Excitotoxic damage to white matter

Abstract

Glutamate kills neurons by excitotoxicity, which is caused by sustained activation of glutamate receptors. In recent years, it has been shown that glutamate can also be toxic to white matter oligodendrocytes and to myelin by this mechanism. In particular, glutamate receptor-mediated injury to these cells can be triggered by activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-d-aspartate glutamate receptor types. Thus, these receptor classes, and the intermediaries of the signal cascades they activate, are potential targets for drug development to treat white matter damage in acute and chronic diseases. In addition, alterations of glutamate homeostasis in white matter can determine glutamate injury to oligodendrocytes and myelin. Astrocytes are responsible for most glutamate uptake in synaptic and non-synaptic areas and consequently are the major regulators of glutamate homeostasis. Activated microglia in turn may secrete cytokines and generate radical oxygen species, which impair glutamate uptake and reduce the expression of glutamate transporters. Finally, oligodendrocytes also contribute to glutamate homeostasis. This review aims at summarizing the current knowledge about the mechanisms leading to oligodendrocyte cell death and demyelination as a consequence of alterations in glutamate signalling, and their clinical relevance to disease. In addition, we show evidence that oligodendrocytes can also be killed by ATP acting at P2X receptors. A thorough understanding of how oligodendrocytes and myelin are damaged by excitotoxicity will generate knowledge that can lead to improved therapeutic strategies to protect white matter.

Introduction

Excitotoxic cell death can occur in virtually all neurons which express ionotropic glutamate receptors (GluRs) and it has been implicated in acute injury to the central nervous system (CNS) and in chronic neurodegenerative disorders (Choi, 1988; Lipton & Rosenberg, 1994; Lee et al. 1999). In addition, glutamate can also be toxic to glial cells including astrocytes (Haas & Erdo, 1991) and oligodendrocytes (Yoshioka et al. 1995; Matute et al. 1997; McDonald et al. 1998). This review will focus on excitotoxicity in oligodendrocytes, as this cell type is particularly vulnerable to glutamate insults. We will first summarize current knowledge of GluRs and glutamate transporters (GluTs) and then the mechanisms of glutamate excitoxicity to white matter and its relevance to CNS diseases.

Glutamate signalling in oligodendrocytes

Glutamate signalling is carried out by GluRs and GluTs. Glutamate activates ionotropic and metabotropic receptors (for reviews, see Dingledine et al. 1999; Cull-Candy & Leszkiewicz, 2004; Swanson et al. 2005). Glial cells express most of these receptors (for recent reviews see, Belachew & Gallo, 2004; Kettenmann & Steinhäuser, 2005; Matute et al. 2006; Matute, 2006). In particular, cells of the oligodendrocyte lineage express functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate-type receptors throughout a wide range of developmental stages and species, including humans (Figs 1 and 2). In addition, immature and mature oligodendrocytes express N-methyl-d-aspartate (NMDA) receptors which can be activated during injury (Karadottir et al. 2005; Salter & Fern, 2005; Micu et al. 2006). Moreover, oligodendrocytes also express receptors of all three groups of metabotropic GluRs but their levels are developmentally regulated and are very low in mature cells of this lineage (Deng et al. 2004).

Fig. 1.

Functional AMPA and kainate receptors in cultured human oligodendrocytes. (A) Cells originated from human cerebral cortex excised at surgery for tumour removal and were immunostained for the oligodendrocyte marker O1. Scale bar = 20 µm. (B) Sample whole-cell recordings of O1⁺ cells of the responses to AMPA and kainate (both at 1 mm). GYKI53655 (100 µm) was added in conjunction with kainate to activate selectively kainate receptors. (C) Increase in [Ca²⁺]_i in oligodendrocytes exposed to AMPA (10 µm) with CTZ (100 µm) and kainate (3 µm) (arrow) in the presence of GYKI53655 (100 µm) from two to five O1⁺ cells.

Fig. 2.

Expression of AMPA and kainate receptors in oligodendrocytes of the human optic nerve. Double immunofluorescence labelling of AMPA (GluR2/3) and kainate (GluR5/6/7) receptor subunits in oligodendrocytes of the human optic nerve. Green and red fluorescence reveal subunit localization and oligodendrocyte labelling by the anti-APC antibody, an oligodendroglial marker, respectively. Arrows point to some double immunolabelled oligodendrocytes. Scale bar = 20 µm.

