Studi di caratterizzazione del meccanismo di azione del proteoglicano NG2/CSPG4 nella sclerosi multipla

Abstract

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS). Although the events triggering MS are still unknown, T lymphocytes reactive toward the myelin sheath are thought to have a prominent role. The myelin sheath is the most abundant membrane structure in the vertebrate nervous system. Its unique composition and its unique segmental structure is responsible for the saltatory conduction of nerve impulses which allow the myelin sheath to support the fast nerve conduction in the thin fibers of the vertebrate system. Oligodendrocytes (OLs), a special class of glial cells, are essential for myelin production and organization; in fact, myelin is a spiral extension of the plasma membrane of OLs. OLs are terminally differentiated and originate from neuro-epithelial stem cells localized in the sub-ventricular zone (SVZ). The OLs lineage is well characterized, SVZ stem cells proliferate and lead to glial-restricted (GR) progenitors and then to oligodendrocyte precursor cells (OPCs), finally to mature oligodendrocytes. The OPCs, are activated during inflammation and mediate remyelination in MS patients even if in an inefficient way. Characterization by biochemical markers such as NG2 proteoglycan has been a specific tool to study the OLs maturation steps in both in vivo and in vitro experiment. Nerve-glial antigen 2 (NG2), the product of the CSPG4 gene, is a single membrane-spanning chondroitin sulphate proteoglycan with a large extracellular domain and a short cytoplasmic tail. NG2 is expressed not only by OPCs but also by vascular mural cells, including pericytes in the CNS, that form the Neuro-vascular unit (NVU) together with blood-brain barrier (BBB). There are numerous functions attributed to NG2: several studies have shown the involvement in cell-cell and cell-extracellular matrix adherence. In contrast, little is known about NG2 function in the CNS and in particular on OPC cells. This work was focused on the study of NG2 in the CNS and our hypotheses are: • Since the pathology is characterized by a relevant autoimmune component, we hypothesize that NG2 proteoglycan may be involved in the abnormal immune response typical of MS; • Regarding NG2 expression in OPCs and pericyte cells, we hypothesize that these cells are involved in disease progression. First of all, to investigate the role of NG2 antigen, we analyzed CSF samples of MS patients appropriately collected and classified, to find auto-antibodies against NG2 through ELISA (enzyme-linked immunosorbent assay) experiments. We verified the presence of auto-antibodies by western blot and correlated the experimental data with the MS patients clinical values. Again, we found a MS patient subset positive for NG2 autoantibodies and these patients show a more severe disease. Finally, we propose autoantibodies against NG2 as a prognostic biomarker. To characterize the role of NG2 in OPCs and pericyte cells, we utilized an animal model of MS (EAE, Experimental Autoimmune Encephalomyelitis, obtained with MOG immunization), in NG2 knock-out (NG2-KO) mice. In NG2 KO, MOG immunization largely failed to produce a substantial demyelinization phenotype and manifestation of disease was overall markedly attenuated, compared to WT. To understand the mechanisms involved in NG2-KO protection from EAE, several approaches were performed. Histological analysis was done and infiltrated cells (macrophages and T-cells) were counted. Compared to WT EAE affected mice, analysis showed an important reduction in demyelination in NG2-KO EAE affected mice. Further histological analysis of CNS spinal cord sections demonstrated a reduced number of IBA1-positive macrophages in NG2KO mice. About T-cells, no significant difference was observed in the number of infiltrated, but proliferation of stimulated (anti-CD3) T-cells from NG2-KO was significantly lower, compared to WT. Cytokines production in in vitro analysis of stimulated T-cells showed different expression in NG2 KO compared to WT, in fact INFγ, IL-17 and IL-4 mRNA levels were up-regulated. About mRNA levels analysis on dendritic cells (DCs), IL-12 was down-regulated and TNFα was up-regulated, but no differences was show on DCs maturation after LPS stimulation. The OPCs analysis in SNC of NG2 KO EAE affected mice, showed the same number of cells compared to in naïve NG2, while in WT-EAE affected mice, the number of OPCs was dramatically lower than the naïve WT. We also analyzed the NVU structure, studying the expression of Claudin-5 and Occludin BBB proteins. Prior to MOG immunization, the expression of all two markers was decreased in naïve NG2-KO mice, compared to wild type mice. However, during the disease phase we observed an increase in the two markers in NG2 null mice to a level comparable to WT mice prior to MOG challenge. On the basis of results from our laboratories, we believe that the absence of NG2 can influence the course of disease in a protective way.