Laboratory of Molecular Neuroembryology

Santa Lucia di Serino, Italy

Laboratory of Molecular Neuroembryology

Santa Lucia di Serino, Italy
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Pagliarini V.,National Institute For Infectious Diseases Irccs L Spallanzani | Wirawan E.,Molecular Signalling and Cell Death Unit | Wirawan E.,Ghent University | Romagnoli A.,National Institute For Infectious Diseases Irccs L Spallanzani | And 12 more authors.
Cell Death and Differentiation | Year: 2012

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program. © 2012 Macmillan Publishers Limited All rights reserved.

Nazio F.,University of Rome Tor Vergata | Nazio F.,Laboratory of Molecular Neuroembryology | Strappazzon F.,University of Rome Tor Vergata | Strappazzon F.,Laboratory of Molecular Neuroembryology | And 13 more authors.
Nature Cell Biology | Year: 2013

Autophagy is important in the basal or stress-induced clearance of bulk cytosol, damaged organelles, pathogens and selected proteins by specific vesicles, the autophagosomes. Following mTOR (mammalian target of rapamycin) inhibition, autophagosome formation is primed by the ULK1 and the beclin-1-Vps34-AMBRA1 complexes, which are linked together by a scaffold platform, the exocyst. Although several regulative steps have been described along this pathway, few targets of mTOR are known, and the cross-talk between ULK1 and beclin 1 complexes is still not fully understood. We show that under non-autophagic conditions, mTOR inhibits AMBRA1 by phosphorylation, whereas on autophagy induction, AMBRA1 is dephosphorylated. In this condition, AMBRA1, interacting with the E3-ligase TRAF6, supports ULK1 ubiquitylation by LYS-63-linked chains, and its subsequent stabilization, self-association and function. As ULK1 has been shown to activate AMBRA1 by phosphorylation, the proposed pathway may act as a positive regulation loop, which may be targeted in human disorders linked to impaired autophagy. © 2013 Macmillan Publishers Limited. All rights reserved.

Cianfanelli V.,Danish Cancer Society | D'Orazio M.,University of Rome Tor Vergata | Cecconi1 F.,Danish Cancer Society | Cecconi1 F.,University of Rome Tor Vergata | Cecconi1 F.,Laboratory of Molecular Neuroembryology
Cell Cycle | Year: 2015

Autophagy-promoting proteins and stimuli are often associated with inhibition of cell proliferation; in this context, we recently described a key role for the pro-autophagic protein AMBRA 1. Indeed, AMBRA1, through its direct interaction with the protein phosphatase PP2A, tightly regulates the stability of the oncoprotein and promitotic factor c-Myc. Moreover, the AMBRA1-mediated regulation of c-Myc affects both cell proliferation rate and tumorigenesis. Interestingly, AMBRA1/ PP2A activity is under the control of the master regulator of autophagy and cell growth, the protein kinase mTOR. Besides the mechanistic details of this regulation pathway which we dissected previously, any possible interplay(s) between AMBRA1 and its interactor BECLIN 1 was not investigated in this scenario. Here we show that both AMBRA1 and BECLIN 1 affect c-Myc regulation, but through two different pathways. Nevertheless, these two proautophagic proteins are, together with PP2A, in the same macromolecular complex, whose functional significance of which will be addressed in future studies © 2015 Taylor & Francis Group, LLC

Strappazzon F.,Laboratory of Molecular Neuroembryology | Vietri-Rudan M.,Laboratory of Molecular Neuroembryology | Vietri-Rudan M.,University of Rome Tor Vergata | Campello S.,University of Geneva | And 9 more authors.
EMBO Journal | Year: 2011

BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis. © 2011 European Molecular Biology Organization. All Rights Reserved.

De Zio D.,University of Rome Tor Vergata | De Zio D.,Laboratory of Molecular Neuroembryology | Bordi M.,University of Rome Tor Vergata | Bordi M.,Laboratory of Molecular Neuroembryology | And 2 more authors.
International Journal of Cell Biology | Year: 2012

Oxidative DNA damage is produced by reactive oxygen species (ROS) which are generated by exogenous and endogenous sources and continuously challenge the cell. One of the most severe DNA lesions is the double-strand break (DSB), which is mainly repaired by nonhomologous end joining (NHEJ) pathway in mammals. NHEJ directly joins the broken ends, without using the homologous template. Ku70/86 heterodimer, also known as Ku, is the first component of NHEJ as it directly binds DNA and recruits other NHEJ factors to promote the repair of the broken ends. Neurons are particularly metabolically active, displaying high rates of transcription and translation, which are associated with high metabolic and mitochondrial activity as well as oxygen consumption. In such a way, excessive oxygen radicals can be generated and constantly attack DNA, thereby producing several lesions. This condition, together with defective DNA repair systems, can lead to a high accumulation of DNA damage resulting in neurodegenerative processes and defects in neurodevelopment. In light of recent findings, in this paper, we will discuss the possible implication of Ku in neurodevelopment and in mediating the DNA repair dysfunction observed in certain neurodegenerations. Copyright © 2012 Daniela De Zio et al.

