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Pierluigi Nicotera

Publications and source records attributed to Pierluigi Nicotera.

16 recordsLinked to original sources
PubMed2006

RX871024 reduces NO production but does not protect against pancreatic beta-cell death induced by proinflammatory cytokines.

The imidazoline compound RX871024 reduces IL-1beta-induced NO production thereby protecting against IL-1beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1beta, IFNgamma, and TNFalpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and beta-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and beta-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-1beta, IFNgamma, and TNFalpha, conditions modelling those that take place in type 1 diabetes, induces pancreatic beta-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.

Animals↗
PubMed2005

Ca2+ signals and death programmes in neurons.

Cell death programmes are generally defined by biochemical/genetic routines that are linked to their execution and by the appearance of more or less typical morphological features. However, in pathological settings death signals may engage complex and interacting lethal pathways, some of which are common to different cells, whereas others are linked to a specific tissue and differentiation pattern. In neurons, death programmes can be spatially and temporally segregated. Most importantly physiological Ca2+ signals are essential for cell function and survival. On the other hand, Ca2+ overload or perturbations of intracellular Ca2+ compartmentalization can activate or enhance mechanisms leading to cell death. An imbalance between Ca2+ influx and efflux from cells is the initial signal leading to Ca2+ overload and death of ischaemic neurons or cardiomyocytes. Alterations of intracellular Ca2+ storage can integrate with death signals that do not initially require Ca2+, to promote processing of cellular components and death by apoptosis or necrosis. Finally, Ca2+ can directly activate catabolic enzymes such as proteases, phospholipases and nucleases that directly cause cell demise and tissue damage.

Apoptosis↗
PubMed2005

Neurodevelopment on route p63.

All known members of the p53 gene family, including the two homologs p73 and p63, have multiple biological functions. In neurons, p53 and p73 are known to regulate cell death in the developing and adult nervous system. A report by Jacobs et al. in this issue of Neuron shows that the more ancestral member of this gene family, p63, is an essential proapoptotic protein during neuronal development.

Animals↗
PubMed2005

Heme deficiency is associated with senescence and causes suppression of N-methyl-D-aspartate receptor subunits expression in primary cortical neurons.

Heme is a crucial component of many pharmacological and toxicological processes, and studies have suggested that heme deficiency may play a role in cellular ageing. A model of ageing neurons was established using prolonged cultures of BALB/c mouse primary cortical neurons. Aged neurons displayed a senescent phenotype and a marked up-regulation of cathepsin-L expression. Down-regulation of the candidate neuron-specific genes for N-methyl-D-aspartate (NMDA) receptor subunits (NMDAzeta1 and -epsilon2) and neurofilament light peptide (NF-L) were found to be characteristic of the aging process as reported in vivo (Brain Res 907:71-83, 2001; Brain Res Mol Brain Res 99:40-45, 2002). In contrast, the genes for the controlling enzymes of heme synthesis and degradation (5-aminolevulinate synthase 1 and heme oxygenase 1, respectively) were up-regulated, implying depletion of a regulatory heme pool. Inhibition of heme synthesis (by 70-80%) at different enzymic steps by succinyl acetone and N-methylprotoporphyrin IX resulted in the earlier lowered expression of NMDAzeta1 and -epsilon2 and NF-L. Exogenous hemin added to heme-depleted cells rescued the expression of these neuron-specific genes. Culture of cortical neurons from BALB/c Fech(m1Pas) mutant mice demonstrating depressed heme synthesis showed premature senescence and reduced expression of NMDAzeta1 and -epsilon2 receptor subunits and NF-L compared with wild-type cells. Our findings suggest that reduced availability of heme in neurons associated with senescence may have significant effects on synaptic function.

5-Aminolevulinate Synthetase↗
PubMed2005

Calpain cleavage regulates the protein stability of p73.

The function of p73, a transcription factor belonging to the p53 family, is finely regulated by its steady-state protein stability. p73 protein degradation/stabilization can be regulated by mechanisms in part dependent on the ubiquitin proteasome system (UPS): (i) Itch/NEDD4-like UPS degradation, (ii) NEDD8 UPS degradation, and (iii) NQO1 20S proteasome-dependent (but ubiquitin-independent) breakdown. Here, we show that, in vitro, Calpain I can cleave p73 at two distinct sites: the first proline-rich region and within the oligomerization domain. Consequently, different p73 isoforms can be degraded by calpains, i.e., both N-terminal isoforms (TAp73 and DeltaNp73) as well as the C-terminal isoforms (alpha, beta, gamma, delta). Moreover, overexpression of the specific endogenous calpain inhibitor, calpastatin, in cultured cells increased the steady-state p73 level. This suggests that calpains may play a physiological role in the regulation of p73 protein stability.

