The Role of Excitotoxicity in the Pathogenesis of Amyotrophic Lateral Sclerosis ⁎ L

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Biochimica et Biophysica Acta 1762 (2006) 1068–1082 www.elsevier.com/locate/bbadis

Review The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis ⁎ L. Van Den Bosch , P. Van Damme, E. Bogaert, W. Robberecht

Neurobiology, Campus Gasthuisberg O&N2, PB1022, Herestraat 49, B-3000 Leuven, Belgium Received 21 February 2006; received in revised form 4 May 2006; accepted 10 May 2006 Available online 17 May 2006

Abstract

Unfortunately and despite all efforts, amyotrophic lateral sclerosis (ALS) remains an incurable neurodegenerative disorder characterized by the progressive and selective death of motor neurons. The cause of this process is mostly unknown, but evidence is available that excitotoxicity plays an important role. In this review, we will give an overview of the arguments in favor of the involvement of excitotoxicity in ALS. The most important one is that the only drug proven to slow the disease process in humans, riluzole, has anti-excitotoxic properties. Moreover, consumption of excitotoxins can give rise to selective motor neuron death, indicating that motor neurons are extremely sensitive to excessive stimulation of glutamate receptors. We will summarize the intrinsic properties of motor neurons that could render these cells particularly sensitive to excitotoxicity. Most of these characteristics relate to the way motor neurons handle Ca2+, as they combine two exceptional characteristics: a low Ca2+-buffering capacity and a high number of Ca2+-permeable AMPA receptors. These properties most likely are essential to perform their normal function, but under pathological conditions they could become responsible for the selective death of motor neurons. In order to achieve this worst- case scenario, additional factors/mechanisms could be required. In 1 to 2% of the ALS patients, mutations in the SOD1 gene could shift the balance from normal motor neuron excitation to excitotoxicity by decreasing glutamate uptake in the surrounding astrocytes and/or by interfering with mitochondrial function. We will discuss point by point these different pathogenic mechanisms that could give rise to classical and/or slow excitotoxicity leading to selective motor neuron death. © 2006 Elsevier B.V. All rights reserved.

Keywords: ALS; Neurodegeneration; Motor neuron; Ca2+ metabolism; Glutamate; AMPA receptors; GluR2

1. What is excitotoxicity? contribute to neuronal damage in stroke, neurotrauma, epilepsy, and a number of neurodegenerative disorders including During glutamatergic neurotransmission, glutamate released amyotrophic lateral sclerosis (ALS) [2–4]. from the presynaptic neuron activates ionotropic glutamate Rather artificially, two types of excitotoxicity are distin- receptors present on the postsynaptic neuron (Fig. 1A). guished and denoted with the terms classical and slow Activation of these glutamate receptors results in the influx of excitotoxicity [4]. Classical excitotoxicity refers to neuronal Na+ and Ca2+ ions into the cell, leading to depolarization and degeneration that occurs after an increase of the extracellular ultimately to the generation of an action potential. Excitotoxi- glutamate concentration (Fig. 1B), while slow excitotoxicity is city is neuronal degeneration caused by over-stimulation of the the death of a weakened postsynaptic neuron in the presence of glutamate receptors. This concept was formulated in 1978 by normal synaptic glutamate levels (Fig. 1C). Olney [1] and was based on the observation that those amino In order to obtain classical excitotoxicity, an elevation of the acids that induce neuronal death were the ones known to extracellular glutamate concentration to 2–5 μM is considered activate excitatory amino acid receptors. Since then, experi- sufficient to cause degeneration of neurons through excessive mental evidence became available that excitotoxicity could stimulation of glutamate receptors [5,6]. Acute elevations of glutamate are thought to induce neuronal damage in conditions ⁎ Corresponding author. Tel.: +32 16 34 57 85; fax: +32 12 33 07 70. such as stroke, status epilepticus and neurotrauma. More E-mail address: [email protected] chronic and milder elevations of glutamate are believed to (L. Van Den Bosch). underly excitotoxicity in neurodegenerative diseases. Elevated

