Valeria Manganelli

Prion protein is a component of the multimolecular signaling complex involved in T cell activation.

In this study we analyzed the interaction of prion protein PrPC with components of glycosphingolipid‐enriched microdomains in lymphoblastoid T cells. PrPC was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrPC is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrPC and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP‐70 was immunoprecipitated by anti‐PrP after T cell activation. These results demonstrate that PrPC interacts with ZAP‐70, suggesting that PrPC is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

Cardiolipin‐enriched raft‐like microdomains are essential activating platforms for apoptotic signals on mitochondria

Cardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase‐8 on mitochondria. We hypothesize that these platforms may correspond to “raft‐like” microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in “raft‐like” microdomains could be to anchor caspase‐8 at contact sites between inner and outer membranes, facilitating its self‐activation, Bid cleavage and apoptosis execution. The role played by “raft‐like” microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

ABSTRACT Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.

Do mitochondria act as "cargo boats" in the journey of GD3 to the nucleus during apoptosis?

Plasma membrane lipid rafts have been considered as a sort of “chamber”, where several subcellular activities, including CD95/Fas‐mediated pro‐apoptotic signaling, can take place. Recently, we demonstrated that, after CD95/Fas triggering, raft‐like microdomains could be detected in mitochondrial membranes. The mitochondrion appears as a dynamic and subcompartmentalized organelle in which microdomains might act as controllers of apoptosis‐associated fission that results in the release of apoptogenic factors. Here, we hypothesize that some “small” mitochondria, possibly derived from their fission process, can reach the nuclear envelope and strictly interact with this. Mitochondria could act as a signaling “device” contributing to molecular trafficking of molecules, including raft‐like components, during apoptosis.

Evidence for the involvement of GD3 ganglioside in autophagosome formation and maturation

Sphingolipids are structural lipid components of cell membranes, including membrane of organelles, such as mitochondria or endoplasmic reticulum, playing a role in signal transduction as well as in the transport and intermixing of cell membranes. Sphingolipid microdomains, also called lipid rafts, participate in several metabolic and catabolic cell processes, including apoptosis. However, the defined role of lipid rafts in the autophagic flux is still unknown. In the present study we analyzed the role of gangliosides, a class of sphingolipids, in autolysosome morphogenesis in human and murine primary fibroblasts by means of biochemical and analytical cytology methods. Upon induction of autophagy, by using amino acid deprivation as well as tunicamycin, we found that GD3 ganglioside, considered as a paradigmatic raft constituent, actively contributed to the biogenesis and maturation of autophagic vacuoles. In particular, fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses revealed that this ganglioside interacts with phosphatidylinositol 3-phosphate and can be detected in immature autophagosomes in association with LC3-II as well as in autolysosomes associated with LAMP1. Hence, it appears as a structural component of autophagic flux. Accordingly, we found that autophagy was significantly impaired by knocking down ST8SIA1/GD3 synthase (ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 1) or by altering sphingolipid metabolism with fumonisin B1. Interestingly, exogenous administration of GD3 ganglioside was capable of reactivating the autophagic process inhibited by fumonisin B1. Altogether, these results suggest that gangliosides, via their molecular interaction with autophagy-associated molecules, could be recruited to autophagosome and contribute to morphogenic remodeling, e.g., to changes of membrane curvature and fluidity, finally leading to mature autolysosome formation.

Raft component GD3 associates with tubulin following CD95/Fas ligation

In a previous investigation, we demonstrated that after CD95/Fas triggering, raft‐associated GD3 ganglioside, normally localized at the plasma membrane of T cells, can be detected in mitochondria, where they contribute to apoptogenic events. Here, we show the association of the glycosphingolipid GD3 with microtubular cytoskeleton at very early time points following Fas ligation. This was assessed by different methodological approaches, including fluorescence resonance energy transfer, immunoelectron microscopy, and coimmunoprecipitation. Furthermore, docking analysis also showed that GD3 has a high affinity for the pore formed by 4 tubulin heterodimers (type I pore), thus suggesting a possible direct interaction between tubulin and GD3. Finally, time‐course analyses indicated that the relocalization of GD3 to the mitochondria was time related with the alterations of the mitochondrial membrane potential. Hence, microtubules could act as tracks for ganglioside redistribution following apoptotic stimulation, possibly contributing to the mitochondrial alterations leading to cell death.—Sorice, M., Matarrese, P., Tinari, A., Giammarioli, A. M., Garofalo, T., Manganelli, V., Ciarlo, L., Gambardella, L., Maccari, G., Botta, M., Misasi, R., Malorni, W. Raft component GD3 associates with tubulin following CD95/Fas ligation. FASEB J. 23, 3298–3308 (2009). www.fasebj.org

Increased HMGB1 expression and release by mononuclear cells following surgical/anesthesia trauma