Di Bartolomeo S.,University of Rome Tor Vergata | Di Bartolomeo S.,Laboratory of Molecular Neuroembryology | Nazio F.,University of Rome Tor Vergata | Nazio F.,Laboratory of Molecular Neuroembryology | And 2 more authors.
Traffic | Year: 2010

Autophagy is a lysosome-mediated degradation pathway used by eukaryotes to recycle cytosolic components in both basal and stress conditions. Several genes have been described as regulators of autophagy, many of them being evolutionarily conserved from yeast to mammals. The study of autophagy-defective model systems has made it possible to highlight the importance of correctly functioning autophagic machinery in the development of invertebrates as, for example, during the complex events of fly and worm metamorphosis. In vertebrates, on the other hand, autophagy defects can be lethal for the animal if the mutated gene is involved in the early stages of development, or can lead to severe phenotypes if the mutation affects later stages. However, in both lower and higher eukaryotes, autophagy seems to be crucial during embryogenesis by acting in tissue remodeling in parallel with apoptosis. An increase of autophagic cells is, in fact, observed in the embryonic stages characterized by massive cell elimination. Moreover, autophagic processes probably protect cells during metabolic stress and nutrient paucity that occur during tissue remodeling. In light of such evidence, it can be concluded that there is a close interplay between autophagy and the processes of cell death, proliferation and differentiation that determine the development of higher eukaryotes. © 2010 John Wiley & Sons A/S.

D'Amelio M.,University of Rome Tor Vergata | D'Amelio M.,Laboratory of Molecular Neuroembryology | Cavallucci V.,University of Rome Tor Vergata | Cavallucci V.,Laboratory of Molecular Neuroembryology | And 2 more authors.
Cell Death and Differentiation | Year: 2010

Caspases are a family of cysteinyl aspartate-specific proteases that are highly conserved in multicellular organisms and function as central regulators of apoptosis. A member of this family, caspase-3, has been identified as a key mediator of apoptosis in neuronal cells. Recent studies in snail, fly and rat suggest that caspase-3 also functions as a regulatory molecule in neurogenesis and synaptic activity. In this study, in addition to providing an overview of the mechanism of caspase-3 activation, we review genetic and pharmacological studies of apoptotic and nonapoptotic functions of caspase-3 and discuss the regulatory mechanism of caspase-3 for executing nonapoptotic functions in the central nervous system. Knowledge of biochemical pathway(s) for nonapoptotic activation and modulation of caspase-3 has potential implications for the understanding of synaptic failure in the pathophysiology of neurological disorders. Fine-tuning of caspase-3 lays down a new challenge in identifying pharmacological avenues for treatment of many neurological disorders. © 2010 Macmillan Publishers Limited All rights reserved.

De Zio D.,University of Rome Tor Vergata | Cianfanelli V.,University of Rome Tor Vergata | Cecconi F.,University of Rome Tor Vergata | Cecconi F.,Laboratory of Molecular Neuroembryology
Antioxidants and Redox Signaling | Year: 2013

Significance: When lesions are unrepaired or there are defects in the DNA repair system, DNA damage is often correlated to apoptosis. However, different kinds of lesions and different degrees of lesion severity can trigger numerous signaling responses. Recent Advances: DNA repair proteins involved in specific DNA repair pathways can modulate the function or activity of some apoptotic factors, further emphasizing the crosstalk between DNA damage and cell death. Critical Issues: Here, we discuss the signaling networks that link DNA damage to apoptosis, and we focus on post-translational modifications, leading to crucial changes in protein behavior, following various kinds of DNA damage. Moreover, we analyze the existence of apoptosis-related functions of typical repair proteins, leading to diverse, often-overlapping, DNA damage responses. Future Directions: The better understanding of the regulation and the functionality of key DNA repair proteins, also involved in apoptosis regulation, has the potential of modulating the cell outcomes on DNA damage, particularly in the context of cancer treatment. Antioxid. Redox Signal. 19, 559-571. © 2013, Mary Ann Liebert, Inc.

Tatti M.,Instituto Superiore Of Sanita | Motta M.,Instituto Superiore Of Sanita | Motta M.,University of Rome La Sapienza | Di bartolomeo S.,University of Rome Tor Vergata | And 5 more authors.
Human Molecular Genetics | Year: 2012

Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function. © The Author 2012. Published by Oxford University Press. All rights reserved.

Fidaleo M.,Laboratory of Molecular Neuroembryology | Zuorro A.,University of Rome La Sapienza | Lavecchia R.,University of Rome La Sapienza
World Journal of Microbiology and Biotechnology | Year: 2013

Triclosan (TCS), an antimicrobial agent widely used in consumer and medical products, was complexed with 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD). Phase-solubility studies indicated that inclusion complexes of 1:1 stoichiometry were formed and allowed estimation of the associated equilibrium constants and free-energy changes. At the highest cyclodextrin concentrations investigated, an almost 20-fold increase in the apparent water solubility of TCS was determined. Susceptibility tests against Escherichia coli and Staphylococcus aureus showed that the TCS-HPβCD and TCS-MβCD complexes exhibited antibacterial properties higher than those of uncomplexed TCS. The two complexes were also found capable of interfering with cell-to-cell communication mechanisms in the C. violaceum model system relying on N-acylhomoserine lactone autoinducers. The inhibitory activity of TCS increased significantly upon inclusion of the drug in HPβCD or MβCD, with small differences between the two CDs. The results obtained suggest that the investigated complexes could be used for treating infections caused by TCS-susceptible pathogens or for preventing biofilm formation on indwelling medical devices such as catheters, stents and orthopedic implants. © 2013 Springer Science+Business Media Dordrecht.

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