Fig. 1. Glutamatergic neurotransmission and excitotoxicity. Under normal conditions, glutamate released from the presynaptic neuron activates the NMDA and AMPA receptors (R). This results in an influx of both Na+ and Ca2+ ions, the depolarization of the postsynaptic neuron and ultimately in an action potential. (B) Classical excitotoxicity is induced by an elevation of the extracellular glutamate concentration. This can be caused by an increased release of glutamate or a deficient re-uptake of glutamate into the astrocytes by the EAAT2/GLT-1 transporter. The excessive stimulation of the glutamate receptors gives rise to an increased intracellular concentration of Na+ and Ca2+ ions and this can result in neuronal death. The disintegration of neuronal cells causes a further increase of extracellular glutamate and amplifies the excitotoxic damage. (C) Slow excitotoxicity is characterized by the fact that the postsynaptic neuron becomes more sensitive to glutamate stimulation, while the extracellular glutamate concentration is not increased. This sensitization of the neuron can be caused by changes in the properties of the glutamate receptors, resulting in a higher increase of the intracellular Ca2+ concentration. Alternatively, the vulnerability of neurons to normal glutamate stimulation can be increased due to mitochondrial damage, resulting in energetically compromised neurons. extracellular glutamate concentrations can occur when the [9,10]: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole pro- release from presynaptic terminals is augmented or when the re- pionic acid), NMDA (N-methyl-D-aspartate) and KA (kainate) uptake from the synaptic cleft is insufficient. This re-uptake is receptors. Although, KA has a high affinity for KA receptors assured by glutamate transporters present in neurons and resulting in rapidly desensitizing currents, it is mostly used as an astrocytes. By far the most important glutamate transporter is agonist of AMPA receptors since it also induces non- EAAT2/GLT-1 as it is widely expressed in astrocytes through- desensitizing currents through these receptors. Functionally, out the central nervous system (CNS) and as it has the highest AMPA receptors are the most important glutamate receptors to affinity for glutamate. Elevated glutamate concentrations can mediate fast excitatory transmission. On the other hand, NMDA also arise if the intracellular glutamate content is released from receptors mediate the late component of excitatory transmission injured neurons. This release can result in excitotoxic death of [11] and play a key role in the induction of synaptic plasticity surrounding neurons and as a consequence could be involved in [12]. The role of KA receptors in physiological and pathological the spread of the neurodegeneration. conditions is less clear. In contrast to the classical excitotoxicity, no upregulation of At first, the NMDA receptor was considered to be uniquely the synaptic glutamate concentration is needed to cause slow responsible for excitotoxicity [13]. More recently, it became excitotoxicity. In this case, normal glutamate receptor stimula- apparent that the activation of AMPA receptors is at least as tion of a weakened postsynaptic neuron is sufficient to damage important [14]. The predominant mediator of neuronal injury is and kill the cell. A well known example that could result in this Ca2+ influx through NMDA receptors, Ca2+-permeable AMPA form of excitotoxicity is the disturbance of the mitochondrial receptors or voltage-gated Ca2+ channels [15–17]. How Ca2+ function resulting in an impaired energy state of the neuron. entry can induce neuronal death is not yet completely This causes a drop in the intracellular ATP level. As a understood, but several possible mechanisms have been consequence, the neuron cannot longer meet the demands of reported. Excessive influx of Ca2+ ions can result in the its many ATP dependent processes, becomes damaged and activation of several enzymes, such as lipases, phospholipases, ultimately dies [7,8]. proteases, endonucleases, protein phosphatases, protein kinase In both forms of excitotoxicity, the glutamatergic neuro- C, xanthine oxidase and NO synthase. In addition, mitochon- transmission plays a pivotal role. Glutamate released from the drial dysfunction due to increased Ca2+ uptake in mitochondria presynaptic neuron activates different types of glutamate and subsequent formation of reactive oxygen species could also receptors present on the postsynaptic neuron. These glutamate contribute to excitotoxic cell death [18–20]. receptors are divided into ionotropic and metabotropic recep- While NMDA receptors consist of NR1 and NR2(A–D) tors. The ionotropic glutamate receptors are ligand-gated cation subunits, AMPA receptors are tetramers composed of a variable channels, while the metabotropic glutamate receptors are G- association of four subunits (GluR1–4) [21–25]. Both NMDA protein coupled receptors that influence second messenger and AMPA receptors are ligand-gated cation channels that systems. The ionotropic receptors can further be subdivided into permit the influx of Na+ and the efflux of K+. In addition, the three classes according to their preferred synthetic agonist NMDA receptor is always Ca2+ permeable, while he AMPA 1070 L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 receptor is permeable for Ca2+ to a variable degree. The Ca2+ against disease progression in patients, has anti-excitotoxic permeability of the AMPA receptor is determined by the properties [36,37]. Riluzole slows the disease progression and absence/presence of the GluR2 subunit in the receptor complex significantly increases survival by a few months. It was shown (Fig. 2). Receptors containing GluR2 have a very low Ca2+ that this drug inhibits