GM1 structural requirements to mediate neuronal functions.

Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc. Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside's oligosaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.

Gangliosides in the differentiation process of primary neurons: the specific role of GM1-oligosaccharide.

It has been recently reported by our group that GM1-oligosaccharide added to neuroblastoma cells or administered to mouse experimental model mimics the neurotrophic and neuroprotective properties of GM1 ganglioside. In addition to this, differently from GM1, GM1-oligosaccharide is not taken up by the cells, remaining solubilized into the extracellular environment interacting with cell surface proteins. Those characteristics make GM1-oligosaccharide a good tool to study the properties of the endogenous GM1, avoiding to interfere with the ganglioside natural metabolic pathway. In this study, we show that GM1-oligosaccharide administered to mice cerebellar granule neurons by interacting with cell surface induces TrkA-MAP kinase pathway activation enhancing neuron clustering, arborization and networking. Accordingly, in the presence of GM1-oligosaccharide, neurons show a higher phosphorylation rate of FAK and Src proteins, the intracellular key regulators of neuronal motility. Moreover, treated cells express increased level of specific neuronal markers, suggesting an advanced stage of maturation compared to controls. In parallel, we found that in the presence of GM1-oligosaccharide, neurons accelerate the expression of complex gangliosides and reduce the level of the simplest ones, displaying the typical ganglioside pattern of mature neurons. Our data confirms the specific role of GM1 in neuronal differentiation and maturation, determined by its oligosaccharide portion. GM1-oligosacchairide interaction with cell surface receptors triggers the activation of intracellular biochemical pathways responsible for neuronal migration, dendrites emission and axon growth.

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

Ganglioside GM1 (GM1) has been reported to functionally recover degenerated nervous system in vitro and in vivo, but the possibility to translate GM1's potential in clinical settings is counteracted by its low ability to overcome the blood-brain barrier (BBB) due to its amphiphilic nature. Interestingly, the soluble and hydrophilic GM1-oligosaccharide (OligoGM1) is able to punctually replace GM1 neurotrophic functions alone, both in vitro and in vivo. In order to take advantage of OligoGM1 properties, which overcome GM1's pharmacological limitations, here we characterize the OligoGM1 brain transport by using a human in vitro BBB model. OligoGM1 showed a 20-fold higher crossing rate than GM1 and time-concentration-dependent transport. Additionally, OligoGM1 crossed the barrier at 4 °C and in inverse transport experiments, allowing consideration of the passive paracellular route. This was confirmed by the exclusion of a direct interaction with the active ATP-binding cassette (ABC) transporters using the "pump out" system. Finally, after barrier crossing, OligoGM1 remained intact and able to induce Neuro2a cell neuritogenesis by activating the TrkA pathway. Importantly, these in vitro data demonstrated that OligoGM1, lacking the hydrophobic ceramide, can advantageously cross the BBB in comparison with GM1, while maintaining its neuroproperties. This study has improved the knowledge about OligoGM1's pharmacological potential, offering a tangible therapeutic strategy.

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

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.

Role of the GM1 ganglioside oligosaccharide portion in the TrkA-dependent neurite sprouting in neuroblastoma cells.

GM1 ganglioside (II3 NeuAc-Gg4 Cer) is known to promote neurite formation in neuroblastoma cells by activating TrkA-MAPK pathway. The molecular mechanism by which GM1 is involved in the neurodifferentiation process is still unknown, however, in vitro and in vivo evidences have suggested that the oligosaccharide portion of this ganglioside could be involved. Here, we report that, similarly to the entire GM1 molecule, its oligosaccharide II3 NeuAc-Gg4, rather than its ceramide (Cer) portion is responsible for the neurodifferentiation process by augmenting neurite elongation and increasing the neurofilament protein expression in murine neuroblastoma cells, Neuro2a. Conversely, asialo-GM1, GM2 and GM3 oligosaccharides are not effective in neurite elongation on Neuro2a cells, whereas the effect exerted by the Fuc-GM1 oligosaccharide (IV2 αFucII3 Neu5Ac-Gg4 ) is similar to that exerted by GM1 oligosaccharide. The neurotrophic properties of GM1 oligosaccharide are exerted by activating the TrkA receptor and the following phosphorylation cascade. By photolabeling experiments performed with a nitrophenylazide containing GM1 oligosaccharide, labeled with tritium, we showed a direct interaction between the GM1 oligosaccharide and the extracellular domain of TrkA receptor. Moreover, molecular docking analyses confirmed that GM1 oligosaccharide binds the TrkA-nerve growth factor complex leading to a binding free energy of approx. -11.5 kcal/mol, acting as a bridge able to increase and stabilize the TrkA-nerve growth factor molecular interactions.

