Abstract

Apoptosis is a distinctive feature in the physiology of the developing brain, but also a key event in pathological conditions of the adult brain. The basic mechanisms executing cell death by apoptosis are conserved among different tissues and in different conditions, while the initiating event(s) may be more specific. Magnesium appears to be an important player in the process, though it might exert opposite actions depending on extra/intracellular availability. Extracellular magnesium deficiency induces apoptosis, mainly through increased oxidative stress, while intracellular magnesium mobilization from intracellular stores and consequent increase of cytosolic free magnesium seem to act in the effector phase. The molecular mechanism and the physio-pathological meaning of these findings await further characterization. The issue is even more complex in the context of the brain, where many concurring factors may determine a pro- or anti-apoptotic environment. A deeper understanding of the yin-yang role of magnesium in apoptosis may cast light on the basic processes that regulate cell fate, and consequently may open up novel opportunities for a successful therapeutic intervention for all the pathological conditions where excessive and undue apoptosis takes place.

Cell death by apoptosis

Multicellular organisms maintain their homeo- stasis thanks to a tightly regulated mode of cell death. Programmed cell death, or apoptosis, is an internally controlled suicide program consisting in a stereotyped sequence of biochemical and morphological changes that allow the cell to die without adversely affecting its neighbours, i.e. without causing inflammation. As such, apoptosis is distinct from necrosis, the traumatic form of cell death whereby massive disruption of the surrounding tissue takes place. A cell may be doomed to die either by apoptosis or necrosis depending on the intensity and duration of the stimulus, the rapidity of the death process, and the extent of ATP depletion suffered by the cell.

Apoptosis occurs in numerous physiological, adaptive and pathological events: 1) during development; 2) as a homeostatic mechanism to maintain cell populations in proliferating tissues; 3) as a defence mechanism such as in immune reactions; 4) following cell injury from various agents; and 5) in aging. There is general agreement that this form of cell death is an energy-dependent cascade of molecular events leading to cell shrinkage with maintenance of organelle integrity; long-recognized features of apoptosis are protein cleavage and cross-linking, chromatin condensation, DNA fragmentation and, ultimately, phagocytic recognition and engulfment of the dying cell. The process of apoptosis is triggered by a diverse range of cell signals, which may originate either extracellularly (i.e. death receptor ligation with their cognate ligands, extrinsic pathway) or intracellularly (e.g. DNA damage or organelle dysfunction, intrinsic pathway).

The apoptotic cascade consists of three parts: first, cells process the receptor-mediated death signal (initiation phase, somewhat more cell- specific), then get ready to implement apoptosis (effector phase, more conserved among different tissues) and finally commit suicide (degradation phase). During the initiation phase, the receptor- mediated death signal activates an intracellular cascade of events including: 1) activation of initiator caspases (caspase-8, for example), that can act early in the cell death process before, or independently of, mitochondrial changes; 2) increase in levels of oxyradicals and Ca²⁺; 3) transcription and translocation of pro-apoptotic Bcl-2 family members (Bax and Bad) to the mitochondrial membrane. The execution phase involves: 1) increased mitochondrial Ca²⁺ and oxyradical levels; 2) opening of permeability transition pores (PTP) in the mitochondrial membrane; 3) release of cytochrome c and many other apoptosis-inducing factors from the mitochondrial matrix into the cytosol; 4) formation of a molecular complex (the apopto- some) between cytochrome c and apoptotic protease-activating factor 1 (Apaf-1), that recruits and activates pro-caspase-9. Activated caspase-9, in turn, activates caspase-3, which begins the degradation phase: proteins are cleaved by various caspases and packed by trans- glutaminases, while nucleic acids are cleaved by activated Ca²⁺/Mg²⁺ endonucleases, which results in the formation of apoptotic bodies and subsequent phagocytosis by elicited macro- phages or neighbouring cells. It is interesting to underline the role of mitochondria in triggering apoptosis. Whether or not a direct injury jeopardizing the efficiency of these energy producing organelles occurs, mitochondria play a pivotal role in the cell death decision, as apoptotic factors are released by active mechanisms, which can be unrelated to mitochondrial damage (Tait and Green, 2010).

