Autophagic flux and autophagosome morphogenesis require the participation of sphingolipids.

Apoptosis and autophagy are two evolutionary conserved processes that exert a critical role in the maintenance of tissue homeostasis. While apoptosis is a tightly regulated cell program implicated in the removal of damaged or unwanted cells, autophagy is a cellular catabolic pathway that is involved in the lysosomal degradation and recycling of proteins and organelles, and is thereby considered an important cytoprotection mechanism. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including lipid rafts and caveolae, and contribute to a number of cellular functions such as cell proliferation, apoptosis and, as suggested more recently, autophagy. For instance, SLs are hypothesized to be involved in several intracellular processes, including organelle membrane scrambling, whilst at the plasma membrane lipid rafts, acting as catalytic domains, strongly contribute to the ignition of critical signaling pathways determining cell fate. In particular, by targeting several shared regulators, ceramide, sphingosine-1-phosphate, dihydroceramide, sphingomyelin and gangliosides seem able to differentially regulate the autophagic pathway and/or contribute to the autophagosome formation. This review illustrates recent studies on this matter, particularly lipid rafts, briefly underscoring the possible implication of SLs and their alterations in the autophagy disturbances and in the pathogenesis of some human diseases.

Raft-like microdomains play a key role in mitochondrial impairment in lymphoid cells from patients with Huntington's disease.

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by an exceedingly high number of contiguous glutamine residues in the translated protein, huntingtin (Htt). The primary site of cell toxicity is the nucleus, but mitochondria have been identified as key components of cell damage. The present work has been carried out in immortalized lymphocytes from patients with HD. These cells, in comparison with lymphoid cells from healthy subjects, displayed: i) a redistribution of mitochondria, forming large aggregates; ii) a constitutive hyperpolarization of mitochondrial membrane; and iii) a constitutive alteration of mitochondrial fission machinery, with high apoptotic susceptibility. Moreover, mitochondrial fission molecules, e.g., protein dynamin-related protein 1, as well as Htt, associated with mitochondrial raft-like microdomains, glycosphingolipid-enriched structures detectable in mitochondria. These findings, together with the observation that a ceramide synthase inhibitor and a raft disruptor are capable of impairing the peculiar mitochondrial remodeling in HD cells, suggest that mitochondrial alterations occurring in these cells could be due to raft-mediated defects of mitochondrial fission/fusion machinery.

Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production.

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies.

Ammonium Glycyrrhizinate Prevents Apoptosis and Mitochondrial Dysfunction Induced by High Glucose in SH-SY5Y Cell Line and Counteracts Neuropathic Pain in Streptozotocin-Induced Diabetic Mice.

Glycyrrhiza glabra, commonly known as liquorice, contains several bioactive compounds such as flavonoids, sterols, triterpene, and saponins; among which, glycyrrhizic acid, an oleanane-type saponin, is the most abundant component in liquorice root. Diabetic peripheral neuropathy is one of the major complications of diabetes mellitus, leading to painful condition as neuropathic pain. The pathogenetic mechanism of diabetic peripheral neuropathy is very complex, and its understanding could lead to a more suitable therapeutic strategy. In this work, we analyzed the effects of ammonium glycyrrhizinate, a derivate salt of glycyrrhizic acid, on an in vitro system, neuroblastoma cells line SH-SY5Y, and we observed that ammonium glycyrrhizinate was able to prevent cytotoxic effect and mitochondrial fragmentation after high-glucose administration. In an in vivo experiment, we found that a short-repeated treatment with ammonium glycyrrhizinate was able to attenuate neuropathic hyperalgesia in streptozotocin-induced diabetic mice. In conclusion, our results showed that ammonium glycyrrhizinate could ameliorate diabetic peripheral neuropathy, counteracting both in vitro and in vivo effects induced by high glucose, and might represent a complementary medicine for the clinical management of diabetic peripheral neuropathy.

Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study.

BACKGROUND: Cathepsins represent a group of proteases involved in determining the metastatic potential of cancer cells. Among these are cysteinyl- (e.g. cathepsin B and cathepsin L) and aspartyl-proteases (e.g. cathepsin D), normally present inside the lysosomes as inactive proenzymes. Once released in the extracellular space, cathepsins contribute to metastatic potential by facilitating cell migration and invasiveness. RESULTS: In the present work we first evaluated, by in vitro procedures, the role of cathepsins B, L and D, in the remodeling, spreading and invasiveness of eight different cell lines: four primary and four metastatic melanoma cell lines. Among these, we considered two cell lines derived from a primary cutaneous melanoma and from a supraclavicular lymph node metastasis of the same patient. To this purpose, the effects of specific chemical inhibitors of these proteases, i.e. CA-074 and CA-074Me for cathepsin B, Cathepsin inhibitor II for cathepsin L, and Pepstatin A for cathepsin D, were evaluated. In addition, we also analyzed the effects of the biological inhibitors of these cathepsins, i.e. specific antibodies, on cell invasiveness. We found that i) cathepsin B, but not cathepsins L and D, was highly expressed at the surface of metastatic but not of primary melanoma cell lines and that ii) CA-074, or specific antibodies to cathepsin B, hindered metastatic cell spreading and dissemination, whereas neither chemical nor biological inhibitors of cathepsins D and L had significant effects. Accordingly, in vivo studies, i.e. in murine xenografts, demonstrated that CA-074 significantly reduced human melanoma growth and the number of artificial lung metastases. CONCLUSIONS: These results suggest a reappraisal of the use of cathepsin B inhibitors (either chemical or biological) as innovative strategy in the management of metastatic melanoma disease.

