UNIVERSITÀ DEGLI STUDI DI CATANIA DIPARTIMENTO DI SCIENZE BIOMEDICHE E BIOTECNOLOGICHE DOTTORATO DI RICERCA IN BASIC AND APPLIED BIOMEDICAL SCIENCES XXIX CICLO ------------------------------------------------------------------------------------- _______________ TESI DI DOTTORATO _____________________ Evaluation of the PI3K/Akt/mTOR pathway in Multiple Sclerosis Dott.ssa Santa Mammana Coordinatore e Tutor: Chiar.mo Prof. Ferdinando Nicoletti --------------------------------------------------------------------------------------------------- Anno accademico 2015-2016 Sommario INTRODUCTION ............................................................................................................................................. 3 Multiple sclerosis ........................................................................................................................................... 3 Clinical Course And Diagnosis ..................................................................................................................... 3 Figure 1. ......................................................................................................................................................... 4 Etiology ......................................................................................................................................................... 6 Role of infectious Agents .............................................................................................................................. 6 Immuno-Pathogenesis ................................................................................................................................... 7 Possible Mechanisms of Injury and Repair in Multiple Sclerosis. ................................................................ 7 Treatment ....................................................................................................................................................... 8 Current therapies ........................................................................................................................................... 9 The Experimental Allergic Encephalomyelitis (EAE) as the animal model for MS ................................... 10 The PI3K/Akt/mTOR pathway .................................................................................................................... 10 Figure 2. ....................................................................................................................................................... 13 Figure 3. ....................................................................................................................................................... 14 The PI3K/Akt/mTOR pathway in the adaptive immune response .............................................................. 15 The PI3K/Akt/mTOR pathway in autoimmune diseases............................................................................. 17 Inhibitors targeting the PI3K/Akt/mTOR Pathway ..................................................................................... 18 mTORC1 Inhibitors ..................................................................................................................................... 19 mTOR Inhibitors ......................................................................................................................................... 19 PI3K Inhibitors ............................................................................................................................................ 20 Dual PI3K/mTOR Inhibitors ....................................................................................................................... 21 Akt Inhibitors............................................................................................................................................... 22 AIM OF THE STUDY .................................................................................................................................... 24 Figure 4. ....................................................................................................................................................... 26 MATERIALS AND METHODS .................................................................................................................... 27 In silico analysis .......................................................................................................................................... 27 Generation of the MS “Disease gene signature” and of Rapamycin “Drug regulated genes” ................ 27 Generation of the mTOR “Regulatory molecular network” .................................................................... 27 Dataset selection and analysis of oligodendrocyte damage and remyelination processes ....................... 28 Gene enrichment analysis ........................................................................................................................ 28 Ex vivo and in vitro analysis ....................................................................................................................... 28 Animals.................................................................................................................................................... 28 Induction of MOG-induced EAE in C57BL⁄ 6 mice. .............................................................................. 29 1 Ex vivo restimulation with MOG35-55 ...................................................................................................... 29 Determination of Cytotoxicity ................................................................................................................. 30 Supernatant cytokine levels ..................................................................................................................... 30 In vitro effects of Rapamycin .................................................................................................................. 30 qRT-PCR. ................................................................................................................................................ 30 Statistical analysis ....................................................................................................................................... 31 RESULTS ........................................................................................................................................................ 32 Generation of the mTOR network ............................................................................................................... 32 Figure 5. ....................................................................................................................................................... 33 Figure 6. ....................................................................................................................................................... 34 Involvement of the mTOR network in MS .................................................................................................. 35 Figure 7 ........................................................................................................................................................ 36 Rapamycin effects in MS ............................................................................................................................ 37 Figure 8. ....................................................................................................................................................... 38 Involvement of the mTOR pathway in MS cortical lesions ........................................................................ 39 Figure 9. ....................................................................................................................................................... 40 Involvement of the mTOR pathway in oligodendrocyte function ............................................................... 41 Figure 10. ..................................................................................................................................................... 41 Figure 11. ..................................................................................................................................................... 42 Effects of PI3K/mTOR inhibitors in MS ..................................................................................................... 43 Figure 12. ..................................................................................................................................................... 44 Figure 13. ..................................................................................................................................................... 45 Figure 14. ..................................................................................................................................................... 46 Effects of mTORC1 inhibition on T cells under a Th17 polarizing condition ............................................ 47 Figure 15. ..................................................................................................................................................... 48 Figure 16. ..................................................................................................................................................... 49 Figure 17. ....................................................................................................................................................