Role of Magnesium in Apoptosis

In the attempt to review the role of magnesium in apoptosis, one should first of all keep in mind that the effects of extracellular magnesium availability may differ from those due to intracellular magnesium fluxes or mobilization. In fact, decreased extracellular magnesium availability does not necessarily reflect an intracellular depletion, as demonstrated by the fact that in vivo serum magnesium levels are not a reliable marker of pathological hypo- magnesemia. Sensitivity to extracellular magnesium availability is highly tissue-specific, and can be translated in molecular terms by considering the expression and activity of magnesium-specific cation channels, such as the transient receptor potential melastatin (TRPM) -6 and -7 channels, which, by modulating trans- membrane cation fluxes, regulate the intracellular ion concentration (Schlingmann et al., 2007). Furthermore, intracellular magnesium is tightly buffered to meet specific metabolic requirements. As discussed elsewhere in this book, large changes in total cell magnesium occur with little or no change in cytosolic free magnesium, suggesting that the changes in total magnesium are due to changes in bound or sequestered magnesium (Murphy, 2000). ATP is the most important intracellular magnesium buffer. Following massive ATP hydrolysis intra- cellular free magnesium transiently increases and is subsequently released into the extracellular environment. As a consequence, in a dying cell an increase in ionized magnesium occurs. Therefore, caution should be used also when investigating the role of intracellular magnesium as a player in the apoptotic program, in order to discriminate coincidental from causative events.

In conclusion, whether considering extracellular magnesium availability or intracellular magnesium concentration, the story is more complicated than it appears at first glance. We will attempt to discuss the issue by reviewing the latest available data in view of the complex cellular physiology of magnesium.

Intracellular magnesium and apoptosis

As to intracellular events, several in vitro studies have suggested a promoting role for magnesium in apoptosis. An early increase in intracellular magnesium seems to follow both extrinsic and intrinsic induction of apoptosis (Patel et al., 1994; Chien et al., 1999; Zhang et al., 2005). The most straightforward explanation is that the increase in intracellular magnesium concentration is necessary for stimulating the activity of Ca²⁺/ Mg²⁺dependent endonucleases, which perform the apoptotic event par excellence, i.e. nucleosomal DNA fragmentation. Interestingly, however, the source of intracellular magnesium was hypothesized to be in the mitochondria (Chien et al., 1999), which is particularly appealing, as it is well established that mitochondria play a central role in the onset of the apoptotic program. Other lines of evidence point to the same direction. While the opening of the permeability transition pores appears to be dispensable for the release of cytochrome c, this is not the case for the presence of magnesium, which instead seems an absolute requirement (Eskes et al., 1998; Kim et al., 2000). Another study (Salvi et al., 2004) indicated that an apoptotic compound, gliotoxin, can specifically activate a magnesium efflux system from mitochondria in conditions of preserved mitochondrial integrity (i.e. high membrane potential, no swelling and retention of other ions). Most importantly, it has been shown that mitochondria act in fact as magnesium stores, and that Mg²⁺ release can occur following Ca²⁺ release and prior to ATP hydrolysis (Kubota et al., 2005). It is noteworthy that the same Authors have recently demonstrated that glutamate administration to rat hippocampal neurons triggers the same pathway, whereby Ca²⁺ accumulation in the mitochondria is required for Mg²⁺ release from the organelles (Shindo et al., 2010). The presence of a specific mitochondrial channel for magnesium, Mrs2, (Kolisek et al., 2003) corroborates these findings. As the activity of Mrs2 is dependent from membrane potential, it can be speculated that stored magnesium might be released through the channel upon depolarization. Intriguingly, in a recent paper Mrs2 expression has been associated with resistance to drug-induced apoptosis in cancer cells (Chen et al., 2009): by upregulating magnesium uptake into mitochondria, Mrs2 might counteract the increase in cytosolic magnesium that seems to be necessary for the execution of the apoptotic program via the mitochondrial pathway (Wolf and Trapani, 2009). In conclusion, the latest findings seem to concur to suggest a finer involvement of magnesium in the apoptotic cascade: not just a biochemical factor, but rather a crucial control element in life vs death decisions (see Table 1 for a summary). At present, these are only attractive speculations; most importantly, it has to be clarified whether the increase in intracellular magnesium occurring following an apoptotic stimulus is just a coin- cidental event (for example, due to mitochondrial depolarization) or rather a causative determinant in the downstream signalling cascade.

