Lucrezia Gambardella

Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band‐3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (∼40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence‐associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite‐exposed RBC (∼7%). Active protease cathepsin E and μ‐calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, μ‐calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte “apoptosis.” Altogether, these results indicate that peroxynitrite, a milestone of redox‐mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

Mitochondrial Membrane Hyperpolarization Hijacks Activated T Lymphocytes Toward the Apoptotic-Prone Phenotype: Homeostatic Mechanisms of HIV Protease Inhibitors

A decrease of mitochondrial membrane potential has been hypothesized to be a marker of apoptotic cells, including activated T lymphocytes. It was recently demonstrated that HIV protease inhibitors, independently from any viral infection, can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis. To analyze the mechanisms underlying these effects, a specific study was undertaken in both resting and activated human PBL exposed to either receptor (e.g., anti-Fas)- or nonreceptor (e.g., radiation)-mediated apoptotic stimuli. T cell activation was found to be accompanied by a significant increase in mitochondrial membrane potential, or hyperpolarization, which was undetectable in resting cells. We also detected apoptotic hindering by HIV protease inhibitors only in activated T lymphocytes. This was apparently due to the ability of these drugs to block activation-associated mitochondria hyperpolarization, which, in turn, was paralleled by an impairment of cell cycle progression. Remarkably, protease inhibitors also prevented zidovudine-mediated mitochondrial toxicity. Finally, HIV-infected cells from naive patients behaved identically to activated T cells, displaying hyperpolarized mitochondria, while lymphocytes from patients under highly active antiretroviral therapy (which included HIV protease inhibitors) seemed to react as resting cells. Altogether these results clearly indicate that the hyperpolarization state of mitochondria may represent a prerequisite for the sensitization of lymphocytes to the so-called activation-induced cell death. They also suggest that HIV protease inhibitors, by interfering with induction of the mitochondrial hyperpolarization state, can result in cell survival even independent of any viral infection.

Clostridium difficile Toxin B Causes Apoptosis in Epithelial Cells by Thrilling Mitochondria INVOLVEMENT OF ATP-SENSITIVE MITOCHONDRIAL POTASSIUM CHANNELS

Targeting to mitochondria is emerging as a common strategy that bacteria utilize to interact with these central executioners of apoptosis. Several lines of evidence have in fact indicated mitochondria as specific targets for bacterial protein toxins, regarded as the principal virulence factors of pathogenic bacteria. This work shows, for the first time, the ability of the Clostridium difficile toxin B (TcdB), a glucosyltransferase that inhibits the Rho GTPases, to impact mitochondria. In living cells, TcdB provokes an early hyperpolarization of mitochondria that follows a calcium-associated signaling pathway and precedes the final execution step of apoptosis (i.e. mitochondria depolarization). Importantly, in isolated mitochondria, the toxin can induce a calcium-dependent mitochondrial swelling, accompanied by the release of the proapoptogenic factor cytochrome c. This is consistent with a mitochondrial targeting that does not require the Rho-inhibiting activity of the toxin. Of interest, the mitochondrial ATP-sensitive potassium channels are also involved in the apoptotic response to TcdB and appear to be crucial for the cell death execution phase, as demonstrated by using specific modulators of these channels. To our knowledge, the involvement of these mitochondrial channels in the ability of a bacterial toxin to control cell fate is a hitherto unreported finding.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas‐mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas‐triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane‐associated domains in uropods and co‐localized with ezrin. Co‐immunoprecipitation analyses confirmed this result, indicating a GD3–ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin‐mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas‐mediated apoptosis.

Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study

Markers of oxidative stress in peripheral blood from patients with Alzheimer disease (AD) were analyzed. Thirty‐three AD patients were recruited. Plasma antioxidant power (AOP), plasma Cystatin C as well as Cathepsin D in PBL were evaluated. We found that the AOP levels were significantly decreased in AD patients if compared to healthy donors, while the plasma level of Cystatin C was significantly higher. Importantly, a significantly decreased expression of Cathepsin D in PBL was also observed. These results suggest that oxidative imbalance in the peripheral blood of AD patients could mirror oxidative changes previously described in the central nervous system.

Cell sex determines anoikis resistance in vascular smooth muscle cells

Sexual dimorphism, detectable in vascular smooth muscle cells freshly isolated from aorta of male and female rats, is associated with a different susceptibility to radiation‐induced apoptosis. In this work we investigated the mechanism underlying this difference and discovered that, in comparison with cells from male rats, cells from female rats show adhesion‐associated resistance to apoptosis, the so called anoikis resistance. This is apparently due to a more adhering phenotype, characterized by a well organized actin microfilament cytoskeleton and to an increased phosphorylated focal adhesion kinase, and, more importantly, to a higher propensity to undergo survival by autophagy.

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.

Analyses of T cell phenotype and function reveal an altered T cell homeostasis in systemic sclerosis Correlations with disease severity and phenotypes

We investigated in systemic sclerosis (SSc) patients the T cell homeostasis and its relationship with the clinical course of the disease. Distribution of peripheral T cell subsets, thymic output, lymphocyte proliferation and apoptosis were analyzed by flow cytometry or ELISA. Age inappropriate levels of naive CD4(+) T cells and thymic output were observed. Proliferation of CD4(+) T cells, lymphocyte apoptosis and CD4(+) regulatory T (Treg) cell frequency were significantly higher than those observed in controls and significantly correlated with clinical phenotypes and clinical progression parameters i.e., diffusing capacity of the lung for carbon monoxide (DLCO) and disease activity. These data indicate that the evaluation of the T cell homeostasis can represent a valuable prognostic tool for SSc patients and it is useful to distinguish between limited and diffuse phenotypes. A therapeutic intervention targeted at reversing T cell homeostasis abnormalities would therefore potentially be helpful in counteracting disease progression.

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.

Differential effects of the glycolysis inhibitor 2‐deoxy‐D‐glucose on the activity of pro‐apoptotic agents in metastatic melanoma cells, and induction of a cytoprotective autophagic response

2‐Deoxy‐D‐glucose (2DG) is a synthetic glucose analogue that inhibits glycolysis and blocks cancer cell growth. In this report, we evaluated the role of 2DG in the induction of cell death in human metastatic melanoma cells. We have also examined the effects of 2DG in combined treatments with four different pro‐apoptotic agents: (i) Temozolomide (TMZ), a chemotherapic drug commonly used to treat metastatic melanoma, (ii) Pyrimethamine (Pyr), a pro‐apoptotic antifolate drug recently reappraised in cancer therapy, (iii) Cisplatin (CisPt), a drug capable of directly binding to DNA ultimately triggering apoptosis of cancer cells and (iv) the kinase inhibitor Staurosporine (STS), a prototypical inducer of mitochondria‐mediated apoptosis. We found that 2DG per se: (i) induced a cell cycle arrest in G0/G1, (ii) promoted autophagy, (iii) was ineffective in inducing apoptosis in association with the chemotherapic drug TMZ, whereas (iv) it was synergistic with CisPt and STS pro‐apoptotic drugs through a mechanism involving changes of mitochondrial homeostasis. Conversely, (v) 2DG hindered the pro‐apoptotic effects of Pyr via a mechanism involving either the block of cell cycle in G0/G1 or the modification of the free radical production of the cell, i.e., decreasing the production of reactive oxygen species (ROS) and increasing the production of reactive nitrogen species (RNS). Moreover, a clear‐cut autophagic response involving endoplasmic reticulum remodelling was detectable. Since autophagic cytoprotection has been suggested to contribute to the induction of chemoresistance, these results could provide useful clues as concerns the use of 2DG as anticancer agent in combinatory protocols.