the release of glutamate due to the permeability compared to GluR2-lacking receptors [26]. The inactivation of voltage-dependent Na+ channels on glutamater- impermeability to Ca2+ of GluR2-containing AMPA receptors gic nerve terminals, as well as to activation of a G-protein- is due to the presence of a positively charged arginine at dependent signal transduction process [38]. Moreover, evidence position 586 (Q/R site) of the GluR2 peptide instead of the exists that riluzole can also block some of the postsynaptic genetically encoded neutral glutamine [27–29]. This arginine effects of glutamate by non-competitive inhibition of NMDA residue at the Q/R site is introduced by the editing of GluR2 pre- and AMPA receptors [38,39]. In addition, postsynaptic effects mRNA [30], which is virtually complete in most cell types. of riluzole on the γ-aminobutyric acid A (GABAA) receptor Under pathological conditions, changes in subunit composition have been described that could also contribute to its protective can dramatically affect neuronal survival. The best studied effect [40,41]. paradigm is the downregulation of GluR2 induced by ischemia, We will start this part with an overview of the intrinsic which results in enhanced Ca2+ influx and neuronal degener- properties of motor neurons that could make these cells more ation in vulnerable regions of the brain [31,32]. vulnerable to excitotoxicity. In the second and third part, we will discuss the arguments in favor of the involvement of classical 2. Excitotoxicity and ALS and slow excitotoxicity in the selective motor neuron death during ALS. In this review, we will focus on the role of excitotoxicity in the pathology of ALS. This neurodegenerative disease is 2.1. Selective vulnerability of motor neurons characterized by the progressive and selective loss of motor neurons in the motor cortex, the brainstem and the spinal cord. Several lines of evidence indicate that motor neurons, both in This degeneration of motor neurons results in a progressive vitro and in vivo, are particularly vulnerable to AMPA receptor- paresis of bulbar, respiratory and limb muscles and leads to the mediated excitotoxicity. Intrathecal or intraspinal administra- death of the patients usually 2 to 5 years after the diagnosis. tion of AMPA receptor agonists induced motor neuron Familial ALS occurs in 5 to 10% of cases and predominantly degeneration in animals, while NMDA failed to damage spinal has an autosomal dominant inheritance. In 20% of familial motor neurons [42–48]. Selective loss of motor neurons was cases, mutations in the superoxide dismutase-1 (SOD1) gene on induced, especially when KA was used [43,48]. chromosome 21q were identified [33]. Mutations in SOD1 give In organotypic rat spinal cord cultures (spinal cord slices rise to a toxic ‘gain of function’ of the mutated protein. from postnatal rats, which can be kept in culture for several Transgenic mice and rats that overexpress mutant SOD1 weeks), motor neurons also proved to be particularly vulnerable develop an age-dependent, selective motor neuron death to AMPA receptor-mediated excitotoxicity [49,50].Direct [34,35]. application of AMPA receptor agonists resulted in selective The most important argument for a role of excitotoxicity in motor neuron loss, which could be prevented by antagonists of ALS is that riluzole, the only drug which proved effective AMPA receptors.

Fig. 2. The Ca2+ permeability of AMPA-type glutamate receptors is determined by the presence or absence of GluR2 subunit in the receptor complex. (A) In response to glutamate (Glt) released from the presynaptic neuron, ionotropic AMPA receptors are activated. Most AMPA receptors only allow Na+ to enter the cell, while a special type of AMPA receptors is permeable for incoming Na+ as well as Ca2+ ions. (B) Detailed representation of a Ca2+-permeable (left) and a Ca2+-impermeable AMPA receptor (right). If the receptor complex is composed of a combination of GluR1-3-4, it is permeable to Ca2+ ions. If at least one GluR2 subunit is present in the receptor complex, it is impermeable to Ca2+. This is due to a positively charged arginine at position 586 that is only present in the GluR2 subunit if the GluR2 mRNA is edited. Under normal conditions this editing process that changes a CAG codon into a CGG codon is highly efficient and affects all GluR2 mRNAs. L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 1071