Table 1:

Magnesium and apoptosis

Neuronal Apoptosis

In contrast to the rapid turnover of cells in proliferative/renewing tissues, neurons commonly survive for the entire lifetime of the organism — this enduring nature of neurons is necessary for maintaining the function of those cells within neuronal circuits. However, during development of the central and peripheral nervous systems, many neurons undergo apoptosis during a time window that coincides with the process of synaptogenesis (Oppenheim, 1991). Initial overproduction of neurons, followed by death of some, is probably an adaptive process that provides enough neurons to form nerve cell circuits that are precisely matched to their functional specifications. However, the persistence of neurons throughout life to preserve brain function implies that a considerable evolutionary pressure was placed on the development of mechanisms that guarded against neuronal death and/or promoted neuronal survival and plasticity.

Role of Magnesium in Neuronal Apoptosis

Deciphering the role of magnesium in the apoptotic process is further complicated in the nervous system, as the blood brain barrier may restrain serum magnesium availability within the CNS. Indeed, this has been advocated as the cause for the failure of clinical trials assessing magnesium therapy in acute and chronic brain injury (Vink et al., 2009).

A significant part of the literature on the effects of magnesium on apoptosis in the brain is related to perinatal brain, as magnesium is often used as a treatment for pre-eclampsia/eclampsia and preterm labour. The basis for its use as a tocolytic is due to its observed effects in reducing myometrial contractility through extra- and intracellular mechanisms of action (Fomin et al., 2006). However, the use of magnesium sulphate as a drug in obstetrical medicine for both mother and fetus is highly controversial. A recent meta- analysis of all trials has recommended the use of magnesium for neuroprotection in the preterm fetus, as it was both safe and effective (Doyle et al., 2009). Nevertheless, some caution is mandatory, as several reports point to detrimental effects of magnesium for the fetal brain, depending on the dose (Mittendorf et al., 2006; Dribben et al., 2009), on the period of neurodevelopment when the exposure occurred (Dribben et al., 2009) or on the level of stress experienced (Krueger et al., 2001). In addition, some papers report a pro-apoptotic action of magnesium in placental tissues (Black et al., 2001; Gude et al., 2000). The major hurdle to establishing protective or deleterious effects of tocolytic magnesium to the developing brain is that magnesium’s effects on preterm infant’s brains, and the mechanisms of those effects, are not understood.

As discussed earlier, neuronal apoptosis can be triggered by three main mechanisms: 1) lack of growth factors; 2) overstimulation of glutamate receptors; and 3) oxidative stress. Magnesium could play a (different) role in each of these signalling pathways.

First, most growth factors work through receptor tyrosine kinases (RTKs) which require two magnesium ions for maximal activity. Kinases downstream of these RTKs, such as PI -kinase and the anti-apoptotic kinase Akt require magnesium both for their activation and activity. Therefore, extracellular magnesium could modulate the effect of growth factors on CNS cells, or activate their second messenger systems directly, ultimately affecting viability, proliferation, and apoptosis in these cells.

Secondly, under conditions of normal membrane polarization magnesium is known to block the NMDA glutamate receptor and prevent ion flow through the channel. Therefore, in principle, magnesium should protect against excitotoxicity- induced apoptosis. Nevertheless, as already discussed, normal physiological patterns of NMDA receptor activity promote neuroprotection against both apoptotic and excitotoxic insults; vice versa, NMDA receptor blockade can promote neuronal death outright or render neurons vulnerable to secondary trauma (Hardingham, 2009). Indeed, most NMDA antagonists have failed miserably as neuroprotective agents in clinical trials, in large part because of intolerable side effects (Lipton, 2006). Moreover, it has been shown that various drugs that inhibit neuronal activity, including NMDA antagonists, trigger widespread apoptotic neurodegeneration in the developing brain and cause long-term neurobehavioral deficits; in the infant rodent brain, peak sensitivity to this effect is in the P3 to P7 period (Ikonomidou et al., 1999). Thus, although other mechanisms may play a role, it seems likely that the neuroapoptogenic action demonstrated for magnesium in some studies could be mediated by its action at NMDA receptors.