Glutamate uptake from the extracellular space by specific GluTs is essential for the shaping of excitatory postsynaptic currents and for the prevention of excitotoxic death due to overstimulation of GluRs (Rothstein et al. 1996). At least five GluTs have been cloned (Danbolt, 2001; Huang & Bergles, 2004). Of these, glutamate transporter 1 [GLT-1; excitatory amino acid transporter 2 (EAAT2) in the modern nomenclature] exhibits the highest level of expression and is responsible for most glutamate transport (Danbolt, 2001). GluTs are expressed by astrocytes and oligodendrocytes. The main transporter expressed by oligodendrocytes is glutamate aspartate transporter (GLAST; EAAT1 in the modern nomenclature; Fig. 3). The neuronal transporter, termed excitatory amino acid carrier 1 (EAAC1; EAAT3 in the modern nomenclature), is present in a subpopulation of adult oligodendrocyte progenitor cells (Domercq et al. 1999). It thus appears that all macroglial cells differentially express the three major GluTs present in the CNS. These transporters maintain basal levels of extracellular glutamate in the range of 1–2 µm and thus prevent over-activation of GluRs under physiological conditions.

Fig. 3.

Expression of glutamate transporter EAAT1 in oligodendrocytes of the human optic nerve. Photomicrographs depict same fields in each row with immunofluorescence labelling of EAAT1 transporter and of APC⁺ oligodendrocytes and GFAP⁺ astrocytes. Overlays (right column) show that EAAT1 co-localizes with the oligodendrocyte marker APC but not with GFAP⁺ cells. Arrows point to same somata in each row of photographs. Scale bar = 30 µm. Abbreviations: APC, adenomatous polyposis coli antigen; GFAP, glial fibrillary acidic protein. Modified from Vallejo-Illarramendi et al. (2006).

Oligodendrocyte vulnerability to excitotoxic insults by glutamate

Numerous studies carried out over the last few years have shown that, in addition to neurons, glial cells can die by excitotoxicity. The glial cell types which are most vulnerable to excitotoxicity are those of the oligodendrocyte lineage. However, there is evidence that sustained activation of ionotropic GluRs can also kill astrocytes and microglia.

The first evidence that oligodendrocytes are highly vulnerable to glutamate was obtained in primary cultures more than 10 years ago (Oka et al. 1993). After a 24-h exposure to glutamate, oligodendroglial death was comparable with that described in neurons. However, oligodendroglial toxicity was not mediated by GluRs, as in neurons, but rather by a transporter-related mechanism involving the inhibition of cystine uptake, which results in glutathione depletion and cellular vulnerability to toxic-free radicals (Oka et al. 1993). More recently, it was shown that prolonged activation of GluRs is toxic to cells of an oligodendroglial cell line (Yoshioka et al. 1996) and to oligodendrocytes in vitro and in vivo (Matute et al. 1997; McDonald et al. 1998; Li & Stys, 2000). This toxicity is directly related to Ca²⁺ influx subsequent to receptor activation, and it is greatly attenuated in the absence of Ca²⁺ in the culture medium (Sánchez-Gómez & Matute, 1999).

Glutamate can also cause glial demise indirectly by inducing the release of toxic agents. In microglia, activation of AMPA and kainate receptors results in the release of tumour necrosis factor-α (TNF-α), which can potentiate glutamate neurotoxicity and kill oligodendrocytes, destroy myelin and damage axons (Merrill & Benveniste, 1996). In turn, stimulation of the mGluR2 receptor on primary rat microglia induces microglial activation and results in a neurotoxic phenotype which secretes TNF-α (Taylor et al. 2005). This toxicity can be neutralized by the activation of the mGluR3 receptor, which is also present in microglia (Taylor et al. 2005). Moreover, inflammatory cytokines including TNF-α and interleukin-1β, which are commonly released by reactive microglia, can impair glutamate uptake and trigger excitotoxic oligodendrocyte death (Takahashi et al. 2003). Indeed, inhibition of the expression and functioning of GluTs in axonal tracts is sufficient to induce oligodendroglial loss and demyelination, which demonstrates that the integrity of oligodendrocytes and white matter depends on proper GluT function (Domercq et al. 2005).

Mechanisms of glutamate damage to oligodendrocytes

Activation of AMPA, kainate or NMDA receptors in oligodendrocytes leads to Ca²⁺ influx (Fig. 4), an effect which is totally abolished by selective receptor antagonists or by removing this cation from the culture medium. The mechanisms triggered by NMDA receptor-mediated insults to oligodendrocytes have not yet been clarified. However, the types of excitotoxic oligodendrocyte death induced by activation of AMPA and kainate receptors are known to depend on the intensity and duration of glutamate exposure. A central event to this process is accumulation of Ca²⁺ within mitochondria, which leads to the depolarization of this organelle, increased production of oxygen free radicals, and release of proapoptotic factors which activate caspases (see Fig. 4).