Similarly, it was demonstrated that motor neurons in culture expressing mice were shown to be more resistant to excitotoxic are susceptible to glutamate receptor agonists, especially those stimuli. These cells showed a lower increase of intracellular stimulating AMPA receptors [16,51–55]. Using motor neurons Ca2+ and an increased survival after AMPA receptor cultured on an astrocytic feeder layer, we showed that Ca2+ stimulation compared to normal motor neurons [66]. Cross- influx through Ca2+-permeable AMPA receptors was crucial for breeding of parvalbumin overexpressing mice with SOD1G93A inducing motor neuron death [17,56]. Other neurons were mice delayed disease onset with 17% and prolonged survival resistant to AMPA receptor over-stimulation [17]. Little or no by 11% [67]. effect of agonists of NMDA receptors was apparent in these A direct consequence of the lower amount of Ca2+-buffering cultured motor neurons [57], Moreover, depolarizing these proteins in motor neurons is that mitochondria have to play a motor neurons by exposing them to a high extracellular K+ more prominent role in Ca2+ buffering in these cells (Fig. 3A). concentration was not sufficient to induce significant cell death We found evidence for activation of mitochondrial Ca2+ [56]. As illustrated in Fig. 3, motor neurons seem to have a buffering during and after the inward current through AMPA number of intrinsic properties related to their Ca2+ handling that receptors, which saturated on repetitive KA application and lead makes them particularly vulnerable to AMPA receptor mediated to a persistent increase in cytosolic Ca2+ concentration in motor excitotoxicity and that we will discuss below. neurons, but not in other neurons [68]. Moreover, evidence exists that excessive entry of Ca2+ into motor neurons results in 2.1.1. Low Ca2+-buffering capacity and role of mitochondria mitochondrial Ca2+ overload leading to depolarization and in The capacity of motor neurons to buffer increases in the generation of reactive oxygen species, at least in vitro [19]. intracellular Ca2+ is limited due to the low expression level of Ca2+-buffering proteins (Fig. 3). This low Ca2+-buffering 2.1.2. High number of Ca2+-permeable AMPA receptors capacity could be essential under physiological conditions as it Motor neurons appear to have a high proportion of Ca2+- allows rapid relaxation times of Ca2+ transients in motor permeable AMPA receptors (Fig. 3A) and stimulation of these neurons during high-frequency rhythmic activity [58]. How- receptors leads to selective motor neuron death [16,17,56].In ever, this characteristic could make motor neurons more vitro, we showed that Ca2+ entry via Ca2+-permeable AMPA susceptible to an excessive influx of Ca2+ ions. receptors is responsible for selective motor neuron death [17]. Immunostaining revealed that spinal motor neurons do not These cells were killed by a short exposure to KA, while other express the Ca2+-binding proteins parvalbumin and calbindin neurons were unaffected. This selective motor neuron death was D28K, while less vulnerable motor neurons such as those in the completely dependent on extracellular Ca2+ and was insensitive oculomotor, trochlear, abducens and Onuf's nucleus clearly to inhibitors of voltage-operated Ca2+ or Na+ channels. It was express Ca2+-binding proteins [59–61]. Similarly, parvalbumin also completely inhibited by the specific AMPA antagonist mRNA expression was high in ALS-resistant motor neuron LY300164 and by Joro spider toxin, a selective blocker of pools, while no measurable parvalbumin expression was found AMPA receptors that lack the GluR2 subunit. KA selectively in ALS-sensitive motor neurons [62]. Electrophysiological killed those motor neurons that stained positive for the Co2+ estimation of the Ca2+-buffering capacity in murine brainstem histochemical staining, a measure for the presence of Ca2+- and spinal cord slices confirmed a much higher Ca2+-buffering permeable AMPA receptors. We also quantified the electro- capacity of oculomotor than hypoglossal or spinal motor physiological properties of AMPA receptors dependent on the neurons [63–65]. Motor neurons from parvalbumin over- relative abundance of GluR2: sensitivity to external polyamines,

Fig. 3. Difference in Ca2+ metabolism between different neurons. The entry pathway and fate of Ca2+ is different in motor neurons (A) as compared to other neurons (B). Motor neurons contain a high amount of Ca2+-permeable AMPA receptors in combination with a low quantity of Ca2+-buffering proteins. As a consequence, glutamatergic stimulation leads to a high influx of Ca2+. As this Ca2+ is not buffered by Ca2+-binding proteins, the cytoplasmic Ca2+ concentration will rise and the low affinity uptake system of mitochondria will start to transport Ca2+ into the mitochondria. In non-motor neurons, most AMPA receptors are Ca2+ impermeable as they contain GluR2 and only Na+ will enter the cell through these receptors. This will lead to the depolarization of the neuron, to the removal of the Mg2+ block at the NMDA receptor and to Ca2+ entry into the cell. Moreover, after depolarization of the cell Ca2+ can also enter the neuron through voltage-operated Ca2+ channels. In contrast to motor neurons, in most neurons this Ca2+ will be buffered by the Ca2+-binding proteins present in the cytoplasm and Ca2+ will not enter the mitochondria. 