Third, as mentioned earlier, magnesium deprivation may induce or exacerbate oxidative stress, another trigger of neuronal death, especially in pathological conditions. In this regard, magnesium supplementation might well have a neuroprotective action.

Apoptosis in neurodegenerative diseases

As already mentioned, neuronal death by apoptosis is a distinctive physiological feature of the developing brain; once synaptogenesis is over, the remaining neurons will usually survive throughout life to preserve brain function. Unfortunately, however, many people experience excessive death of specific neuronal sub- populations as a result of chronic diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis. Neuronal death may also occur as a consequence of acute conditions, such as stroke or traumatic brain and spinal cord injury. For each of these disorders, apoptosis has been implicated as the main form of cell death, though it is very difficult to demonstrate it for both technical and ethical reasons. Much of the evidence supporting an apoptotic mode of neuronal death comes from studies of animal and cell-culture models of neurodegenerative disorders (Mattson, 2000).

Physiological apoptosis in the developing brain and pathological apoptosis in the adult brain share similar molecular mechanisms in the effector phase, but may differ in the initiation. Whereas trophic factor withdrawal has a prominent role in apoptosis during development, toxic insults resulting from biochemical or genetic accidents seem to be the triggering event in neurodegenerative disorders. An emerging theme is the toxicity of abnormal protein structures or aggregates (Yuan and Yankner, 2000). Indeed, a defining feature of Alzheimer’s disease is accumulation of amyloid plaques formed by aggregates of amyloid-β peptide (Aβ). Parkinson’s disease is characterized by the appearance of α-synuclein oligomers. Huntington’s disease is caused by expansions of a trinucleotide (CAG) sequence in the huntingtin gene producing a protein containing increased polyglutamine repeats. The neuropathological signature of amyotrophic lateral sclerosis (ALS) is the presence of ubiquitinated inclusions immuno- reactive for the proteins TDP-43 and/or FUS/TLS in the cytoplasm of motor neurons. Moreover, mutations in specific genes may predispose to neuronal degeneration, e.g. presenilin in Alzheimer's disease, parkin in Parkinson's disease, huntingtin in Huntington's disease, and Cu/Zn- superoxide dismutase (SOD) in ALS (please refer to the dedicated chapters in this book for further details).

The identification of specific genetic and environmental factors responsible for chronic neurodegenerative disorders has bolstered evidence for a shared pathway of neuronal apoptosis involving oxidative stress, perturbed calcium homeostasis and mitochondrial dysfunction ultimately converging on caspase activation (Zündorf and Reiser, 2010; Gibson et al., 2010).

In Alzheimer's disease, toxic forms of Aβ may induce Ca²⁺ influx into neurons by formation of an oligomeric pore in the plasma membrane, thereby rendering neurons vulnerable to excitotoxicity and apoptosis (Supnet and Bezprozvanny, 2010). Aβ also generates hydrogen peroxide and hydroxyl radicals, producing membrane lipid peroxidation and consequently membrane depolarization and excitotoxicity through NMDA receptor channels and voltage- gated Ca²⁺ channels (Sultana and Butterfield, 2010; Butterfield et al., 2010). In addition, Aβ accumulates in mitochondria, impairs the activity of complexes III and IV of the respiratory chain, causes elevated cytoplasmic Ca²⁺ levels and oxidative stress, and reduces ATP synthesis, further increasing Ca²⁺ overload and oxidative stress (Moreira et al., 2010).

Mitochondrial dysfunction is a defect occurring early in the pathogenesis of both sporadic and familial Parkinson's disease. Mitochondrial association of α-synuclein in cells was linked to impairment of respiratory complex I activity, oxidative modification of mitochondrial proteins, and increased levels of Ca²⁺ and nitric oxide (Navarro and Boveris, 2009).

