doi: 10.1161/01.STR.0000256294.46009.29
(Stroke. 2007;38:674.)
© 2007 American Heart Association, Inc.
Glutamate-Independent Calcium Toxicity: Introduction |
Ca2+ Signals and Neuronal Death in Brain Ischemia
From the Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, Leicester, UK.
Correspondence to Pierluigi Nicotera, MD, PhD, Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Rd LE1 9HN, Leicester, UK. E-mail pn10{at}le.ac.uk
Abstract
Although Ca2+ signals are necessary for cell communication and survival, abnormal cellular Ca2+ load can trigger different cell death programs. Ca2+ mediates cell death by activating proteases (ie, calpains), by reinforcing signals leading to caspase activation or by triggering other catabolic processes mediated by lipases and nucleases. Failure in the clearance of excitatory amino acid is a critical determinant of neuronal loss in the ischemic brain. Glutamate activates glutamate-ionotropic receptors at synaptic and extra-synaptic sites, causing prolonged neuronal depolarization and triggering deregulation of cellular ion homeostasis, mainly intracellular calcium and sodium. The mechanisms leading to the sustained calcium deregulation in excitotoxic conditions are only in part elucidated. Recently, we have shown that calpains mediate the inhibition of calcium efflux in primary dissociated neurons challenged with excitotoxic glutamate concentrations. Calpains cleave the sodium-calcium exchanger (NCX) and inhibit its capability to remove calcium accumulated as a consequence of the excitotoxic stimulus. Our findings highlight the link between calcium-dependent proteases, calcium overload and neuronal degeneration after an excitotoxic insult.
Key Words: calcium transporter • cell cultures • neurochemistry • neuroprotection
It is well established that genetically encoded programs decide the fate of individual cells or organs during development and in normal tissue-cell turnover. However, in recent years it has also become clear that cells execute one or more biochemical programs to signal or execute cell death, particularly under pathological conditions. In many instances, developmental cell death and death under pathological conditions share similar morphological features as well as signals and execution systems. For example, the characterization of the main signals, modulators and executioners of programmed cell death in Caenorhabditis elegans has led to the understanding of very important death pathways in pathological cell death of mammalian organisms. This is not a singularity of the death program that we call apoptosis, because the concept can be extended to other paradigms of cell death. For example, autophagic cell death, whose main feature is the presence of cytoplasmic lysosomes–derived vacuoles, is frequent in both neuronal development and neurodegenerative disease.1 The existence of conserved biochemical pathways to signal and execute cell death has somewhat reduced the emphasis on the morphological characterization of cell death. Despite efforts to define individual forms of cell death based on their appearance, it is clear that in all circumstances cell disassembly involves nuclear fragmentation/dissolution, organelle disruption (sooner or later) and eventually membrane lysis and phagocytosis. Thus, condensation and eventual fragmentation of the nucleus occurs in apoptosis as well as in necrosis and autophagic cell death. In apoptosis, as well as in necrosis, the mitochondria can be partially or totally damaged and, again, in both apoptosis and necrosis the cytoskeletal structure is compromised and molecules are exposed at the cell surface to promote recognition and scavenging. Therefore, the concept of a death program is not necessarily linked to a morphological appearance. It seems more likely that several death executing routines may be activated at once within injured cells, one or the other becoming predominant depending on the stimulus and the tissue metabolic conditions. For example, there is increasing evidence that apoptotic-like features can also be found when the main executioners of apoptosis, the caspases, are inhibited,2 whereas caspase-mediated cleavage of relevant substrates may be involved in cell lysis/ necrosis.3 Thus, under pathological conditions several protease families may cooperate to disassemble cells targeting different organelles or cellular substructures. It is, for example, remarkable that caspases and calpains share many substrates. Notably the cleavage sites for the 2 protease families are in several cases located on very close regions within the same protein.3 Whereas the predominance of one or another death executing mechanism may be dictated by factors as different as energy requirement, signaling molecules and the intensity of a given insult, in many instances the differentiation program within a given cell tissue dictates the way to die. This is particularly true of neurons, where spatial selectivity of death signals and promiscuity of execution systems can result in the complex and relatively slow demise, which occurs in neurodegenerative disease. The promiscuity of death subroutines is evident in brain ischemia, where the intensity of the insult and the death triggers activate both caspase-dependent and independent death pathways. The latter involve the Ca2+ activation of the calpain family of proteases.
