Molecular Switches Deciding the Death of Injured Neurons

Pierluigi Nicotera1

MRC Toxicology Unit, Hodgkin Building, University of Leicester, LE 1 9HN Leicester, United Kingdom

Received November 6, 2002; accepted April 6, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 REFERENCES
 
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 postdevelopmental 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.

Key Words: apoptosis; caspases; neurodegeneration; ATP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 REFERENCES
 
The raison d’être of the neuronal populations within the nervous system is to establish contact with their targets during development and refine it throughout their entire life span. To achieve the scope of forming and retaining the functional nervous system architecture, neuronal differentiation and death are ruthlessly regulated in development and strictly kept under control in postmitotic systems. A true utilitarian principle, therefore, governs the harmonic development and survival of the nervous system. Derangements in neural networks would affect neuronal populations at large. Therefore, individual neurons that fail to retain most of their synaptic connections have no reason to further exist and die. This is accomplished by the activation of several signaling pathways, which activate proteolysis and promote cell recognition by macrophage or other scavenging cells.

Caspases and Cell Death
The apoptotic death program seems to converge mainly on one class of proteases, the caspases (cysteine aspartases), which can execute death signals in virtually every cell (see Earnshaw et al., 1999Go, and Nicholson, 1999Go, for reviews). Many caspase substrates are responsible for the appearance of the characteristic apoptotic features (i.e., DNA laddering, nuclear envelope breakdown, cell shrinkage, and exposure of phosphatidylserines). However, inhibition of the effector caspases, while preventing the appearance of the apoptotic morphology, does not always preclude the occurrence of cell death that may be triggered by other programs or alternative subroutines (Nicotera et al., 1999Go; Volbracht et al., 2001Go).

Caspases do not generally execute death forms other than apoptosis. Nevertheless, caspase inhibition reduces the overall rate of cell death in ischemic stroke models (Hara et al., 1997aGo,bGo; Robertson et al., 2000Go), where the necrotic morphology is predominant (Choi, 1996Go; Linnik et al., 1993). Clearly, caspase inhibition can stave off the breakdown of the cell membrane, which occurs if dying cells are not scavenged prior to membrane dissolution. Cells can indeed lyse after many of the typical apoptotic features have been acquired (Künstle et al., 1997Go).

Different Killing Subroutines: The "Calcium Death"
The idea that Ca2+ may be cytotoxic dates back to Fleckenstein’s suggestion in 1968 that excessive entry of Ca2+ into myocytes would be the underlying mechanism of cardiac pathology following ischemia. Subsequent studies showed that agonist stimulation (Leonard and Salpeter, 1979Go) or cytotoxic xenobiotics could cause lethal Ca2+ entry into cells (Schanne et al., 1979Go). From the early 80s on, the role of Ca2+ in cell death was examined intensively, especially in isolated hepatocytes, in the kidney, and in the brain (Nicotera et al., 1986Go; Siesjö, 1981Go; Siesjö and Bengtsson, 1989Go; Trump and Berezesky, 1995Go). It became soon evident that cellular Ca2+-overload involved multiple intra- and extracellular routes, most of which are also used for physiological signaling. Along with the understanding of the role of Ca2+ as physiological regulator, it soon became clear that not only alterations of the normal Ca2+-homeostasis but also changes in Ca2+-signaling would have adverse effects. In a large number of experimental paradigms it has now been shown that: (1) Direct sustained elevation of [Ca2+]i, for example by exposure of cells to ionophores, causes cell death. (2) A [Ca2+]i elevation precedes cell death induced by pathophysiological stimuli. (3) Prevention of [Ca2+]i elevation during such experiments can inhibit cell death. (4) Alterations of Ca2+-signaling pathways (e.g., potentiation or inhibition of Ca2+-currents) can result in cytotoxicity.

Intracellular Ca2+ overload can set off cell demise via Ca2+-dependent processes (Nicotera and Orrenius, 1994Go), change the balance of neuronal death from apoptosis to necrosis by depleting energy stores (Ankarcrona et al., 1995Go), or potentiate other subroutines of the death program (Wang et al., 1999Go). The interplay between Ca2+-activated proteases and caspases may also amplify apoptotic signals (Nakagawa and Yuan, 2000Go). Among several possible effectors of calcium overload, hydrolytic enzymes are the best characterized. The main classes include proteases, DNAses, and lipases. Several of these protein families have been implicated as effectors of Ca2+-elicited toxicity: calpains are Ca2+ activated cysteine proteases (Saido et al., 1994Go; Wang and Yuen, 1997Go). They have been implicated in toxic cell death in the liver (Nicotera et al., 1986Go), and in excitotoxic neuronal death in the brain (Siman and Noszek, 1988Go).