Fig. 4.

Molecular events triggered by glutamate receptor-mediated oligodendrocyte demise. Selective activation of AMPA receptors (AMPA-R) and kainate receptors (Kai-R) leads to Na⁺ and Ca²⁺ influx through the receptor channel complex. Subsequent depolarization activates voltage-gated Ca²⁺ channels (VGCC), which contributes to [Ca²⁺]_i increase. Ca²⁺ overload induces rapid uptake by mitochondria, which results in attenuation of mitochondrial potential and an increase in the production of reactive oxygen species (ROS). Cytochrome c (Cyt c) is released from depolarized mitochondria, interacts with apoptotic protease-activating factor 1 (Apaf-1) and activates caspases. Other pro-apoptotic factors include apoptosis-inducing factor (AIF), which activates poly(ADP-ribose)polymerase-1 (PARP-1). In oligodendrocytes, insults channelled through Kai-R activate caspases 9 and 3, whereas those activating AMPA-R induce apoptosis by recruiting caspase 8, which truncates Bid, caspase 3 and PARP-1, or cause necrosis. In addition, Ca²⁺ influx triggered by Kai-R stimulation but not by AMPA-R activates calcineurin (CdP), which dephosphorylates Bad and facilitates apoptosis. Finally, activation of NMDA receptors (NMDA-R) also initiates oligodendrocyte death, which is entirely dependent on Ca²⁺ influx; however, the molecular mechanisms activated by these receptors are not yet known. Abbreviations: FADD, Fas-associated death domain; 14-3-3, phosphoserine-binding protein 14-3-3. Scheme based on Sánchez-Gómez et al. (2003).

Glutamate at non-toxic concentrations can also induce oligodendrocyte death by sensitizing these cells to complement attack (Alberdi et al. 2006). Thus, a brief incubation with glutamate followed by exposure to complement is lethal to oligodendrocytes in vitro and in freshly isolated optic nerves (Fig. 5A). Intriguingly, complement toxicity is induced by activation of kainate, but not of AMPA, NMDA or metabotropic GluRs, and is abolished by removing Ca²⁺ from the medium during glutamate priming. Dose–response studies show that sensitization to complement attack is induced by two distinct kainate receptor populations displaying high and low affinities for glutamate. Oligodendrocyte death by complement required the formation of the membrane attack complex, which in turn increased membrane conductance, induced Ca²⁺ overload and mitochondrial depolarization as well as a rise in the level of reactive oxygen species. Treatment with the antioxidant Trolox and inhibition of poly(ADP-ribose) polymerase-1, but not of caspases, protected oligodendrocytes against damage induced by complement (Fig. 5B,C). This novel mechanism of glutamate-induced toxicity to oligodendrocytes is also shared by neurons and may be relevant to glutamate injury in acute and chronic neurological disease with primary or secondary inflammation.

Fig. 5.

Glutamate sensitizes cultured oligodendrocytes to complement attack by activating kainate receptors. (A) Oligodendrocyte viability (green calcein fluorescence) and death (propidium iodide red fluorescence) 24 h after pretreatment with vehicle or glutamate (Glu; 10 µm) for 10 min, followed by exposure to culture medium or complement (LTC). The histogram on the right illustrates oligodendrocyte death. Note that the culture medium alone (SATO) and heat-inactivated LTC (HI-LTC) were ineffective. *P < 0.01 compared with SATO, paired Student's t-test. Scale bar = 40 µm. (B) Complement causes oxidative stress in glutamate (Glu)-sensitized oligodendrocytes. * and #: P < 0.05 compared with vehicle + LTC or Glu-pre + LTC, respectively, paired Student's t-test. (C) PARP-1 inhibition with DPQ (30 µm) reduces cell death, whereas the pan-caspase inhibitor ZVAD-F (50 µm) was ineffective. In turn, the antioxidant Trolox (10 µm) fully protected oligodendrocytes from LTC toxicity. *P < 0.05, **P < 0.01 compared with Glu pre + LTC, paired Student's t-test. Modified from Alberdi et al. (2006).