1072 L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 rectification index, and relative Ca2+ permeability. Motor Ca2+ driving force through a partial repolarization. As a neurons had a higher sensitivity to external polyamines, a consequence, Cl− ions could play a vital role in glutamate- lower rectification index, and a higher relative Ca2+ permeabi- mediated excitotoxicity. Although Cl− influx suppresses lity ratio than other neurons [69]. These findings confirm that neuronal excitability and thereby counteracts the action of motor neurons are relatively deficient in GluR2. Moreover, excitatory neurotransmitters such as glutamate, Cl− influx single cell RT-PCR showed that the relative amount of GluR2 during exposure to pathological amounts of glutamate could mRNA was significantly lower in motor neurons compared to amplify the excitotoxic action of glutamate on motor neurons. other neurons, which indicates that the GluR2 regulation Co-administration of GABA enhanced the Cl− influx during underlying the GluR2 difference is at the transcription level AMPA receptor stimulation and this resulted in an increased (unpublished results). Ca2+ influx and enhanced cell death, suggesting that concom- In vivo evidence for a possible crucial role of GluR2 in itant GABAergic stimulation may aggravate excitotoxic motor motor neuron viability comes from studies with transgenic neuron death [77]. This effect of Cl− influx on excitotoxicity mice. Transgenic mice that lack the GluR2 subunit do not does not seem to be unique to motor neurons, as similar results suffer from a motor neuron disease [70]. This suggests that a were found in cerebellar granule cells [78]. However, even if it low GluR2 level is not sufficient to cause ALS, but it could is a general mechanism it could have more pronounced effects be a modifier of motor neuron degeneration in ALS. In line on motor neurons as they contain a high number of AMPA with this, we observed that GluR2 deficiency accelerated receptors [79] in combination with a very high inhibitory significantly the motor neuron degeneration and shortened the GABAergic innervation [80]. Moreover, a disturbance of the life span of mutant SOD1G93A mice with 15% [71]. Providing GABAergic function was proposed as the mechanism contrib- motor neurons with extra GluR2 subunits did also not induce uting to excitotoxic upper motor neuron death. Pathological a phenotype. Crossbreeding these GluR2 overexpressing mice studies support the loss of cerebral inhibitory interneuronal cells with mutant SOD1 mice resulted in a 14% increase of the life in ALS [81] and an alteration in the expression of GABAA span of these double transgenic mice [72]. All together, these receptor unit subtypes in the motor cortex was reported [82].In data confirm that the expression level of GluR2 is an addition, clinical neurophysiology using transcranial magnetic important parameter determining the life span of mutant stimulation (TMS) has revealed changes in cortical excitability SOD1 mice. [83,84]. These hyperexcitability changes reflect both degener- The importance of edited GluR2 in neuronal survival is also ation of the corticomotoneuronal system as well as a loss of indicated by the phenotypes of transgenic mice in which the inhibition due to an impaired function of inhibitory interneu- extent of RNA editing at the Q/R site is reduced. This generates ronal circuits in the motor cortex that renders the motor neurons a lethal phenotype with seizures and acute neurodegeneration hyperexcitable. In conclusion, these data indicate that both a [73,74]. However, transgenic mice carrying a minigene with the normal GABAergic stimulation as well as the loss of inhibitory upstream region of the mouse GluR2 gene that encodes an GABAergic innervation could contribute to excitotoxic motor asparagine (GluR2-N) at the Q/R site, which makes editing neuron death. impossible, are viable and fertile [75]. AMPA receptor channels incorporating GluR2-N are permeable to Ca2+ [28].The 2.1.4. Role of metabotropic glutamate receptors combined expression of the GluR2-N transgene and endoge- Metabotropic glutamate receptors (mGluRs) appear to nous GluR2 alleles caused a moderately (2-fold) increased Ca2+ modulate excitotoxicity in motor neurons, but their exact role permeability of the AMPA receptors in neurons of these remains to be elucidated. The expression pattern of mGluRs transgenic mice [75]. Such transgenic mice were reported to differs between motor neuron populations that are vulnerable develop motor neuron degeneration late in their life [75]. More and motor neuron populations that are resistant in ALS. Spinal recently, it was reported that Glu2-N overexpression induced a and hypoglossal motor neurons mainly express mGluR1, 4 and progressive decline in the functions of the spinal cord, as well as 7, while motor neurons from Onuf's, oculomotor and trochlear late-onset degeneration of spinal motor neurons [76]. Moreover, nucleus also express mGluR5 [85–88]. Stimulation of mGluRs crossbreeding heterozygous GluR2-N and mutant SOD1 mice has been shown to attenuate excitotoxic motor neuron death in aggravated the course of the motor neuron disease in the mutant vitro [89,90]. Furthermore, in spinal cords from familial and SOD1 mice and reduced survival by 8% [76]. sporadic ALS patients an upregulation of mGluRs in reactive astrocytes has been described [86]. These findings suggest a 2.1.3. AMPA receptor mediated excitotoxicity is aggravated by regulating role for mGluRs