Calcium-dependent DNases can be responsible for DNA-degradation (Hewish and Burgoyne, 1973Go), although the nature of the Ca2+-dependent enzyme(s) responsible for the typical oligonucleosomal DNA-cleavage has remained elusive.

Among lipases, the Ca2+-dependent phospholipase A2 (PLA2) has been implicated in neurotoxicity. Its activation results in the release of arachidonic acid and related polyunsaturated fatty acids, which are further metabolized by lipoxygenases or cyclooxygenases with concomitant generation of reactive oxygen species (ROS). In addition, PLA2 activation generates lysophosphatids that alter the membrane structure. This may facilitate Ca2+ influx and Ca2+ release from internal stores (Traystman et al., 1991Go). In neurons, there is a close correlation between Ca2+-influx through N-methyl-D-aspartate receptors (NMDA-Rs) and PLA2-activation (Dumuis et al., 1988Go). The release of arachidonic acid following activation of PLA2 inhibits glutamate uptake into neurons and glial cells and may therefore prolong the excitotoxic action of this amino acid on its receptors (Volterra et al., 1992Go).

Calcium and Excitotoxicity
Excitotoxicity is a phenomenon typically encountered in neurons or myocytes following a stimulation that exceeds the physiologic range with respect to duration or intensity. Typical excitotoxic stimulators are capsaicin, acetylcholine or—most important in the central nervous system—glutamate. A large variety of chronic neurodegenerative diseases seem to have an excitotoxic component (Choi, 1992Go; Meldrum and Garthwaite, 1990Go). Early observations showed that direct injection of glutamate was selectively neurotoxic in vivo (Olney, 1969Go). The contribution to neurotoxicity of synaptic activity, i.e., the release of glutamate and stimulation of its receptors on postsynaptic membranes, was deduced from experiments showing that inhibition of neurotransmission by Mg2+ or glutamate antagonists protected neurons from hypoxia (Rothman, 1983Go). A causal contribution of excitotoxicity to neuronal damage was then established in stroke or head trauma (Bullock, 1995Go; Myseros and Bullock, 1995Go). Generally, excitotoxicity is induced by conditions favoring glutamate-accumulation in the extracellular space. Typical conditions leading to increased extracellular glutamate concentration are depolarization of neurons, energy depletion due to hypoglycemia or hypoxia (Cheng and Mattson, 1991Go; Wieloch, 1985Go), or defects in the glutamate reuptake systems (Rothstein et al., 1996Go; Volterra et al., 1992Go). More recently, the link between glutamate-release and increased [Ca2+]i has been established within individual neurons; it has been shown that neurotransmitter-release triggered by electrical stimulation of hippocampal neurons with autosynapses was sufficient to cause glutamate-induced increases of [Ca2+]i within the same cell. This close reciprocal relationship between increased extracellular glutamate concentrations and raised [Ca2+]i results in neuronal death under excitotoxic conditions.

Overall, the key role of Ca2+ in excitotoxicity is suggested by three different lines of evidence: (1) There is an obvious increase in [Ca2+]i in in vivo and in in vitro models of excitotoxic cell death. This has been observed in ischemic brain (Simon et al., 1984Go) or in brain slices exposed to NMDA-R agonists or anoxia (Garthwaite and Garthwaite, 1986Go). In addition, glutamate-stimulated Ca2+-influx in cultured neurons has been shown by the 45Ca-technique (Eimerl and Schramm, 1994Go), and increased [Ca2+]i after NMDA-R stimulation has been observed repeatedly using fluorescent probes (Dubinsky and Rothman, 1991Go; Murphy et al., 1987Go; Tymianski et al., 1993Go). (2) Prevention of Ca2+-entry into the cell by removal of extracellular Ca2+ (Garthwaite and Garthwaite, 1986Go), depletion of NMDA-Rs, or by pharmacological inhibition of glutamate receptors or VDCCs (Choi, 1995Go; Lipton and Rosenberg, 1994Go) prevents neuronal death in many paradigms of excitotoxicity. (3) A prevention of neurotoxicity by inhibition of downstream effects of Ca2+ overload most strongly suggests a causal role of Ca2+ in excitotoxicity. Intracellular Ca2+ chelators can prevent ischemic damage in vivo and excitotoxic neuronal damage in vitro (Tymianski et al., 1993Go). Also, inhibition of effectors of Ca2+ toxicity such as calmodulin, calcineurin (Dawson et al., 1993Go), or bNOS (Dawson et al., 1993Go) protects neurons from the toxicity of excitatory amino acids. Xenobiotics such as Hg+ or lead can recruit Ca2+ transport systems to exert their toxic effects via apoptosis or necrosis. Enhancement of NMDA signaling, for example, can facilitate neuron elimination by excitotoxic stimuli (Rossi et al., 1997Go)