Clinical relevance of glutamate damage to white matter

In humans white matter constitutes about 50% of the brain volume and consequently glutamate-induced oligodendrocyte death is highly relevant to the pathophysiology of CNS diseases. In addition, primary and/or secondary glutamate damage to oligodendroglia in grey matter may also contribute to the onset and progression of acute and chronic brain and spinal cord disorders. Thus, loss of oligodendrocytes and/or damage to white matter occurs in stroke, traumatic injury, neurodegenerative diseases, multiple sclerosis (MS) (Matute et al. 2006) as well as in psychiatric diseases (Davis et al. 2003). Here, we will only illustrate the putative relevance of oligodendrocyte excitotoxicity to ischaemia [stroke and periventricular leukomalacia (PVL)] and MS.

Stroke

In humans, most cases of focal ischaemia and occlusion of major cerebral arteries damage both grey and white matter. Energy deprivation causes neuronal death, axonal dysfunction and loss of oligodendrocytes which are very sensitive to transient oxygen and glucose deprivation (Dewar et al. 2003; Goldberg & Ransom, 2003; Stys, 2004). After 1 h under these conditions, the viability of oligodendrocytes in mixed glial cultures is severely impaired, an effect which is attenuated by AMPA/kainate antagonists (McDonald et al. 1998). In turn, immature oligodendrocytes are even more sensitive to ischaemic injury than their mature counterparts (Fern & Möller, 2000). Thus, these cells may release glutamate under ischaemic conditions by reverse functioning of their GluTs (Domercq et al. 1999, 2005).

In vivo models of stroke and cardiac arrest such as permanent middle cerebral artery occlusion and brief transient global ischaemia induce rapid oligodendroglial death (Mandai et al. 1997; Petito et al. 1998). Interestingly, a few days after the insult, there is an increase in the number of oligodendroglial cells in areas bordering affected regions (Mandai et al. 1997), as well as in the number of immature oligodendrocytes surrounding the lateral ventricles (Gottlieb et al. 2000), indicating that ischaemic damage to oligodendroglia can be compensated for, at least in part, by the generation and migration of new oligodendrocytes.

Preterm and perinatal ischaemia

PVL, the main substrate for cerebral palsy, is characterized by diffuse injury of deep cerebral white matter, accompanied in its most severe form by focal necrosis. The classical neuropathology of PVL has given rise to several hypotheses about its pathogenesis, largely relating to hypoxia-ischaemia and reperfusion in the sick premature infant. These include free radical injury, cytokine toxicity (especially given the epidemiological association of PVL with maternofetal infection) and excitotoxicity (Folkerth, 2006).

Injury to oligodendrocyte progenitors, caused in part by glutamate and the subsequent derailment of Ca²⁺ homeostasis, contributes to the pathogenesis of myelination disturbances in this illness (Back & Rivkees, 2004). In addition to this mechanism, glutamate-induced depletion of glutathione and the subsequent oxidative stress in PVL also contributes to damage to oligodendrocytes (Haynes et al. 2003), which are sensitive to oxidative stress in part because of their high lipid and iron content. Notably, the vitamin K deficiency in preterm infants is a risk factor for developing PVL, and in turn its presence is protective against oxidative injury to immature oligodendrocytes (Li et al. 2003).

Multiple sclerosis

In MS, the immune system attacks the white matter of the brain and spinal cord, leading to disability and/or paralysis. Myelin and oligodendrocytes are lost due to the release by immune cells of cytotoxic cytokines, autoantibodies and toxic amounts of glutamate (Matute et al. 2001; Srinivasan et al. 2005). Excitotoxins such as kainate infused onto the optic nerve cause glutamate receptor-mediated MS-like lesions (Matute, 1998). In turn, experimental autoimmune encephalomyelitis (EAE), an animal model which exhibits the clinical and pathological features of MS, is alleviated by AMPA and kainate receptor antagonists (Pitt et al. 2000; Smith et al. 2000; Groom et al. 2003). Remarkably, blockade of these receptors in combination with anti-inflammatory agents is effective even at an advanced stage of unremitting EAE, as assessed by increased oligodendrocyte survival and remyelination, and corresponding decreased paralysis, inflammation, CNS apoptosis and axonal damage (Kanwar et al. 2004).