The Neuroprotective Role of the GM1 Oligosaccharide, II3Neu5Ac-Gg4, in Neuroblastoma Cells.

Recently, we demonstrated that the GM1 oligosaccharide, II3Neu5Ac-Gg4 (OligoGM1), administered to cultured murine Neuro2a neuroblastoma cells interacts with the NGF receptor TrkA, leading to the activation of the ERK1/2 downstream pathway and to cell differentiation. To understand how the activation of the TrkA pathway is able to trigger key biochemical signaling, we performed a proteomic analysis on Neuro2a cells treated with 50 µM OligoGM1 for 24 h. Over 3000 proteins were identified. Among these, 324 proteins were exclusively expressed in OligoGM1-treated cells. Interestingly, several proteins expressed only in OligoGM1-treated cells are involved in biochemical mechanisms with a neuroprotective potential, reflecting the GM1 neuroprotective effect. In addition, we found that the exogenous administration of OligoGM1 reduced the cellular oxidative stress in Neuro2a cells and conferred protection against MPTP neurotoxicity. These results confirm and reinforce the idea that the molecular mechanisms underlying the GM1 neurotrophic and neuroprotective effects depend on its oligosaccharide chain, suggesting the activation of a positive signaling starting at plasma membrane level.

Parkinson's disease recovery by GM1 oligosaccharide treatment in the B4galnt1+/- mouse model.

Given the recent in vitro discovery that the free soluble oligosaccharide of GM1 is the bioactive portion of GM1 for neurotrophic functions, we investigated its therapeutic potential in the B4galnt1+/- mice, a model of sporadic Parkinson's disease. We found that the GM1 oligosaccharide, systemically administered, reaches the brain and completely rescues the physical symptoms, reduces the abnormal nigral α-synuclein content, restores nigral tyrosine hydroxylase expression and striatal neurotransmitter levels, overlapping the wild-type condition. Thus, this study supports the idea that the Parkinson's phenotype expressed by the B4galnt1+/- mice is due to a reduced level of neuronal ganglioside content and lack of interactions between the oligosaccharide portion of GM1 with specific membrane proteins. It also points to the therapeutic potential of the GM1 oligosaccharide for treatment of sporadic Parkinson's disease.

Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium.

An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.

Gastrointestinal digestates of Grana Padano and Trentingrana cheeses promote intestinal calcium uptake and extracellular bone matrix formation in vitro.

In the present work, Grana Padano (GP) and Trentingrana (TN) cheeses at different ripening time were in vitro digested. To study calcium uptake and utilization, the intact digestates (selected doses that do not alter cell viability and Transepithelial Electrical Resistance) were administered to Caco2/HT-29 70/30 cells, cultured on a semipermeable membrane in transwells, as a model of human intestinal epithelium. Intact digestates as well as the whole basolateral solutions (mimicking the passage of digestates through intestinal cells before reaching the blood flow and bone) in parallel were further administered to human osteoblast-like cells SaOS-2 to study the extracellular bone matrix formation. In vitro digestates deriving from GP and TN promoted calcium uptake and extracellular bone matrix formation independently of both the cheese type and its ripening period (13, 19 or 26months). The present study reports the ability of whole digestates of GP and TN cheeses to improve intestinal calcium absorption and bone matrix formation in vitro. Once fully explored at bone level, this finding could better support the role of cheese in ameliorating calcium deficiencies and associated diseases in vivo.