Apoptosis, Necrosis, or Only Different Death Subroutines?
The mode of cell death in excitotoxicity has been and still is largely debated. Apoptosis and necrosis, in their classical definition, are two fundamentally different modes of cell death (Wyllie et al., 1980Go). Whereas apoptosis is characterized by a preservation of membrane integrity until the cell is phagocytosed, this is not the case in necrosis/lysis of cells. The duration and extent of Ca2+-influx may determine whether neurons survive, die by apoptosis, or undergo necrotic lysis (Bonfoco et al., 1995Go; Choi, 1995Go). Very low [Ca2+]i, or the prolonged inhibition of Ca2+-influx may be neurotoxic (Mattson et al., 1995Go). A continuous moderate increase in [Ca2+]i, such as that produced by a sustained slow influx, may cause apoptosis, whereas an exceedingly high influx would cause rapid cell lysis. E.g., stimulation of cortical neurons with high concentrations of NMDA results in necrosis, whereas exposure to low concentrations causes apoptosis (Bonfoco et al., 1995Go). Accordingly, neuronal death in experimental stroke models is necrotic in the ischemic core but delayed and apoptotic in the less severely compromised penumbra or border regions (Beilharz et al., 1995Go; Charriaut-Marlangue et al., 1996Go; Li et al., 1995Go; Pollard et al., 1994Go). The same applies to several other neuropathologic conditions where apoptosis and necrosis have been observed to occur simultaneously (Portera-Cailliau et al., 1995Go; Shimizu et al., 1996Go). One sensor that switches neurons towards one or the other fate may be the ability of mitochondria to generate enough ATP (Ankarcrona et al., 1995Go). A complete de-energization of the cell (e.g., failure of all mitochondria and of glycolysis) may not allow the ordered sequence of changes required for the apoptotic demise. In such a case random processes would result in rapid uncontrolled cell lysis/necrosis. Therefore, it seems likely that apoptosis ensues under conditions of Ca2+ overload or other stress, where there remains sufficient energy production (ATP) to execute the death program. Accordingly, ATP levels are maintained in CGN undergoing apoptosis (Ankarcrona et al., 1995Go), and the cytoplasmic ATP levels indeed decide between different shapes of cell death (Leist et al., 1997Go).

A Subroutine of the Apoptotic Program Can Cause Secondary Necrosis Via Calcium Overload
The signals leading to secondary lysis or necrosis in cells that have initiated the apoptotic program are unknown. Osmotic imbalance, which results from changes in ion channels and pump activity, can intervene in doomed cells and lead to cell rupture. A sustained Ca2+ overload, such as that resulting from dysfunction of the main routes of Ca2+ entry or efflux may be lethal.

Regardless of the source of calcium overload, the integrity of plasma membrane Ca2+ ATPase (PMCA) is essential to rectify changes in intracellular Ca2+ in the long term. Thus, neurons that survive the acute Ca2+ accumulation through the NMDA-R, rapidly pump calcium out and recover a steady state cytosolic calcium. To this end, an important role is also played by the Na+–Ca2+ exchanger, which can effectively contribute to remove large amounts of calcium accumulated in the cytosol.

Independent genes encode the four major PMCA isoforms currently known. PMCA isoforms 1 and 4 are expressed ubiquitously in mammalian cells, whereas isoforms 2 and 3 are expressed in significant amounts only in the brain (Guerini et al., 1999Go). Another Ca2+ pump is located in the endo-/sarcoplasmic reticulum (SERCA).

We have recently investigated the fate of calcium translocases in neurons and nonneuronal cells undergoing apoptosis. We have studied the neuron specific PMCA2, the ubiquitous PMCA4 isoforms, the SERCA pump, and the Na+–Ca2+ exchanger (NCX).