Mitochondrial Ca²⁺ overload is also a decisive commitment step for Huntington's disease. Mutant huntingtin facilitates opening of the PTP and directly impairs the mitochondrial function. Furthermore, huntingtin forms a ternary complex with other proteins, which causes Ca²⁺ release from the ER and renders neurons more sensitive to Ca²⁺-mediated cellular dysfunction (Damiano et al., 2010).

The mutations in SOD responsible for ALS do not decrease antioxidant activity of the enzyme, but result in the gain of an adverse pro-apoptotic activity that may involve increased peroxidase activity. Through interactions with hydrogen peroxide or superoxide anion, the mutant enzyme may induce oxidative damage to membranes and disturbances in mitochondrial function that make neurons vulnerable to excitotoxic apoptosis (Vucic and Kiernan, 2009). Also, stroke and trauma, and the consequent brain ischemia, initiate biochemical and molecular events involving many of the same neurodegenerative cascades that occur in the chronic neurodegenerative diseases described above, namely excitotoxicity, calcium overload and oxidative stress (Arundine and Tymianski, 2004).

In conclusion, despite the concurrence of diverse genetic and environmental factors for acute and chronic neurodegenerative disorders, such conditions possess a common denominator as to the ultimate mechanisms executing neuronal death, which are identical to those involved in normal brain development and all converge to mitochondrial dysfunction. On one hand, these findings testify the evolutionary importance of conserving fundamental processes. On the other hand, they call for further research to understand the basic mechanisms and the key regulators of neuronal fate, as novel opportunities for targeted therapeutic intervention may arise.

Role of magnesium in neurodegenerative diseases

As detailed above, there is increasing evidence that neuronal death by apoptosis is associated with both acute and chronic neurodegenerative disorders. Interestingly, brain free magnesium levels have been shown to decline in a number of such pathologies (Vink et al., 2009). As a consequence, a considerable research effort has been directed toward establishing the mechanisms of such decline and the potential for magnesium administration as a neuroprotective agent.

Brain magnesium decline is a ubiquitous feature of traumatic brain injury and is associated with the development of motor and cognitive deficits. Experimentally, parenteral administration of magnesium up to 12 h post-trauma restores brain magnesium homeostasis and profoundly improves both motor and cognitive outcome. Although the mechanism of action is unclear, magnesium has been shown to attenuate a variety of secondary injury factors, including brain edema, cerebral vasospasms, glutamate excitotoxicity, calcium-mediated events, lipid per- oxidation, mitochondrial permeability transition, and apoptosis. Disappointingly, magnesium therapy has failed in clinical trials. Increase in brain free magnesium concentration seems to be essential to confer neuroprotection, and intravenous magnesium administration only marginally increases CSF magnesium concentration, which suggests that the integrity of the blood- brain barrier and the regulation of magnesium in the cerebrospinal fluid are largely maintained following acute brain injury and limit magnesium bioavailability in the brain (Vink and Nimmo, 2009).

Magnesium therapy has also been described in the clinical stroke literature, with similar negative results, though magnesium was shown to be beneficial in a subgroup of patients with noncortical, or lacunar, strokes (Ginsberg, 2008). Magnesium may protect via multiple mechanisms, including NMDA receptor blockade, inhibition of excitatory neurotransmitter release, blockade of calcium channels, as well as vascular smooth muscle relaxation, and may be more bioavailable, as it is well known that the blood brain barrier around infarcted tissue is highly permeable, thus potentially facilitating local magnesium entry to the injured tissues.

Intriguingly, the cation channel TRPM7, which is crucial for Mg²⁺ homeostasis and cell survival (Schmitz et al., 2003), seems to be a critical mediator of anoxic cell death. It appears that TRPM7 gating lies downstream of NMDA receptor-mediated NO free-radical production. In this paradigm, excitotoxicity is likely the initiating signal in a cell death cascade that involves nNOS activation with subsequent peroxynitrite production, which in turn activates TRPM7 channels. Calcium influx through TRPM7 then creates a positive feedback loop of ROS production, which eventually kills the cell (Aarts and Tymianski, 2005). TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function; also, it prevented ischemia-induced deficits in long-term potentiation and preserved performance in memory tasks (Sun et al., 2009). The role of the magnesium-specific channel TRPM6 in these processes is currently unclear, but, given its localization in the brain, its permeability to Mg²⁺ and its functional interaction with TRPM7, it may be involved in neuronal death as well (Cook et al., 2009). At present, the role of TRPM6/7 channels in magnesium transport seems less important than their facilitation of other cation fluxes, but it cannot be ruled out, especially considering the known involvement of magnesium in excitotoxicity and oxidative stress, as discussed earlier.

