Modulation of calcium signaling depends on the oligosaccharide of GM1 in Neuro2a mouse neuroblastoma cells
- PDF / 2,951,009 Bytes
- 15 Pages / 595.276 x 790.866 pts Page_size
- 34 Downloads / 185 Views
ORIGINAL ARTICLE
Modulation of calcium signaling depends on the oligosaccharide of GM1 in Neuro2a mouse neuroblastoma cells Giulia Lunghi 1
&
Maria Fazzari 1 & Erika Di Biase 1 & Laura Mauri 1 & Sandro Sonnino 1 & Elena Chiricozzi 1
Received: 10 September 2020 / Revised: 2 November 2020 / Accepted: 9 November 2020 / Published online: 17 November 2020 # The Author(s) 2020
Abstract Recently, we demonstrated that the oligosaccharide portion of ganglioside GM1 is responsible, via direct interaction and activation of the TrkA pathway, for the ability of GM1 to promote neuritogenesis and to confer neuroprotection in Neuro2a mouse neuroblastoma cells. Recalling the knowledge that ganglioside GM1 modulates calcium channels activity, thus regulating the cytosolic calcium concentration necessary for neuronal functions, we investigated if the GM1-oligosaccharide would be able to overlap the GM1 properties in the regulation of calcium signaling, excluding a specific role played by the ceramide moiety inserted into the external layer of plasma membrane. We observed, by calcium imaging, that GM1-oligosaccharide administration to undifferentiated Neuro2a cells resulted in an increased calcium influx, which turned out to be mediated by the activation of TrkA receptor. The biochemical analysis demonstrated that PLCγ and PKC activation follows the TrkA stimulation by GM1oligosaccharide, leading to the opening of calcium channels both on the plasma membrane and on intracellular storages, as confirmed by calcium imaging experiments performed with IP3 receptor inhibitor. Subsequently, we found that neurite elongation in Neuro2a cells was blocked by subtoxic administration of extracellular and intracellular calcium chelators, suggesting that the increase of intracellular calcium is responsible of GM1-oligosaccharide mediated differentiation. These results suggest that GM1-oligosaccharide is responsible for the regulation of calcium signaling and homeostasis at the base of the neuronal functions mediated by plasma membrane GM1. Keywords GM1 ganglioside . GM1-oligosaccharide . TrkA neurotrophin receptor . Calcium signaling . Plasma membrane signaling . Neurodifferentiation
Abbreviations A.U. arbitrary units BAPTA-AM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid (acetoxymethyl ester) BSA bovine serum albumin Ca2+ calcium CTRL control
Ganglioside nomenclature is in accordance with IUPAC-IUBB recommendations [56].
DMEM HG DRM DTT EDTA EGTA ERK1/2 FBS F GM1
* Giulia Lunghi * Elena Chiricozzi
1
Department of Medical Biotechnology and Translational Medicine, University of Milano, Segrate, Milano, Italy
HBSS+ HPTLC IP3 N2a NS
Dulbecco’s modified Eagle’s high glucose medium Detergent-resistant membrane 1,4-Dithiothreitol ethylenediamine tetraacetic acid ethylene glycol tetraacetic acid extracellular signal-regulated protein kinases 1 and 2 fetal bovine serum fluorescence I I 3 N e u 5 A c - G g 4 C e r , β - G a l -( 1 – 3 ) - β GalNAc-(1–4)-[α-Neu5Ac-(2–3)]-β-Gal-(1– 4)-β-Glc-Cer Hank’s Balanced Salt Solution containing calc
Data Loading...