To investigate the fate of PMCA2 during brain ischemia and excitotoxicity, we recently carried out studies in collaboration with Drs. Nicholson and Carafoli’s laboratories. In the neonatal, hypoxia-ischemia model we found that PMCA2 (133 kDa) is degraded to a main fragment of 123 kDa. The extent of pump cleavage correlates with the degree of brain injury, as assessed by brain swelling, and to the apoptotic index, which reflects apoptotic DNA fragmentation. Subsequently, we exposed CGN to either low concentrations of glutamate or to the indirect excitotoxins, S-nitrosoglutathione (GSNO) or 1-methyl-4-phenylpyridinium (MPP+), which stimulate synaptic glutamate release and excitotoxicity (Bonfoco et al., 1996Go; Leist et al., 1998Go). Western blots of membrane fractions isolated from CGN undergoing apoptosis showed that PMCA2 (133 kDa) was degraded to the same 123 kDa fragment found in the ischemic brain tissue. We then went on to show that the ubiquitous PMCA4 isoform is analogously cleaved in nonneuronal cells induced to apoptose by staurosporine. The cleavage of PMCAs is operated by caspases. The major effector caspase, caspase-3 cleaves PMCA4 and PMCA2, whereas PMCA2 can also be cleaved by caspase-1. Cleavage of PMCAs results in loss of function and aberrant intracellular Ca2+ transients, with Ca2+ overload. Expression of mutants of human PMCA4 that lack the caspase cleavage site(s) prevents Ca2+ overload during apoptosis and markedly delays secondary cell lysis/necrosis. These findings show that caspases can activate a subroutine of the death program that causes secondary necrosis. They also suggest an explanation for the observation that caspase inhibitors prevent the appearance of secondary necrosis in stroke models and rescue ischemic areas (Schwab et al., 2002Go).

Since cell death is not necessarily linked to a single program or a morphological phenotype, events that confer irreversibility to its execution would seal a cell’s fate. Along with nuclear and cytoskeletal damage, disruption of cell signaling and ion homeostasis could warrant irreversibility to the death sentence. Inactivation of Ca2+-motive ATPases impairs the ability of cells to handle Ca2+ and can lead to cell demise (Chami et al., 2001Go; Nicotera et al., 1986Go). PMCA inhibition has been implicated in neuronal cell death elicited by amyloid-ß peptide (Mark et al., 1995Go) or in the delayed neuronal death following ischemia in CA1 hyppocampal pyramidal cells. Interestingly, in the latter study, PMCA activity transiently recovered after ischemia, to decay irreversibly hours after the insult. Our findings of the cleavage of PMCA2 following ischemic brain injury may explain these early observations. Caspase activation does not necessarily require elevation of intracellular Ca2+. However, Ca2+ chelators or channel blockers can rescue part of the death program in paradigms where Ca2+ is not the initial death signal. Several amplification loops have been suggested between the caspase execution pathway and disturbances in intracellular Ca2+. These include activation of signaling proteins (Wang et al., 1999Go), transcriptional factors (Camandola et al., 2000Go), and other protease families (Leist et al., 1998Go; Nath et al., 1996Go). Rising Ca2+ levels, as seen in cells where Ca2+ extrusion is compromised, may then affect execution of apoptosis, in addition to promotion of secondary lysis.

In addition to PMCAs, the Na+–Ca2+ transporter (NCX) drives Ca2+ efflux in excitable cells (Carafoli et al., 2001Go). NCX has a low Ca2+ affinity, but high Ca2+ transporting velocity, which is required to rapidly eject large amounts of Ca2+ (Linck et al., 1998Go). While the NCX contribution in regulating resting cytosolic Ca2+ may be less important than that of PMCAs, its function may prevent Ca2+ overload in stimulated cells undergoing apoptosis. Preliminary results in our laboratories show that NCX is cleaved by caspase-3 in CGN undergoing apoptosis and suggest that cleavage of plasma membrane Ca2+ transporters is a generally relevant subroutine of the neuronal death program.

Multiple Commitment Points in Neuronal Cell Death
It is clear that neurodegeneration in the course of disease or triggered by toxic agents may interfere with several survival/death pathways. These pathways may not always lead to apoptosis or necrosis of neurons, but may promote loss of connectivity, loss of adhesion, and even developmental regression. When the insult is intense, neuronal demise is usually the outcome. However, under these conditions (i.e., excitotoxicity, in stroke), multiple death pathways can also cooperate to eliminate damaged neurons. Where, then, is the commitment point in neuronal cell death? Death pathways in high organisms have evolved as independent subroutines. Some are restricted to individual tissues as part of their differentiation and developmental programs, whereas others, which are longer conserved in evolution, are active in every organ and cell type. Each subroutine has a commitment point, and often cell death requires the recruitment of many. Neurons may cease to exist when all their relationships are lost and the social marginalization becomes unbearable, when they regress to an infantile state, when extracellular cues de-repress their apoptosis pathways. Thus, endowing a degenerative process with a single commitment end point may be unrealistic. This may prove difficult in toxicological settings, when searching for simple toxicity markers, and may prove even more difficult in the search for therapies based on postinjury intervention. Nevertheless, the understanding of common ancestrally conserved responses to endogenous or toxic insult patterns may provide more significant end points to assess neurotoxicity and to identify relevant therapeutic targets.


    NOTES
 
1 For correspondence via e-mail: pn10{at}le.ac.uk. Back


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