in motor neurons and surrounding chloride influx astrocytes, but at present the impact of these findings remains In cultured rat spinal motor neurons, chloride influx unclear. aggravated Ca2+-dependent AMPA receptor mediated motor neuron death [77]. The membrane depolarization caused by 2.2. Classical excitotoxicity and ALS AMPA receptor stimulation resulted in Cl− influx through 5- nitro-2(3-phenylpropyl-amino) benzoic acid- and niflumic acid- 2.2.1. Exogenous excitotoxins cause motor neuron disease sensitive Cl− channels. This Cl− influx aggravated excitotoxic Specific forms of motor neuron disease are caused by oral motor neuron death by two mechanisms. It increased the AMPA intake of excitotoxins. This does not only support the receptor conductance and it also resulted in an elevation of the hypothesis that an increase of the glutamatergic stimulation L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 1073 can cause neuronal death, but also confirms that motor neurons tion of BMAA in cycad flour in the Western Pacific is low and are particularly sensitive to this over-stimulation (cfr supra). Guam ALS can develop many years after the last exposure to Domoic acid concentrated in mussels was responsible for a this flour [101]. A recent study suggests that human food poisoning in Canada due to the consumption of consumption of flying foxes, which consumed palm seeds and contaminated mussels from Prince Edward Island [91,92]. possibly accumulated the plant toxins, could be the source of This led to gastrointestinal and neurological symptoms. Patients BMAA [102]. developed headache, seizures, hemiparesis, ophtalmoplegia and altered conscious level. In many patients, there was evidence for 2.2.2. Detection of increased concentrations of endogenous a pure motor or sensorimotor neuropathy [92]. excitotoxins in ALS Prolonged consumption of the chickling pea Lathyrus Crucial to classical excitotoxicity is that the extracellular sativus caused lathyrism [93]. This is a toxic upper motor concentration of glutamate is increased (Fig. 4). As a neuron disease characterized by spasticity mainly in the lower consequence, detection of an increased glutamate concentration limbs. The excitotoxin BOAA (β-N-oxalyl-amino-L-alanine), in the extracellular compartment of ALS patients would be a which is a potent AMPA receptor agonist [94] and abundantly strong argument. Abnormalities in the glutamate metabolism present in this pea, causes this motor neuron degeneration. have indeed been observed in these patients. Reductions of Monkeys fed with Lathyrus sativus or BOAA developed upper glutamate (and aspartate) levels were found in brain and spinal motor neuron signs [93,95] reminiscent of lathyrism, and cord tissue of ALS patients [103–107]. Similarly, levels of intrathecal injections with BOAA induced motor neuron NAAG (N-acetylaspartylglutamate), an acidic dipeptide which degeneration in rat and mouse spinal cord [42,96]. can be converted to glutamate and NAA (N-acetylaspartate) by Western Pacific amyotrophic lateral sclerosis-parkinsonism- NAALADase (N-acetylated-α-linked-amino dipeptidase), and dementia (Guam ALS) is a motor neuron syndrome originally NAA was reduced, while NAALADase was elevated in brain thought to be caused by the excitotoxin BMAA (β-methyla- and spinal cord tissue of ALS patients [104,105]. In the medulla mino-L-alanine) present in the seeds of false sago palm Cycas of ALS patients evidence was found using proton magnetic circinalis [97]. BMAA is not only an NMDA receptor agonist resonance spectroscopy that levels of NAA and NAAG were [98], but also activates AMPA receptors [99]. Oral administra- reduced, while glutamate levels were increased [108]. In the tion of BMAA to monkeys resulted in a motor neuron disorder, cortex of mutant SOD1G93A mice, elevated levels of extracel- parkinsonian features and behavioral anomalies with predom- lular glutamate were found in a microdialysis study [109]. inantly degenerative changes in the motor cortex and ventral Elevated levels of glutamate (and NAAG, NAA and aspartate) spinal cord [100]. This finding suggests that excitotoxicity is in the cerebrospinal fluid (CSF) of ALS patients have been involved in the pathogenesis of Guam ALS. However, detected in several [105,110], but not in all studies [111]. alternative mechanisms have been suggested as the concentra- Elevated plasma levels of glutamate during fasting and after oral

