Overexpressed GM1 Suppresses Nerve Growth Factor (NGF) Signals by Modulating the Intracellular Localization of NGF Receptors and Membrane Fluidity in PC12 Cells

Abstract

Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/ GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1؉ -transfectant cells (GM1 ؉ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1 ؉ cells. Binding of 125 I-NGF to PC12 cells was almost equivalent between GM1؉ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1؉ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125 I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1 ؉ cells. p75 NTR and Ras also moved from the raft to non-raft fraction in GM1 ؉ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1 ؉ cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/ TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.

Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues

“…In the case of GEM/rafts, there are no definite morphological features, and ganglioside GM1 has been used as a specific raft marker (19). In our recent studies, GM1 seemed to be a functional molecule to regulate signals via PDGF or NGF (40). In the DKO mice analyzed here, all gangliosides other than GM3 were lost.…”

The Tyrosine Kinase Fyn Determines the Localization of TrkB Receptors in Lipid Rafts

“…A role for lipid rafts in Trk receptor signaling was previously addressed in PC12 cells (Huang et al, 1999;Peiro et al, 2000;Nishio et al, 2004). TrkA was shown to be constitutively present in caveolae, a specific type of lipid rafts (Simons and Toomre, 2000), whereas lipid raft-disrupting compounds markedly inhibited NGF-induced ERK phosphorylation (Peiro et al, 2000;Nishio et al, 2004).…”

“…Ganglioside G M1 is a normal component of lipid rafts, and exogenous application of this sphingolipid to the cells affects the stability and protein composition of lipid rafts (Simons et al, 1999). Both compounds have been used previously to disturb Trk localization at lipid rafts in PC12 cells and rat cortical neurons (Peiro et al, 2000;Nishio et al, 2004;Suzuki et al, 2004). MCD, at a concentration of 3 mM, and ganglioside G M1 , at 100 M, abolished the TrkB recruitment to lipid rafts in response to BDNF (Fig.…”

Ganglioside‐induced amyloid formation by human islet amyloid polypeptide in lipid rafts

“…The amount of cell surface gangliosides and accumulated hIAPP amyloids were estimated by the fluorescence intensity of bound CTX-B and Congo red, respectively [20,21]. The background fluorescence was subtracted from the CTX-B or Congo red fluorescence expressed in 256 tones pixel by pixel.…”