Università degli Studi di Milano, 2018-01-25

Multiple sclerosis (MS), considered the lead disease featuring demyelination, is the most common cause of non traumatic disability in young people. It is characterized by inflammation, progressive demyelination and gliosis, axonal injury and loss. The pathological hallmarks of all the subtypes of this disease are focal areas, called plaques, of demyelination in the CNS, with surrounding inflammation and neurodegeneration. Despite its high prevalence, multiple sclerosis remains a challenging ailment to study. Currently the most widely accepted hypothesis concerning MS pathogenesis is the autoimmune hypothesis: an autoimmune inflammation is proposed to be the cause of demyelination and auto-reactive leukocytes could be the disease initiators. One of the therapeutic approaches that is currently being developed to improve the regenerative outcome involves the use of CNS reactive antibodies to promote remyelination. One of these antibodies, rHIgM22, is able to bind to oligodendrocytes and myelin in vitro. Moreover, rHIgM22 is able to enter the CNS, accumulate at lesion site and promote remyelination in mouse models of chronical demyelination. As a matter of fact, this antibody has recently passed a phase I clinical trial for treatment of MS. Evidence suggests that the binding target of rHIgM22 could be associated with plasma membrane lipid rafts, and that lipid rafts might be involved in the signaling associated with the biological activity of this antibody. Moreover, it has been demonstrated that, isolated OPCs do not respond to rHIgM22 treatment, instead mixed glial cultures consisting of astrocytes, OPCs and microglial cells demonstrate observable rHIgM22-mediated OPC proliferation. The aim of this study was to analyze the plasma membrane lipid rafts composition in MGC in order to evaluate the effects exerted by rHIgM22 on these cells after single dose treatment of various duration. The analysis of the lipids and proteins distribution in MGC fractions, obtained after discontinuous sucrose gradient centrifugation of cells, and the alteration on lipids and protein composition of MGC due to the rHIgM22 treatment have been tested using TLC immunostaining assays and western blot analysis. The results obtained show that the DRM fraction obtained from MGC was enriched in sphingolipids, in particular sphingomyelin and gangliosides together with Lyn, Caveolin 1 and PrP(SAF32). On the contrary, phospholipids, in particular phosphatididylethanolamine and phosphatidic acid are enriched in the HD fraction, together with integrin αv and Akt. Furthermore we observed that rHIgM22 exerted an effect on the expression of P-Src(Y416) family and Lyn, that show a significant decrease, and PDGFRα that shows a significant increase. Moreover, the rHIgM22 treatment also induces a decrease in the activity of the aSMase. We hypothesize that the treatment with IgM22 could elicit biological responses mediated by alterations of lipid-dependent membrane organization which result in a reorganization of Lyn, integrin αvβ3 and PDGFRα at the cell surface to form a signaling complex. The formation of this complex triggers Lyn activation which in turn promotes oligodendrocyte precursor cells (OPCs) survival and proliferation and an inhibition of the pro apoptotic signaling. Moreover, the increased activation of Lyn could determine a decrease in ASMase activity and consequently in ceramide generation, thus inhibiting pro-apoptotic signaling and/or organization of sphingolipid-dependent signaling platforms. The identification of the binding targets of this antibody, able to promote remyelination in validated mouse models of MS, and the characterization of their membrane microenvironment could significantly contribute to the reveal the signaling mechanisms underlying the biological activity of rHIgM22.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/543913
supervisore: A. Prinetti; coordinatore: S. Sonnino
Settore BIO/10 - - Biochimica

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18002978