Fig. 4. Mechanisms of classical excitotoxicity in ALS. (A) Under normal conditions, the glutamate released by the presynaptic neuron stimulates the Ca2+-permeable AMPA receptors present in the postsynaptic motor neuron. This leads to a moderate, non-toxic increase of the cytoplasmic Ca2+ concentration. The action of glutamate is terminated by reuptake into the astrocytes surrounding the synapse. (B) If the glutamate transporter is damaged, the reuptake of glutamate into the astrocytes is diminished, and the concentration of glutamate into the synaptic cleft starts to increase, leading to a higher and/or longer stimulation of the Ca2+-permeable AMPA receptors. The damage to the EAAT2/GLT1 transporter can be due to the presence of mutant (mt) SOD1 but as the decrease in uptake capacity is also observedin sporadic ALS cases, other unknown factors must be involved. Moreover, activated microglial cells can secrete substances that increase the glutamatergic stimulation of the postsynaptic neuron. All together, this results in an increased influx of Ca2+ in the motor neurons. As these neurons are devoid of Ca2+-binding proteins, mitochondria start to buffer Ca2+. (C) A further increase in both the factors released by the microglial cells and in the extracellular glutamate concentration, leads to a further increase in the cytoplasmic Ca2+ concentration in the postsynaptic neuron. This leads to the generation of toxic reactive oxygen species in the mitochondria. Once the buffering capacity of the mitochondria is exceeded, intracellular Ca2+ over-activates different types of Ca2+-dependent enzymes. This interferes with the normal neuronal function and ultimately leads to neuronal death. The intracellular glutamate content is released from these injured neurons and this results in a dramatic increase of the extracellular glutamate concentration and in the spread of neurodegeneration. 1074 L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 glutamate loading were reported in one study [112], but could tion of these antisense oligonucleotides in rats resulted in a rise not be confirmed by others [110,111]. A similar controversy of extracellular glutamate levels and a progressive motor exists about the glutamate dehydrogenase (GDH) activities in syndrome [129]. A loss of EAAT2/GLT1 immunoreactivity has ALS. GDH, a key enzyme in the glutamate metabolism, also been described in the ventral horn of mutant SOD1G93A reversibly converts 2-oxoglutarate into glutamate. GDH and mutant SOD1G37R mice [130,131] and more recently in activities in leukocytes from ALS patients were found to be mutant SOD1G93A rats [35]. However, this finding could not be decreased in one study [113], but normal in another [112]. GDH reproduced by others [132,133], who found no change in activities measured in the lumbar spinal cord from ALS patients EAAT2/GLT1 expression or only a retarded mobility on gels. were increased in several spinal cord areas, but normal in the The mechanism behind the loss of EAAT2/GLT1 protein has ventral horns [103]. Interestingly, a recent report described a been the subject of intense research, at the DNA, mRNA and transgenic mouse model in which the expression of a GDH gene protein level. Mutations in the EAAT2/GLT1 gene have been (GLUD1) was increased. These mice show tremors and hind looked for, but no clear disease-associated mutations were limb weakness and a loss of large motor neurons in the ventral found. Interestingly, in one sporadic ALS patient a point spinal cord [114]. mutation in exon 5 of EAAT2/GLT1 was detected [134]. This More consistent evidence indicates that plasma and CSF of N206S mutation resulted in deficient glycosylation of the ALS patients contain toxic components that can induce protein and lower EAAT2/GLT1 expression in the cell excitotoxicity [115,116]. Couratier et al. [117] showed that membrane [135]. As a consequence, it gave rise to a loss of CSF from ALS patients was toxic for cortical neurons in glutamate transport capacity. In addition, mutated EAAT2/ culture and that this neuronal death could be inhibited by the GLT1 exerted a dominant negative effect on wild-type EAAT2/ AMPA receptor blocker CNQX, while NMDA receptor GLT1. In two patients with familial ALS not linked to SOD1, a antagonists had no effect. A recent study reported toxicity mutation in intron 7 and a silent G to A transition in exon 5 of of ALS-CSF to cultured motor neurons [118]. This treatment EAAT2/GLT1 were found [134]. No such mutations were substantially elevated the intracellular Ca2+ concentration in detected by others [136,137] and therefore their significance motor neurons via AMPA type of glutamate receptors [118]. awaits further clarification. It was also shown that CSF from ALS patients can activate At the mRNA level, an explanation for the loss of EAAT2/ microglia and that toxins released by these activated cells GLT1 could not be identified either. No reductions in EAAT2/ stimulate ionotropic glutamate receptors leading to the death GLT1 mRNA levels were found [138]. Aberrant transcripts of of spinal motor neurons [119] (Fig. 4). Minocycline inhibits EAAT2/GLT1 in disease affected areas were reported in the microglial activation and provides protection against this sporadic ALS patients [139]. CSF toxicity [119,120]. Moreover, we and others have shown Interestingly, a link between ALS-causing SOD1 mutations that this semi-synthetic tetracycline analogue influences and a decreased EAAT2/GLT1 function was found in vitro disease progression and survival of mutant SOD1 mice [140]: catalysis of H2O2 by mutant SOD1 induced oxidative [121–123], indicating that a similar process could be involved damage to the intracellular carboxyl-terminal part of EAAT2/ in vivo. GLT1, resulting in decreased