Death Signals in Brain Ischemia: Calpains Amplify Lethal Ca2+ Signals
Failure in the clearance of excitatory amino acids causes sustained gating of receptor-operated channels,4 with prolonged neuronal depolarization and calcium overload. In ischemic regions, excitotoxic stimulation can trigger an inflammatory response and apoptotic or necrotic cell death, depending on the intensity of the insult.5,6 Subsequent work has shown that synaptic glutamate overstimulation may predominantly elicit apoptosis,7 whereas more widespread excess glutamate causes predominantly necrosis. Thus, treatments that reduce ion imbalance by blocking gating of glutamate-ionotropic receptor and voltage gate channels can protect neurons and reduce brain infarct.8,9 However, as shown recently, after an ischemic insult, other plasma membrane channels can contribute to neuronal depolarization and Ca2+ accumulation.10,11 Hence, the study of downstream events triggered by unphysiological calcium increase and involved in neuronal excitotoxic degeneration can potentially provide a pharmacological target to prevent extensive neuronal demise during stroke. Increasing evidence suggests that unregulated calcium-dependent proteolysis is a key process during excitotoxicity and suppression of calpains activity ameliorates postischemic damage.12,13 We have recently shown that calpains can cleave one of the major isoforms (isoform 3) of the sodium-calcium exchanger (NCX3) that operate Ca2+ efflux from neurons. Lentiviral delivery of calpastatin, the endogenous calpain inhibitor, prevented cleavage of NCX3 and prevented the secondary Ca2+ overload linked to neuronal demise.14 Ca2+ overload was also prevented in the majority of neurons by overexpressing a functional NCX isoform (NCX2), whereas it was exacerbated by siRNA downregulation of NCX3. Replacement of NCX3 with the functional isoform NCX2 also protected neurons from excitotoxicity. Our findings suggest a mechanism that can be downstream of the initial Ca2+ influx through different plasma-membrane channels (Figure) and, as such, is potentially interesting for pharmacological intervention aimed to prevent an irreversible Ca2+ overload.
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As shown by others, NCX is a fundamental player during ischemia15,16 and neuronal degeneration17 and its downregulation leads to massive calcium overload and neuronal demise. Interestingly, NCX3 is required also in the maturation of dissociated neurons18 and in the control of intracellular Ca2+ in skeletal muscle fibers.19 Not surprisingly, sustained and unregulated protease activity has been linked with a wide number of pathological conditions, including neuronal degeneration.20 As shown previously, caspases can affect the activity of the other major Ca2+ extruding system, the plasma membrane Ca2+ ATPase (PMCA), which is internalized after cleavage.3 Recent work has extended these observations showing that during glutamate-mediated neuronal death, PMCAs can be internalized, which contributes to Ca2+ deregulation and further supports a fundamental role for Ca2+ transporters in neuronal demise.21
In conclusion, it is apparent that different death programs/routines have in common at least 2 fundamental features: (a) they are evolutionarily conserved. This may be explained by a dual role of many components as life and death signals; (b) they involve the activation of one or more families of proteases and possibly other degradative enzymes. The diverse actions of intracellular Ca2+ signals provide an optimum example of the fact that the same signal may be physiological or detrimental depending on threshold and cellular conditions. Tight homeostatic mechanisms regulate intracellular Ca2+ concentration to spatially and temporally localize Ca2+ signals22 and to allow multiple Ca2+-mediated signaling cascades to occur independently within the same cell. However, excessive Ca2+ influx, release from intracellular stores or impairment in the Ca2+-extruding machinery can overcome Ca2+-regulatory mechanisms and lead to cell death.20 Ca2+ signals can also reinforce the execution of Ca2+-independent death subroutines.20 Redistribution of Ca2+ within intracellular stores can amplify apoptotic signals,23 but as shown above can also initiate cell death execution by calpains.24
Acknowledgments
Disclosures
None.
accepted December 14, 2006.
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