Nicotera, Pierluigi
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Alteration of the nuclear pore complex in Ca(2+)-mediated cell death
Cell death requires coordinated intracellular signalling before disassembly of cell architecture by degradative enzymes. Although the death signalling cascades that involve the mitochondria, the ER and the plasma membrane have been extensively characterized, only a handful of studies have examined the functional and structural alterations of the nuclear pore complex (NPC) during neuronal death. Here, we show that during excitotoxic neuronal degeneration calpains redistributed across the nuclear envelope and mediated the degradation of NPC components causing altered permeability of the nuclear membrane. In primary dissociated neurons, simultaneous recording of cytosolic [Ca(2+)] and localization of fluorescent proteins showed that the onset of Ca(2+) overload signalled a progressive increase in the diffusion of small reporter molecules across the nuclear envelope. Later, calpain-mediated changes in nuclear pore permeability allowed accumulation of large proteins in the nucleus. Further, in a model of excitotoxic neuronal degeneration in Caenorhabditis elegans, we found similar nuclear changes and redistribution of fluorescent probes across the nuclear membrane in dying neurons. Our findings strongly suggest that increased leakiness of the nuclear barrier affects nucleocytoplasmic transport, alters the localization of proteins across the nuclear envelope and it is likely to be involved in Ca(2+)-dependent cell death, including ischemic neuronal demise.
Cleavage of plasma membrane calcium pumps by caspases : a link between apoptosis and necrosis
Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are centralmediators of the apoptotic program.Herewe demonstrate that caspases cleave and inactivate the plasma membrane Ca2+ pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca2+ handling, which results in Ca2+ overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca2+ handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.
Cascade of Caspase Activation in Potassium-Deprived Cerebellar Granule Neurons : Targets for Treatment with Peptide and Protein Inhibitors of Apoptosis
Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.
Calpain inhibitors prevent nitric oxidetriggered excitotoxic apoptosis
The pathogenesis of some neurodegenerative disorders has been linked to excitotoxicity, excess generation of nitric oxide (NO) and apoptosis. Here, we used a model of NO-triggered neuronal apoptosis that was strictly dependent on autocrine NMDA receptor (NMDA-R) activation and intracellular Ca2 increase. We investigated the efficiency and potentially beneficial effects of calpain inhibition. Three calpain inhibitors that prevented intracellular fodrin proteolysis also blocked apoptotic features such as decrease in mitochondrial membrane potential, chromatin breakdown, and subsequent death of cerebellar granule neurons exposed to NO donors (S-nitroso- L-glutathione, S-nitroso-N-acetyl D,L-penicillamine, and diethylamino- diazenolate-2-oxide). Since inhibitors did not interfere with NMDA-R activation, we suggest that block of calpains blunts NO-triggered neuronal apoptosis by stopping the cascade downstream of primary autocrine excitotoxic events.
The dawning of a new age of toxicology
Die Toxikologie steht in einer Welt, in der wir Tausenden von Chemikalien und Millionen von deren Mischungen ausgesetzt sind, vor enormen Herausforderungen. Als Antwort darauf müssen radikal neue Ansätze entwickelt werden, um die Sicherheit der Bevölkerung zu gewährleisten. Ein Meilenstein in dieser Richtung ist die Vision des Nationalen Forschungsrates der USA Toxizitätstestung im 21. Jahrhundert: Eine Vision und eine Strategie . Gegenwärtig testet eine Allianz, die zwischen dem NTP* und dem NCGC des NIH sowie dem NCCT der EPA gebildet wurde, ob diese neue Strategie realistischerweise eine Basis für künftige Entscheidungen zum Schutz der öffentlichen Gesundheit sein kann. Die Vision setzt einen Paradigmenwechsel im Ansatz von Sicherheitsevaluationen voraus und stellt die traditionell angewandten Verfahren auf den Kopf. Wo bisher Tierexperimente die wichtigste Technologie waren, setzt die Zukunftsvision auf in vitro und in silico Ansätze, die auf menschlichem Material beruhen. Toxizitätstests beginnen heutzutage relativ blind mit einem Tierexperiment, dem dann nur manchmal mechanistische Studien folgen, während die neu vorgeschlagene Strategie die Sicherheitsevaluation von unten her beginnt: Zunächst würden mit in vitro Tests die durch Giftstoffe gestörten Stoffwechsel- und Regulationswege identifiziert; erst in einem möglichen zweiten Schritt kämen dann begrenzte und streng fokussierte Tierversuche dazu, um eventuell noch offene Wissenslücken zu schließen. Dies bedeutet nicht weniger als eine Umkrempelung der Toxikologie von einer hauptsächlich beschreibenden Tätigkeit und Hilfsdisziplin für Behörden wieder hin zu einer Naturwissenschaft mit all ihren Dimensionen. Der Hintergrund und die Konsequenzen werden hier insbesondere für Leser mit einem Interesse auch an parallelen Europäischen Trends beschrieben.
Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis
Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.
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.
Disialoganglioside GD3 induced oligodendrocyte death and GD3 release by microglia
Apoptosis in Caspase-inhibited Neurons
Background: There is growing evidence of apoptosis in neurodegenerative disease. However, it is still unclear whether the pathological manifestations observed in slow neurodegenerative diseases are due to neuronal loss or whether they are related to independent degenerative events in the axodendritic network. It also remains elusive whether a single, caspase-based executing system involving caspases is responsible for neuronal loss by apoptosis. Materials and Methods: Long-term exposure to the microtubule-disassembling agent, colchicine, was used to disrupt the axodendritic network and eventually trigger caspase-3-mediated apoptosis in cultures of cerebellar granule cells. For this model, we investigated the role of Bcl-2 and caspases in neurite degeneration and death of neuronal somata. Results: Early degeneration of the axodendritic network occurred by a Bcl-2 and caspase-independent mechanism. Conversely, apoptosis of the cell body was delayed by Bcl-2 and initially blocked by caspase inhibition. However, when caspase activity was entirely blocked by zVAD-fmk, colchicine-exposed neurons still underwent delayed cell death characterized by cytochrome c release, chromatin condensation to irregularly shaped clumps, DNA-fragmentation, and exposure of phosphatidylserine. Inhibitors of the proteasome reduced these caspase-independent apoptotic-like features of the neuronal soma. Conclusion: Our data suggest that Bcl-2 dependent and caspase-mediated death programs account only partially for neurodegenerative changes in injured neurons. Blockage of the caspase execution machinery may only temporarily rescue damaged neurons and classical apoptotic features can still appear in caspase-inhibited neurons.