With respect to chronic brain degeneration, the first connection between magnesium and three apparently dissimilar neurodegenerative dis- orders (ALS, Parkinson's and Alzheimer's diseases) came from the observation that those conditions occurred at extraordinarily high rates in geographically separate foci among three genetically different, homogenous western Pacific populations (Durlach et al., 1997). Intensive research conducted over the years led to the identification of two candidate environmental factors: 1) severely low levels of Ca²⁺ and Mg²⁺ in the soil and drinking water coupled with abnormal mineral metabolism; and 2) the putative neurotoxin β-methylamino-L- alanine (L-BMAA), derived from the cycad plant, a traditional food source in Guam. Experimental findings supported this hypothesis: animals fed diets that mimic the mineral composition in the disease foci environment showed signs of neuronal damage; of the various combinations of Ca²⁺ and Mg²⁺ contents tested, exposure to low Mg²⁺ (one-fifth of the normal level) was more deleterious, causing significant loss of dopaminergic neurons in the substantia nigra (Oyanagi et al., 2006). Furthermore, motor neurons are selectively more vulnerable to L- BMAA toxicity, and these toxic effects are mediated through [Ca²⁺]_i rises via Ca-A/K channels and other pathways, and subsequent reactive oxygen species generation (Rao et al., 2006).

In the hyperendemic foci in the Western Pacific, the unique mineral composition of the environment creates a condition of increased oxidative stress because low Mg²⁺ levels cause lipid peroxidation while transition metals act as redox catalysts causing increased ROS production. Therefore, the genes whose function is most likely to be affected are ion channels expressed in the CNS and modulated by oxidative stress. TRPM7 and TRPM2 fulfil these conditions; their functional properties are interconnected with calcium and magnesium homeostasis, oxidative stress, mitochondrial dysfunction, and immune mechanisms, all principal suspects in neurodegeneration (Hermosura and Garruto, 2007). Indeed, variants of both genes have been found in Guamanian neurodegenerative disorders and may contribute to their pathogenesis (Hermosura et al., 2008; Hermosura et al., 2005).

Despite the evidence implicating magnesium in the aetiology of neurodegenerative diseases, the therapeutic effects of magnesium supplementation are not well documented. Magnesium administration proved beneficial in an in vitro model of Parkinson's disease, where in particular magnesium concentration seems critical in the onset of the disease (Hashimoto et al., 2008), but there is still much to be done to fully appreciate the therapeutic potential of magnesium.

Conclusion

An extensive and up-to-date review of the literature shows that drawing a clear-cut conclusion on the role of magnesium in neuroapoptosis is not an easy task. Magnesium may play pleiotropic roles in different stages of the apoptotic process, that range from modulating growth factor- and Ca²⁺-dependent signal transduction to affecting oxidative stress- related reactions. From this point of view, increased extracellular magnesium availability should in principle be considered anti- apoptogenic. On the other hand, an increase in cytosolic free magnesium, which seems to be released from mitochondrial stores, appears to accompany the intrinsic apoptotic process. The pathophysiological meaning of this occurrence in the context of apoptosis induction/execution awaits to be defined (see Figure 1 for a summary).

Figure 1.

Potential points of interaction between magnesium and apoptotic factors.

In a more physiological basis, magnesium availability in the brain should be considered within a more complex and interconnected picture, where other conditions, such as developmental stage, inflammation, acute or chronic injuries, blood brain barrier integrity, and so on, may concur in determining a pro or anti- apoptotic environment.

We believe that further investigation into the role of magnesium in the molecular mechanisms of apoptosis is warranted for a deeper understanding of the basic processes that regulate cell fate, and ultimately for a successful therapeutic intervention when these processes go awry.

Acknowledgments

Supported by the Italian Ministry of Education, University and Research PRIN 2007ZT39FN and Linea D1 2004-2009.

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