Base Sequence↗
PubMed2005

Botulinum neurotoxin C initiates two different programs for neurite degeneration and neuronal apoptosis.

Clostridial neurotoxins are bacterial endopeptidases that cleave the major SNARE proteins in peripheral motorneurons. Here, we show that disruption of synaptic architecture by botulinum neurotoxin C1 (BoNT/C) in central nervous system neurons activates distinct neurodegenerative programs in the axo-dendritic network and in the cell bodies. Neurites degenerate at an early stage by an active caspase-independent fragmentation characterized by segregation of energy competent mitochondria. Later, the cell body mitochondria release cytochrome c, which is followed by caspase activation, apoptotic nuclear condensation, loss of membrane potential, and, finally, cell swelling and lysis. Recognition and scavenging of dying processes by glia also precede the removal of apoptotic cell bodies, in line with a temporal and spatial segregation of different degenerative processes. Our results suggest that, in response to widespread synaptic damage, neurons first dismantle their connections and finally undergo apoptosis, when their spatial relationships are lost.

Animals↗
PubMed2005

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity.

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

Amino Acid Sequence↗
PubMed2004

Regulation of the apoptosis-necrosis switch.

Execution of the apoptotic program involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can also be found when caspases are inhibited. Moreover, under pathological conditions, apoptosis and nonapoptotic death paradigms are often interwined, which suggest that, in vivo, cells may use diverging execution pathways. Molecular switches between apoptosis and necrosis include adenosine triphosphate-dependent steps in the activation of caspases or steps sensitive to reactive oxygen/nitrogen species. In turn, caspase activation can cause necrosis by promoting ion overload.

Adenosine Triphosphate↗
PubMed2003

Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein.

The hepatitis B virus X protein (HBx) is a multifunctional protein, acting on different targets (e.g. transcription factors, cytoplasmic kinases, and mitochondrial proteins) and exerting cellular effects as diverse as stimulation of cell proliferation and apoptosis. In its biological effects, the modulation of cellular Ca2+ signals has been proposed to be involved, but the direct assessment of Ca2+ homeostasis in HBx-transfected cells has not been carried out yet. In this work, we have employed for this purpose aequorin-based recombinant probes specifically targeted to intracellular organelles and microdomains. Using these probes, we observed that overexpression of HBx enhanced agonist-evoked cytosolic Ca2+ signals in HepG2 and HeLa cells, without affecting either the steady state of endoplasmic reticulum Ca2+ concentration or the kinetics of Ca2+ release. Rather, caspase-3-dependent cleavage of the plasma membrane Ca2+ ATPase could be demonstrated, and larger rises were detected in the cytoplasmic rim beneath the plasma membrane. In mitochondria, major morphological (fragmentation and swelling) and functional (reduced Ca2+ uptake) alterations were detected in HBx-expressing cells. As to the cellular consequences, we observed that HBx-induced apoptosis was markedly reduced when the alterations in Ca2+ signaling (e.g. by loading a Ca2+ chelator or preventing PMCA cleavage) or the downstream effects (e.g. by inhibiting mitochondrial permeability transition) were prevented. Overall, these results indicate that HBx perturbs intracellular Ca2+ homeostasis, acting on the extrusion mechanisms, and that this effect plays an important role in the control of HBx-related apoptosis.

Apoptosis↗
PubMed2003

Molecular switches deciding the death of injured neurons.

The endpoints used to evaluate neurotoxicity of drugs and chemicals are multiple and reflect the complexity of the nervous system. In many instances, loss of function can result from a temporary impairment of synaptic activity. However, exposure to some neurotoxic conditions may eventually lead to neuronal loss and be fatal to the organism. Execution of the apoptotic program seems to be the mechanism involved in loss of neurons in human neurodegenerative conditions. Apoptosis is a conserved mode of cell death, prominent in developmental conditions, whose main execution pathway converges on the activation of the caspase family of proteases. However, there is increasing evidence that cell death in post developmental conditions is more complex. Other routines or subroutines of cell death can be activated under toxic or pathological conditions, and several protease families may contribute to produce apoptotic-like features or other phenotypically different forms of cells death. This has posed the question as to whether classical apoptosis is a valid endpoint to test the effect of neurotoxic agents and whether inhibitors of the caspase subroutine to cell death may then be used to treat diseases characterized by an excess of apoptosis. The recognition of the molecular switches that toggle between cell death subroutines becomes, therefore, of central importance in biomedicine and toxicology.

