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Charcot-Marie-Tooth disease is the most common neuromuscular disorder, with a prevalence of 1:2500. Despite the phenotypical similarities among patients, it is characterized by a remarkable genetic heterogeneity. Among the different genes that are responsible for CMT, mutations in Myelin Protein Zero (MPZ) gene cause CMT1B and represent the 5% of all CMT cases. MPZ gene encodes for the P0 protein, the principal myelin protein of the peripheral nervous system; at the moment, more than 200 mutations in these gene are known and almost all mutations are pathogenic. The phenotype of MPZ related neuropathies is also variable, implying multiple pathogenic mechanisms. We focused our interest on misglycosylation, which is increasingly recognized as a cause of different diseases, but has never been investigated in inherited neuropathies, even if peripheral myelin is largely composed of glycoproteins. We identified two groups of mutations in MPZ gene: one group is composed by variants introducing a new glycosylation site in the P0 sequence, the other one includes mutations that eliminate the constitutive glycosylation site of the P0 protein. Through transient transfection in different cellular lines (HeLa cells and Schwannoma rat cells), and through different techniques of cell and molecular biology, we founded that mutant protein with a double oligosaccharide are partially retained intracellularly, with consequences on their adhesion capacity; instead, mutant proteins that loose the constitutive glycosylation site were less affected in localization or adhesion capacity. To further investigate how peripheral myelination is affected by misglycosylation, in particular, we generated a mouse model carrying the mutation D61N to evaluate in vivo the effects of hyperglycosylation. This mouse model, that represent the first knock-in model of CMT1B generated using the CRISPR/Cas9 technology, faithfully recapitulates the human pathology and confirms hyperglycosylation as a novel pathomechanism for CMT1B. We characterized the mice with behavioral test and with electrophysiological analysis, finding a severe impairment in nerve functionality; this was further confirmed by morphological analysis that revealed a de/dysmyelinating phenotype with the presence in sciatic nerves of myelin abnormalities similar to tomacula structures. Starting from evidences founded in literature, we evaluated the possibility to test a molecule, belonging to a class of compounds called iminosugars, that are potentially able to inhibits some enzymes involved in glycosylation cascade, and that are already used for the treatment of other pathologies. We obtained encouraging results from the treatment of cells transfected with the D61N mutation, in terms of protein localization and adhesion capacity; also the treatment of mutant DRGs that were characterized by myelin abnormalities, ameliorated the morphology. However, more in depth experiments showed that this molecule didn't act modifying the glycosidic structure but decreasing the amount of myelin proteins. The ineffectiveness of the treatment showed that, before proceeding, it is necessary to know the glycosidic composition of the new oligosaccharide but also of the constitutive one, to find a molecule able to modify glycans in a targeted way.

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    Università degli studi di Genova
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    DDC:    572
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