Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-ß-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation.

Trafficking of PrPc to mitochondrial raft-like microdomains during cell apoptosis.

The cellular form of prion protein (PrP (c)) is a highly conserved cell surface GPI-anchored glycoprotein that was identified in cholesterol-enriched, detergent-resistant microdomains, named "rafts." The association with these specialized portions of the cell plasma membrane is required for conversion of PrP (c) to the transmissible spongiform encephalopathy-associated protease-resistant isoform. Usually, PrP (c) is reported to be a plasma membrane protein, however several studies have revealed PrP (c) as an interacting protein mainly with the membrane/organelles, as well as with cytoskeleton network. Recent lines of evidence indicated its association with ER lipid raft-like microdomains for a correct folding of PrP (c), as well as for the export of the protein to the Golgi and proper glycosylation. During cell apoptosis, PrP (c) can undergo intracellular re-localization, via ER-mitochondria associated membranes (MAM) and microtubular network, to mitochondrial raft-like microdomains, where it induced the loss of mitochondrial membrane potential and citochrome c release, after a contained raise of calcium concentration. We suggest that PrP (c) may play a role in the multimolecular signaling complex associated with cell apoptosis Lipid rafts and their components may, thus, be investigated as pharmacological targets of interest, introducing a novel and innovative task in modern pharmacology, i.e., the development of glycosphingolipid targeted drugs.

Dynamics of mitochondrial raft-like microdomains in cell life and death.

On the basis of the biochemical nature of lipid rafts, composed by glycosphingolipids, cholesterol and signaling proteins, it has been suggested that they are part of the complex framework of subcellular intermixing activities that lead to CD95/Fas-triggered apoptosis. We demonstrated that, following CD95/Fas triggering, cellular prion protein (PrP(C)), which represents a paradigmatic component of lipid rafts, was redistributed to mitochondrial raft-like microdomains via endoplasmic reticulum (ER)-mitochondria associated membranes (MAM) and microtubular network.   Raft-like microdomains appear to be involved in a series of intracellular functions, such as: (1) the membrane "scrambling" that participates in cell death execution pathways, (2) the remodeling of organelles, (3) the recruitment of proteins to the mitochondria; (4) the mitochondrial oxidative phosphorylation and ATP production. IN CONCLUSION, WE SUGGEST THAT LIPID RAFT COMPONENTS CAN EXERT THEIR REGULATORY ACTIVITY IN APOPTOSIS EXECUTION AT THREE DIFFERENT LEVELS: (1) in the DISC formation at the plasma membrane; (2) in the intracellular redistribution at cytoplasmic organelles, and, (3) in the structural and functional mitochondrial modifications associated with apoptosis execution.

Recruitment of cellular prion protein to mitochondrial raft-like microdomains contributes to apoptosis execution.

We examined the possibility that cellular prion protein (PrP(C)) plays a role in the receptor-mediated apoptotic pathway. We first found that CD95/Fas triggering induced a redistribution of PrP(C) to the mitochondria of T lymphoblastoid CEM cells via a mechanism that brings into play microtubular network integrity and function. In particular, we demonstrated that PrP(C) was redistributed to raft-like microdomains at the mitochondrial membrane, as well as at endoplasmic reticulum-mitochondria-associated membranes. Our in vitro experiments also demonstrated that, although PrP(C) had such an effect on mitochondria, it induced the loss of mitochondrial membrane potential and cytochrome c release only after a contained rise of calcium concentration. Finally, the involvement of PrP(C) in apoptosis execution was also analyzed in PrP(C)-small interfering RNA-transfected cells, which were found to be significantly less susceptible to CD95/Fas-induced apoptosis. Taken together, these results suggest that PrP(C) might play a role in the complex multimolecular signaling associated with CD95/Fas receptor-mediated apoptosis.

Paracrine diffusion of PrP(C) and propagation of prion infectivity by plasma membrane-derived microvesicles.

Cellular prion protein (PrP(C)) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1-1 microm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrP(C) diffusion and prion infectivity transmission. We first identified PrP(C) in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrP(Sc) is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrP(Sc)-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrP(C) diffusion and signaling as well as to the process of prion spread and neuroinvasion.

Neurotrophic signalling pathway triggered by prosaposin in PC12 cells occurs through lipid rafts.

Prosaposin is a neurotrophic factor that has been demonstrated to mediate trophic signalling events in different cell types; it distributes to surface membranes of neural cells and also exists as a secreted protein in different body fluids. Prosaposin was demonstrated to form tightly bound complexes with a variety of gangliosides, and a functional role has been suggested for ganglioside-prosaposin complexes. In this work, we provide evidence that exogenous prosaposin triggers a signal cascade after binding to its target molecules on lipid rafts of pheochromocytoma PC12 cell plasma membranes, as revealed by scanning confocal microscopy and linear sucrose gradient analysis. In these cells, prosaposin is able to induce extracellular signal-regulated kinase phosphorylation, sphingosine kinase activation, and consequent cell death prevention, acting through lipid rafts. These findings point to the role of lipid rafts in the prosaposin-triggered signalling pathway, thus supporting a role for this factor as a new component of the multimolecular signalling complex involved in the neurotrophic response.