IntroductionHigh mobility group box 1 (HMGB1) is a key mediator of inflammation that is actively secreted by macrophages and/or passively released from damaged cells. The proinflammatory role of HMGB1 has been demonstrated in both animal models and humans, since the severity of inflammatory response is strictly related to serum HMGB1 levels in patients suffering from traumatic insult, including operative trauma. This study was undertaken to investigate HMGB1 production kinetics in patients undergoing major elective surgery and to address how circulating mononuclear cells are implicated in this setting. Moreover, we explored the possible relationship between HMGB1 and the proinflammatory cytokine interleukin-6 (IL-6).MethodsForty-seven subjects, American Society of Anesthesiologists physical status I and II, scheduled for major abdominal procedures, were enrolled. After intravenous medication with midazolam (0.025 mg/Kg), all patients received a standard general anesthesia protocol, by thiopentone sodium (5 mg/Kg) and fentanyl (1.4 μg/Kg), plus injected Vecuronium (0.08 mg/Kg). Venous peripheral blood was drawn from patients at three different times, t0: before surgery, t1: immediately after surgical procedure; t2: at 24 hours following intervention. Monocytes were purified by incubation with anti-CD14-coated microbeads, followed by sorting with a magnetic device. Cellular localization of HMGB1 was investigated by flow cytometry assay; HMGB1 release in the serum by Western blot. Serum samples were tested for IL-6 levels by ELISA. A one-way repeated-measures analysis ANOVA was performed to assess differences in HMGB1 concentration over time, in monocytes and serum.ResultsWe show that: a) cellular expression of HMGB1 in monocytes at t1 was significantly higher as compared to t0; b) at t2, a significant increase of HMGB1 levels was found in the sera of patients. Such an increase was concomitant to a significant down-regulation of cellular HMGB1, suggesting that the release of HMGB1 might partially derive from mononuclear cells; c) treatment of monocytes with HMGB1 induced in vitro the release of IL-6; d) at t2, high amounts of circulating IL-6 were detected as compared to t0.ConclusionsThis study demonstrates for the first time that surgical/anesthesia trauma is able to induce an early intracellular upregulation of HMGB1 in monocytes of surgical patients, suggesting that HMGB1 derives, at least partially, from monocytes.

Analyzing Morphological and Ultrastructural Features in Cell Death

Diverse forms of cell death have initially been described thanks to their observation at the electron microscope. Morphological and ultrastructural features of necrosis, apoptosis, and autophagy, considered here as prototypic cell death processes, allow one to characterize and quantify early and late cytopathological changes occurring in cells undergoing degeneration. Both light microscopy and scanning electron microscopy can provide useful insights, for example, to quantitatively evaluate cell death or to characterize cell surface changes of the cells, respectively. However, transmission electron microscopy preparation allows distinguishing among different forms of cell death. This chapter describes in brief the methods used to characterize cell death forms, including membrane, nucleus, and organelle changes, and shows paradigmatic micrographs. In particular, morphogenetic changes occurring in mitochondria during apoptosis, that is, fission process or, conversely, vacuole formation during autophagy, are shown. Possible artifacts are also described. Ultrastructural analysis seems still to provide essential information for studies on cell death.

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

PrPC is identified as a new component of mitochondrial raft-like microdomains in T cells undergoing CD95/Fas–mediated apoptosis, and microtubular network integrity and function could play a role in the redistribution of PrPC from the plasma membrane to the mitochondria.

Ganglioside GD3 as a raft component in cell death regulation.

Subcellular organelles such as mitochondria, endoplasmic reticulum and the Golgi complex are involved in the progression of cell death program. Recent evidence unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases. On the basis of the biochemical nature of lipid rafts, composed by sphingolipids, including gangliosides and sphingomyelin, cholesterol and signaling proteins, it has been suggested that they are part of this traffic and can participate in cell remodelling leading to cell death program execution. Although detected in various cell types, the role of lipid rafts in apoptosis has been mostly studied in T cells, where the physiological apoptotic program occurs through CD95/Fas. In this review, the possible contribution of lipid rafts to the cascade of events leading to T cell apoptosis after CD95/Fas ligation is summarized. We focused on the paradigmatic component of rafts GD3, which can proceed from the cell plasma membrane (and/or from trans Golgi network) to the mitochondria via a microtubule-dependent mechanism. This transport may be regulated by CLIPR-59, a new CLIP-170-related protein, involved in the regulation of microtubule dynamics. Particular attention has been given to mitochondrial raft-like microdomains, which may represent preferential sites where key reactions take place. Indeed, GD3, by interacting with mitochondrial raft-like microdomains, may trigger specific events involved in the apoptogenic program, including mitochondria hyperpolarization and depolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These findings introduce an additional task for identifying new molecular target(s) of anti-cancer agents.