5 RIASSUNTO 6 AIMS OF THE WORK 43 MATERIALS AND METHODS 44 Antibodies and reagents 44 Plasmids, oligonucleotides and site-directed mutagenesis 44 Cell culture 45 Cell transfection 46 Cell lysis and fractionation of nuclei/cytosol 47 Protein purification from bacteria extracts 48 Protein pulldown 48 Western blotting 49 Immunolabeling of live cells and cell fixation 49 Immunofluorescence 50 Mouse colony 50 Immunohistochemistry 51 Total RNA purification, retrotranscription and qRT-PCR 51 Image

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... isition and analysis 52 Statistical analysis 53 RESULTS 54 A PDZ-binding motif is specifically required for endosomes to plasma membrane recycling of GPR17 54 SNX27 interacts with GPR17 at the level of endosomal compartments 59 SNX27 regulates GPR17 recycling and levels of expression 64 SNX27 is required for the correct kinetics of OL differentiation 71 Altered OL differentiation and myelin defects in Ts65Dn mouse model of DS correlate with a decrease of SNX27 levels 76 mir-155 overexpression inhibits Oli-neu differentiation 83 DISCUSSION 85 SNX27 interacts with the PDZ-binding motif of GPR17 and is required for receptor recycling to the plasma membrane 86 Loss of SNX27 accelerates OL differentiation kinetics 88 OL development in SNX27-deficient Ts65Dn brains 89 mir-155 up-regulation inhibits OL differentiation 91 CONCLUSIONS 92 ACKNOWLEDGMENTS 93 REFERENCES 94 TABLES 106 3