GM1 promotes TrkA‐mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA

Abstract

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA–GM1 oligosaccharide complex at the cell surface. To study the TrkA–GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross‐linked TrkA–GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA–GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent‐resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by ‘flopping’ down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1.

GM1 Oligosaccharide Crosses the Human Blood–Brain Barrier In Vitro by a Paracellular Route

“…Subsequently, N2a cells were incubated with the basolateral solution containing the hBLEC-crossed OligoGM1. As shown in Figure 9a, hBLEC-crossed OligoGM1 induced a neuron-like morphology after 24 h. In accordance with the OligoGM1 behavior reported in previous studies [22,23], the N2a receiving hBLEC-crossed OligoGM1 showed a clear neurite sprouting that was instead absent in the round-shaped control cells.…”

“…Additionally, we previously reported that OligoGM1 induces N2a neuritogenesis by eliciting the activation of the TrkA-ERK1/2 pathway [22,23]. Importantly here, by immunoblotting, we highlighted the activation of the TrkA-ERK1/2 pathway by hBLEC-crossed OligoGM1, as shown in Figure 9b.…”

“…To date, although the missing association between GM1 injection and Guillain-Barré syndrome development seems to have been accepted, the disadvantage of GM1 s inability to cross the BBB still means its potential is underexploited. Importantly, our studies to disclose the mechanism of action underlying the neurotrophic and reparative properties of GM1 have revealed that the bioactive portion of the molecule is the oligosaccharide chain [22][23][24]26,27], which surprisingly, if administered peripherally into mice, is detectable within the brain [25]. Even more noteworthy is the finding that OligoGM1 injection in a sporadic Parkinson's disease model, the B4galnt1 +/mouse, in which the neuronal GM1 content is decreased due to a reduced expression of B4galnt1 glycosyltransferase, promoted a recovery of both behavioral and biochemical features, reaching healthy conditions [25].…”

“…To further verify if OligoGM1 maintains its bioactive properties after crossing the BBB model, we evaluated the hBLEC-crossed OligoGM1 neurodifferentiative potential on N2a cells, as previously reported [22,23]. Thus, we performed an apical to basolateral transport experiment (A → B), employing 300 µM OligoGM1 applied to the upper compartment.…”

“…As reported in recent reviews, many efforts are still ongoing and seem to be concentrated on the possibility of using vehicles (i.e., liposomes) to drive the GM1 into the brain [2,7,8], however without any reported success to date at the best of our knowledge.In this context, which seems to be disadvantaging the therapeutic use of GM1, our studies have investigated the molecular mechanism underlying the neurotrophic properties of GM1. Indeed, we recently recognized that GM1 exerts its neurotrophic functions by interacting with and activating plasma membrane receptors throughout its oligosaccharide portion-GM1-oligosaccharide (OligoGM1) [22][23][24][25]. We demonstrated that OligoGM1, alone and without entering into the cells, perfectly replicates the neuronal differentiative properties of GM1 in neuroblastoma Neuro2a (N2a) cells and in primary neurons [22,26].…”