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\u2013GM1 oligosaccharide complex at the cell surface. To study the TrkA\u2013GM1 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\u2013GM1 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\u2013GM1 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 \u2018flopping\u2019 down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1. (Figure presented.)

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 \u3b1-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

The Neurotrophic properties of GM1 oligosaccharide: a new promising story

Experimental evidence, both in vitro and in vivo, highlight the neurotrophic and neurodifferentiative effects of ganglioside GM1. So far, the molecular mechanism underlining the GM1 neuro activities is still unknown. In order to clarify the molecular mechanism by which GM1 exerts its neurotrophic action we investigated the role of its oligosaccharide portion. GM1 oligosaccharide was added to the culture medium of neuronal cells. To study the neurodifferentiation, cells were followed over the time to assess both morphological parameters and biochemical markers. In murine neuroblastoma cell lines we found that the oligosaccharide chain of GM1 is directly involved in the processes of neuronal differentiation by inducing TrkA-MAPK pathway activation. In this cell line, the GM1 oligosaccharide chain and TrkA receptor showed a direct interaction at the plasma membrane level. Using mouse primary neurons, we highlighted that GM1 oligosaccharideisabletoinfluencethedifferentiationprocesses byacceleratingtheneuronaldifferentiationfrombothmorphological and biochemical view. We surmise that the neurotrophic effect of GM1 is due to a direct interaction between the oligosaccharide chain and TrkA receptor. Trying to define the molecular mechanism of GM1, we have potentially found a new neurotrophic player, which invitro is able to influence differentiation of mice primary neurons

Investigating the hydrogen-bond acceptor site of the nicotinic pharmacophore model : a computational and experimental study using epibatidine-related molecular probes

The binding mode of nicotinic agonists has been thoroughly investigated in the last decades. It is now accepted that the charged amino group is bound by a cation-\u3c0 interaction to a conserved tryptophan residue, and that the aromatic moiety is projected into a hydrophobic pocket deeply located inside the binding cleft. A hydrogen bond donor/acceptor, maybe a water molecule solvating this receptor subsite, contributes to further stabilize the nicotinic ligands. The position of this water molecule has been established by several X-ray structures of the acetylcholine-binding protein. In this study, we computationally analyzed the role of this water molecule as a putative hydrogen bond donor/acceptor moiety in the agonist binding site of the three most relevant heteromeric (\u3b14\u3b22, \u3b13\u3b24) and homomeric (\u3b17) neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Our theoretical investigation made use of epibatidine 1 and deschloroepibatidine 2 as molecular probes, and was then extended to their analogues 3 and 4, which were subsequently synthesized and tested at the three target receptor subtypes. Although the pharmacological data for the new ligands 3 and 4 indicated a reduction of the affinity at the studied nAChRs with respect to reference agonists, a variation of the selectivity profile was clearly evidenced

Design of novel \u3b17-subtype-preferring nicotinic acetylcholine receptor agonists: application of docking and MM-PBSA computational approaches, synthetic and pharmacological studies

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric \u3b17 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the \u3b17 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal \u3b17 as well as \u3b14\u3b22 nAChRs

Novel ligands for neuronal nicotinic receptor subtypes: synthetic, pharmacological and molecular docking studies

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric proteins made up of homomeric or heteromeric combinations of \u3b1 and \u3b1/\u3b2 subunits, whose differential association confers specific structural and functional properties to the resulting subtypes. The \u3b14\u3b22 and the \u3b17 nAChRs are by far the most expressed in the central nervous system (CNS), whereas the \u3b13\u3b24 subtype is predominant in the sensory and autonomic ganglia and in a subpopulation of neurons in the medial habenula (MHN) and interpeduncular nucleus (IPN) in the CNS [1]. Recently, behavioral and functional studies have further indicated the crucial role of nAChRs in nicotine reward, addiction and expression of withdrawal. The \u3b14 and \u3b22 subunits appear to be crucial for nicotine dependence and the \u3b13\u3b24 nAChR subtype seems to be implicated in addiction to nicotine and other drugs of abuse [2]. As an extension of our research in this field [3], we aimed at deepening the investigation on the involvement of nAChR subtypes in each of the aspects of tobacco addiction. To such an end, we designed and synthesized the set of novel compounds 4-9, which may be related to Nicotine 1, Epibatidine 2 and Anabaseine 3. In the new derivatives, the pyridine ring featuring the parent ligands was replaced by a 3-hydroxybenzene or a 3-hydroxymethylbenzene moiety. The synthetic approach to the target compounds, the data of their pharmacological screening as well as the results of molecular modeling investigations will be presented and discussed

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