GM1 OLIGOSACCHARIDE MODULATION OF CALCIUM SIGNALLING IN NEURONAL FUNCTIONS

It has been already demonstrated that the oligosaccharide chain (OligoGM1) of the ganglioside GM1, β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc-(1-1)-Ceramide, promotes neurodifferentiation in the Neuro2a murine neuroblastoma cells, used as a model, by directly interacting with the NGF specific receptor TrkA, leading to the activation of ERK1/2 downstream pathway. In this context, my PhD work aimed to investigate which other biochemical pathways, in addition to TrkA-MAPK cascade activation, are prompted by OligoGM1, with an emphasis on Ca2+ modulating factors. A proteomic analysis (nLC-ESi-MS-MS) performed on Neuro2a cells treated with 50 µM OligoGM1 for 24 hours led to the identification and quantification of 324 proteins exclusively expressed by OligoGM1-treated cells. Interestingly, some of these proteins are involved in the regulation of Ca2+ homeostasis and in Ca2+-dependent differentiative pathways. In order to evaluate if OligoGM1 administration was able to modulate Ca2+ flow, we performed calcium-imaging experiments on Neuro2a cells using the Ca2+-sensitive Fluo-4 probe. Starting from 5 minutes upon OligoGM1 administration to undifferentiated Neuro2a, a significant increase in Ca2+ influx occurs. At the same time an increased activation of TrkA membrane receptor was observed and, importantly, the addition of a specific TrkA inhibitor abolished the OligoGM1 mediated increase of the cytosolic Ca2+, suggesting that the opening of the cell Ca2+ channels following OligoGM1 administration depends on the activation of TrkA receptor. To unveil which cellular pathway activated by OligoGM1 could lead to the increase of intracellular Ca2+, time-course immunoblotting analyses were performed. The data revealed that following TrkA activation, OligoGM1 induced the activation of phospholipase PLCγ1 which converts phosphatidylinositol 4,5-bisphosphate (PIP2) to diacyglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), the second messengers that propagate cellular signalling via Ca2+ mobilization. Moreover, we observed a hyperphosphorylation of the DAG substrate, protein kinase C (PKC), which is a priming event that enables its catalytic activation in response to lipid second messengers, and we found its enrichment in lipid rafts, events that consolidate its activation. When calcium-imaging experiments where performed in the presence of xestospongin C, a potent inhibitor of IP3 receptors on endoplasmic reticulum, a reduction of about 50% of Ca2+ influx was observed, suggesting that the Ca2+ flows moved by the OligoGM1 come not only from intracellular storages, but probably also from the extracellular environment. Accordingly, in the presence of both extracellular (EGTA) and intracellular (BAPTA-AM) Ca2+ chelators the neuritogenic effect induced by OligoGM1 was abolished. The work described in this thesis confirms that the effects of GM1 ganglioside on neuronal differentiation are mediated by its oligosaccharide portion. In particular, here I highlight that the oligosaccharide, initiating a signalling cascade on the cell surface, is responsible alone for the balancing of the intracellular Ca2+ levels that underlie neurite sprouting, which have historically been attributed to the whole GM1 ganglioside and its role as lipid inserted into the plasma membrane. In this way, these data give additional information on the molecular characterization of the mechanisms by which GM1 exerts its neuronal functions.