Cell Line↗
PubMed2003

To die or to sleep, perhaps to dream.

Establishing social contacts is the raison d'être of neurons throughout their entire life span. To form and retain functional connections, neuronal differentiation and death are ruthlessly regulated in development and kept strictly under control in post-mitotic systems. Derangements in neural networks affect neuronal populations at large. Therefore, failure to retain synaptic connectivity is linked to dysfunction and often followed by neuronal death. Loss of neurons is a predominant feature of neurodegenerative disease. Nevertheless, neuronal cell death is not an obligate requirement for neural dysfunction at the level of distributed circuits or local circuits. Although more or less wide spread neuronal loss can occur after acute insults such as brain ischemia or invasion of the brain by pathogens, neuronal death is a hallmark of end-stage neurodegenerative and psychiatric disease. The relative contributions made by loss of synaptic connectivity versus cell death for these diseases are still debated. Here these processes are discussed in relation to acute and chronic CNS disorders.

Apoptosis↗
PubMed2003

Regulation of cell death: the calcium-apoptosis link.

To live or to die? This crucial question eloquently reflects the dual role of Ca2+ in living organisms--survival factor or ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.

Animals↗
PubMed2002

Apoptosis and age-related disorders: role of caspase-dependent and caspase-independent pathways.

The execution of the apoptotic program involves a relatively limited number of pathways that converge on the activation of the caspase family of proteases. However, there is increasing evidence that other protease families may contribute to produce apoptotic-like features. This has posed the question as to whether caspase inhibitors may then be used to treat diseases characterised by an excess apoptosis. In several neurodegenerative diseases including acute neuronal loss as in stroke or slowly developing diseases at least two major events contribute to neurodegeneration: the loss of neuronal connectivity and cell loss. In many of these conditions, mitochondrial dysfunction and the resulting ATP depletion may preclude caspase activation, and consequently switch execution of cell death towards necrosis. A block or partial inhibition of the typical apoptotic demise may have profound implications in vivo, as persistence within the nervous system of damaged, but 'undead' cells, followed by delayed lysis may favour neuroinflammatory reactions. Furthermore, caspases may be involved in loss of neurons, but not in the loss of connectivity that seems to initiate degenerative processes in the nervous system. Some recent findings, which suggest that degenerating neurons may use multiple execution pathways will be discussed.

Age Factors↗
PubMed2002

A simple cell line based in vitro test system for N-methyl-D-aspartate (NMDA) receptor ligands.

The generation of cell lines stably expressing the functional recombinant N-methyl-D-aspartate (NMDA) receptors (NRs) and their use for ligand testing in a simple excitotoxicity model is described. The mouse fibroblast cell line L(tk-) was co-transfected stably with cDNAs encoding the human NR subunits, NR1-1a/NR2A or NR1-1a/NR2B, respectively. The NR expression and functionality in resulting clones have been verified by RT-PCR, Western blotting, immunocytochemistry and fluo-4 calcium imaging. Stimulation of NR expressing clones with L-glutamate and glycine resulted in necrosis of cultures within 1 h. Therefore, a lactate dehydrogenase-based excitotoxicity assay was used for the pharmacological characterisation. The two selected clones exhibited pharmacological properties corresponding to the distinct NR subunit assemblies. Both cell lines showed proton inhibition of cell death in the range of physiological pH. EC50-values for L-glutamate under saturated D-serine concentrations were 3.7 microM for L12-G10 (NR1-1a/NR2A) and 2.8 microM for L13-E6 (NR1-1a/NR2B), respectively. Competitive antagonists (RS)-APV and (RS)-CPP as well as glycine B site antagonist DCKA prevented L-glutamate/glycine-induced cell death. NR2B selective antagonists such as ifenprodil or haloperidol did only protect L13-E6 cells. Spermine (300 microM) triggered cell death selectively in the L13-E6 clone in a pH-dependent manner.

Aniline Compounds↗