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.

Potent, Efficacious, and Stable Cyclic Opioid Peptides with Long Lasting Antinociceptive Effect after Peripheral Administration.

Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for µ-, δ-, and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands, MACE3 demonstrated a better activity than biphalin after intravenous administration, and its corresponding analogue incorporating the hydrazine linker (MACE2) was able to induce longer lasting analgesia following subcutaneous administration. An analogue of MACE2 containing 2,6-dimethyl-l-tyrosine (MACE4) showed the best potency and in vivo antinociceptive activity of this series.

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.

Recruitment of mitofusin 2 into "lipid rafts" drives mitochondria fusion induced by Mdivi-1.

The regulation of the mitochondrial dynamics and the balance between fusion and fission processes are crucial for the health and fate of the cell. Mitochondrial fusion and fission machinery is controlled by key proteins such as mitofusins, OPA-1 and several further molecules. In the present work we investigated the implication of lipid rafts in mitochondrial fusion induced by Mdivi-1. Our results underscore the possible implication of lipid "rafts" in mitochondrial morphogenetic changes and their homeostasis.

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.

Estrogen receptor ß ligation inhibits Hodgkin lymphoma growth by inducing autophagy.

Although Hodgkin lymphoma (HL) is curable with current therapy, at least 20% of patients relapse or fail to make complete remission. In addition, patients who achieve long-term disease-free survival frequently undergo infertility, secondary malignancies, and cardiac failure, which are related to chemotherapeutic agents and radiation therapies. Hence, new therapeutic strategies able to counteract the HL disease in this important patient population are still a matter of study. Estrogens, in particular 17ß-estradiol (E2), have been suggested to play a role in lymphoma cell homeostasis by estrogen receptors (ER) ß activation. On these bases, we investigated whether the ligation of ERß by a selective agonist, the 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), could impact HL tumor growth. We found that DPN-mediated ERß activation led to a reduction of in vitro cell proliferation and cell cycle progression by inducing autophagy. In nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice engrafted with HL cells, ERß activation by DPN was able to reduce lymphoma growth up to 60% and this associated with the induction of tumor cell autophagy. Molecular characterization of ERß-induced autophagy revealed an overexpression of damage-regulated autophagy modulator 2 (DRAM2) molecule, whose role in autophagy modulation is still debated. After ERß activation, both DRAM2 and protein 1 light chain 3 (LC3), a key actor in the autophagosome formation, strictly interacted each other and localized at mitochondrial level.Altogether these results suggest that targeting ERß with selective agonists might affect HL cell proliferation and tumor growth via a mechanism that brings into play DRAM2-dependent autophagic cascade.

Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells.

We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts.

Low expression of estrogen receptor ß in T lymphocytes and high serum levels of anti-estrogen receptor α antibodies impact disease activity in female patients with systemic lupus erythematosus.

BACKGROUND: Current evidence indicates that estrogens, in particular 17ß-estradiol (E2), play a crucial role in the gender bias of autoimmune diseases although the underlying molecular mechanisms have not yet been fully elucidated. Immune cells have estrogen receptors (ERs), i.e., ERα and ERß, that play pro- and anti-inflammatory functions, respectively, and the presence of one estrogen receptor (ER) subtype over the other might change estrogen effects, promoting or dampening inflammation. In this study, we contributed to define the influences of E2 on T cells from female patients with systemic lupus erythematosus (SLE), a representative autoimmune disease characterized by a higher prevalence in women than in men (female/male ratio 9:1). Particularly, our aim was to evaluate whether alterations of ERα and ERß expression in T cells from female SLE patients may impact lymphocyte sensitivity to E2 and anti-ERα antibody (anti-ERα Ab) stimulation interfering with cell signaling and display a direct clinical effect. METHODS: Sixty-one premenopausal female patients with SLE and 40 age-matched healthy donors were recruited. Patients were divided into two groups based on the SLE Disease Activity Index 2000 (SLEDAI-2K) (i.e., <6 and ≥6). ER expression was evaluated in T lymphocytes by flow cytometry, immunofluorescence, and Western blot analyses. Serum anti-ERα Ab levels were analyzed by enzyme-linked immunosorbent assay (ELISA). ER-dependent signaling pathways were measured by a phosphoprotein detection kit. RESULTS: Intracellular ERß expression was significantly lower in T cells from patients with SLEDAI-2K ≥6 as compared with healthy donors and patients with SLEDAI-2K <6 and negatively correlated with disease activity. The expression of intracellular and membrane-associated-ERα was similar in SLE and control T cells. ER-dependent signaling pathways were activated in T cells from SLE patients with SLEDAI-2K ≥6, but not with SLEDAI-2K <6, when both membrane and intracellular ERs were stimulated by co-treatment with E2 and anti-ERα Abs. CONCLUSIONS: Our results demonstrate an altered ER profile in SLE patients, possibly contributing to SLE pathogenesis and interfering with clinical activity, and highlight the potential exploitation of T cell-associated ERß as a biomarker of disease activity.

Schwann cell autophagy counteracts the onset and chronification of neuropathic pain.

Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

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.

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.