The concentration of glutamate in cerebrospinal fluid (CSF) is higher in patients with acute rather than silent MS and in controls (Stover et al. 1997) and it is associated with the severity and course of the disease (Barkhatova et al. 1998). Notably, glutamate levels are increased in acute MS lesions and in normal-appearing white matter in MS patients (Srinivasan et al. 2005). Potential cellular sources contributing to enhanced glutamate levels in CSF include activated microglia, which can release glutamate via the reversal of GluT function, a process which is potentiated under pathological conditions (Noda et al. 1999). In addition, oxidative stress may also contribute to the increase in glutamate concentrations in the extracellular space, as free radicals reduce the efficiency of GluTs (Volterra et al. 1994). Other factors which may contribute to perturbing glutamate homeostasis include altered activity of the glutamate-producing enzyme glutaminase in activated macrophages/microglia in close proximity to dystrophic axons (Werner et al. 2001), and altered expression of the glutamate transporters EAAT-1 and EAAT-2 in oligodendrocytes as a consequence of enhanced exposure to the proinflammatory cytokine TNFα (Pitt et al. 2003; Vallejo-Illarramendi et al. 2006). Overall, these alterations probably lead to high extracellular glutamate levels and an increased risk of oligodendrocyte excitotoxicity in MS.

ATP can also induce oligodendrocyte death

Like glutamate, extracellular ATP is a major excitatory neurotransmitter in the CNS, activating ionotropic (P2X) and metabotropic (P2Y) receptors (Ralevic & Burnstock, 1998; North, 2002). ATP-gated P2X channels are formed by P2X1–P2X7 subunits and have marked Ca²⁺ permeability (North, 2002). P2X receptors are expressed in CNS neurons, where they participate in fast synaptic transmission and modulation. In addition, P2X receptors mediate signalling cascades leading to neurodegeneration after ischaemia (Le Feuvre et al. 2003). Recently, it has also been shown that spinal cord injury is associated with prolonged P2X7 receptor activation, which results in neuronal excitotoxicity (Wang et al. 2004).

Differentiated oligodendrocytes in vitro express functional P2X and P2Y receptors, the former with high permeability to Ca²⁺ (Alberdi et al. 2005). As a result, we tested whether activation of these receptors can kill these cells. Incubation of oligodendrocytes with ATP or P2X agonists such BzATP, but not P2Y agonists, was toxic to oligodendrocytes (Fig. 6). Toxicity was prevented by removal of Ca²⁺ from the culture media, and by the non-selective P2X receptor antagonist PPADS, as well as by oATP and BBG at concentrations that selectively block P2X7 receptors (Fig. 6). This is consistent with a predominant expression of P2X7 receptors in differentiated oligodendrocytes, as shown by immunochemistry (Matute et al. 2003).

Fig. 6.

Activation of purinergic P2X receptor kills oligodendrocytes in vitro. Acute exposure to ATP (A) or to BzATP (B) induces cell death of differentiated oligodendrocytes obtained from optic nerves of 14-day-old rats. This is prevented by co-application of the broad-spectrum P2 antagonist PPADS, the P2X7 antagonist O-ATP and by omitting Ca²⁺ in the culture medium. Data represent the means ± SEM. *P < 0.05 and **P < 0.001, one-way anova, Fischer's PDLS test.

The relevance of ATP excitoxicity to CNS damage to the aetiology of acute and chronic diseases is unknown, but the release of this neurotransmitter from dying cells may well contribute to aggravate the extent of ongoing damage in numerous pathological conditions.

Concluding remarks

Oligodendrocytes display great vulnerability to over-activation of AMPA, kainate and NMDA receptors. The proper functioning of glutamate uptake is critical to prevent glutamate-induced damage to oligodendrocytes, and drugs that regulate the function and expression of GluTs have the potential to attenuate glutamate insults to glial cells. Likewise, positive regulators of the expression of GluTs also have a protective potential, as they contribute to ischaemic tolerance after ischaemic preconditioning (Romera et al. 2004). These include tumour growth factor-α and epidermal growth factor (EGF), which by signalling through EGF receptors and activation of phosphoinositol-3-kinase and nuclear factor-κB, strongly enhance EAAT2 expression (Su et al. 2003). Remarkably, clinically used beta-lactam antibiotics are also potent activators of GluT expression and thus hold great therapeutic potential (Rothstein et al. 2005).

Another set of molecular targets to prevent glutamate insults to oligodendrocytes lie downstream of GluRs activation. For instance, tetracyclines, which attenuate mitochondrial damage subsequent to insults including excitotoxicity, protect oligodendrocytes and white matter, making these antibiotics promising candidates for the treatment of acute and chronic diseases with oligodendrocyte loss (Domercq & Matute, 2004). By contrast, drugs supporting the management of Ca²⁺ overload subsequent to GluRs and P2X receptor activation may improve oligodendrocyte viability.

In summary, knowledge about the mechanisms leading to glutamate and ATP receptor-mediated oligodendrocyte injury will facilitate new pharmacological strategies for the treatment of CNS disorders which cause white matter damage.