$CC A 'B B F CB CCAAB B BBACBCA"BBBACBCA"B AE F CC C$A (B(BB )$)$EE "C CACA"BEBBACCAB$CCCA BBAABBAA*BAC+,EC*BAC+,ECAAAA A"BACCBBBBACC-CBC-CB CC*.AA,*.AA,*.AA,CCABACCCABACCCABACBBBB BADA/CB0$ABCCCCCABCCAB ABAB %ABACCC&C%ABACCC&C1111%C%C%C2222CAAABCAAAB BB$$CC%CB BBCB CC 3ACBCCC4EBCACA 'BABBBEABAAAAEFCB ACCACACCAC 3 C0E'C5AC%ECBAABC B6BACABEACCAB78)EABACA BBBB 3 C C 44EEEE 9AC B 5BAB C C A "CE AB B BB A B C A C B *B,ABABBBACCAFCBA FBBFBB %%B:EBBCAB:EBBCA ACC ACC $A ' A B C CE A BB A - AFCB ACBCACBC %CCA%CCA000EBCEBC C-BBCABC-BBCABBABABABAACB$$CB*BCACB$$CB*BC BBFACB+,EAAFCBBABCCACBABCCAC 3CCBECBBA ACBE A CC!!!!0A"CE0A"CE B CA B BB BB &B B AAB BBAAACBABBBAAACBAB % B"A A CCC % C B BB BB A B AACAB"CCCABBCB*CB "";;;,;;;,;;;, CAABBAAB-CA"C- A"BA"B<E%BBE7"CBEC<E%BBE7"CBEC<E%BBE7"CBEC!!!!E:AAE0CE-E(BE:AAE0CE-E(B :B"BF-BCBA1:B"BF-BCBA1.CE=">C.CE=">C.CE=">C????2222 C AAB B A - A A BAA<< .E 05CE EE4"FE@BABACB4"FE@BABACB4"FE@BABACB CAABAABCBCACAC"A AA1AA1CACACA2C=C+2C=C+2C=C+ 4 % B"A BCA CCC BBA B B 3CBA CC A C 0C0CEE A B CACCACACACA C %%!!!!00 .AECAAABABACAACA %CCA@BAAA5BC7"B&AE CB5B$BCA % AB A CCC BBCB .CAC )C)CAEAE # .BB>E BB 'CA A CD A C A $CCAFC5A"B$CCAFC5A"B $EFABCCCAACACACBBC CAABBEB&CEBAABBAAB-B-B AAAA 5 6 7 8 LISTE DES ABREVIATIONS --------------------------------------------------------------------------------- 13 INTRODUCTION ------------------------------------------------------------------------------------------------ 19 I. LE MUSCLE SQUELETTIQUE ----------------------------------------------------------------------- 23 I.1. GENERALITES SUR LE MUSCLE SQUELETTIQUE ---------------------------------------------------------- 23 a. Origine embryonnaire des précurseurs du muscle ---------------------------------------------- 23 b. Voies moléculaires et engagement dans la voie myogénique ---------------------------------- 24 c. Structure du muscle squelettique------------------------------------------------------------------- 27 d. Structure des fibres musculaires ------------------------------------------------------------------- 27 e. Structure des myofibrilles --------------------------------------------------------------------------- 28 I.2. REGENERATION MUSCULAIRE ET CELLULES SATELLITES ---------------------------------------------- 30 a. Cellules satellites : définition ---------------------------------------------------------------------- 30 b. Activation des cellules satellites ------------------------------------------------------------------- 31 c. Devenir des cellules satellites activées ------------------------------------------------------------ 33 d. Différenciation des myoblastes --------------------------------------------------------------------- 35 e. La fusion des myoblastes ---------------------------------------------------------------------------- 36 f. Modèle in vitro de cellules satellites -------------------------------------------------------------- 37 II. CAPACITE PROLIFERATIVE ET ACTEURS DU CYCLE CELLULAIRE --------------- 41 II.1. CAPACITE PROLIFERATIVE DES CELLULES SATELLITES -------------------------------------------- 41 a. Capacité proliférative ------------------------------------------------------------------------------- 41 b. Le cycle cellulaire : les cyclines ------------------------------------------------------------------- 43 c. Le cycle cellulaire : les kinases dépendantes des cyclines (CdK) ----------------------------- 46 d. La régulation de l’activité des CdK --------------------------------------------------------------- 47 e. Le rôle de la protéine du Rétinoblastome --------------------------------------------------------- 48 II.2. LES INHIBITEURS DES KINASES DEPENDANTES DES CYCLINES (CKI) ---------------------------- 49 a. Les inhibiteurs CIP/KIP----------------------------------------------------------------------------- 50 b. Les inhibiteurs INK4 -------------------------------------------------------------------------------- 52 III. LA SENESCENCE REPLICATIVE --------------------------------------------------------------- 59 III.1. GENERALITES --------------------------------------------------------------------------------------------- 59 a. L’arrêt prolifératif ----------------------------------------------------------------------------------- 59 b. La résistance au processus d’apoptose ----------------------------------------------------------- 60 c. Les modifications morphologiques ---------------------------------------------------------------- 61 d. Les modifications métaboliques -------------------------------------------------------------------- 62 III.2. LA VOIE « TELOMERES DEPENDANTE » --------------------------------------------------------------- 62 INK4A III.3. LA VOIE P16 ---------------------------------------------------------------------------------------- 64 a. Locus INK4A/ARF (CDKN2A) --------------------------------------------------------------------- 64 b. Mécanismes d’activation de la voie p16 ---------------------------------------------------------- 66 9 III.4. INDUCTION DE LA SENESCENCE PAR UN STRESS ---------------------------------------------------- 69 a. Modification du taux d’oxygène dans les cultures ----------------------------------------------- 69 b. Addition de substances oxydantes
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