glutamate transport (Fig. 4). Crossbreeding EAAT2/GLT1 overexpressing mice with mutant 2.2.3. Decreased clearance of glutamate SOD1 mice delayed disease onset as measured by grip strength, Rothstein et al. [124] reported diminished glutamate but did not prolong survival [141]. transport in synaptosomes from affected brain areas and Recently, β-lactam antibiotics were discovered as potent spinal cord of sporadic ALS patients. In addition, the number stimulators of EAAT2/GLT1 expression after a blinded 3 of [ H]D-aspartate binding sites, a measure for the density of screen of 1040 FDA approved drugs and nutritionals glutamate transporters, was reduced in spinal cords from [142]. Both, in vitro and in vivo administration of sporadic ALS patients [125]. This decreased glutamate ceftriaxone, one of these β-lactam antibiotics, led to a 3- transport was found to be due to a selective loss of the fold increase in protein levels and a comparable increase in astroglial glutamate transporter, EAAT2/GLT1, in the motor EAAT2/GLT-1 specific biochemical and electrophysiological cortex and spinal cord [126]. This loss of EAAT2/GLT1 was glutamate transport. The mechanism of this overexpression is not only observed in sporadic ALS patients [126–128], but the activation of the EAAT2/GLT1 promoter. Ceftriaxone also in familial cases [126]. treatment of mutant SOD1 mice starting at the onset of the AMPA receptor-mediated excitotoxicity of motor neurons disease significantly increased the life span of the mutant induced by a loss of EAAT2/GLT1 in ALS-affected areas of the SOD1 mice from 122 to 132 days. When the drug was CNS is an attractive hypothesis (Fig. 4). Both in vitro and in given somewhat earlier, a maximal prolongation of the life vivo experiments demonstrated that loss of EAAT2/GLT1 span of the mutant SOD1 mice by 11% was observed [142]. function may give rise to selective motor neuron degeneration. Moreover, it was shown that ceftriaxone prevented motor Treatment of organotypic spinal cord cultures with antisense neuron loss and astrogliosis [142]. In conclusion, these data oligonucleotides to EAAT2/GLT1, resulting in diminished indicate that an induction of the EAAT2/GLT1 expression transporter protein expression, was shown to induce progressive and a higher clearance of glutamate from the synaptic cleft motor neuron loss, which could be prevented by an AMPA can protect motor neurons during ALS, at least in the mutant receptor antagonist [129]. Chronic intraventricular administra- SOD1 mouse model. L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 1075

2.3. Slow excitotoxicity and ALS benzo[f]quinoxaline-7-sulfonamide (NBQX, [148])and ZK187638 [149] increased the life span of these mice by 2.3.1. Presence of mutant SOD1 increases sensitivity of motor 13%, 10% and 11%, respectively. Moreover, interference with neurons to excitotoxicity glutamate metabolism by inhibiting glutamate carboxypepti- In vitro, the sensitivity of motor neurons to the toxicity of dase II with 2-MPPA also increased significantly (by 15%) the mutant SOD1 was studied by intranuclear injections of mutant life span of the late onset SOD1G93A mouse model [150]. SOD1 in mixed motor neuron cultures [143]. These injections Furthermore, it was shown that the motor neuron degeneration induced formation of SOD1 aggregates and motor neuron death, induced by intrathecal administration of the excitotoxin β-N- while no aggregates or cell death was observed after injection in oxalylamino-L-alanine (BOAA) was more pronounced in spinal other cultured neurons. This selective motor neuron death was cords of mutant SOD1G93A mice than in those of non-transgenic dependent on extracellular glutamate as it could be inhibited by mice [42]. All together, these results strongly indicate that both a general AMPA receptor antagonist (6-cyano-7-nitroqui- excitotoxicity plays a role in mutant SOD1 induced motor noxaline-dione disodium; CNQX) and a selective antagonist of neuron death and suggest that SOD1 mutations may render Ca2+-permeable AMPA receptors (joro spider toxin) [144].A motor neurons more sensitive to excitotoxicity. crucial role for intracellular Ca2+ in this process was further indicated by the protection offered to mutant SOD1-induced 2.3.2. Mutant SOD1 and mitochondrial function motor neuron death by coexpression of the Ca2+-binding As mentioned above, slow excitotoxicity has often been protein, calbindin D28K [144]. These results indicate that the linked to mitochondrial dysfunction. As a consequence, it is selective toxicity of mutant SOD1 to motor neurons is induced interesting to note that there is growing evidence that mutant or at least can be aggravated by glutamatergic stimulation. The SOD1 is not only localized in mitochondria, but also interferes exact mechanism is not yet clear, although it was suggested that with their function (Fig. 5). The presence of human mutant Ca2+ influx through Ca2+-permeable AMPA receptors could be SOD1 in vacuolated mitochondria was first demonstrated by responsible for aggregation of mutant SOD1 and subsequent electron microscopy in mutant SOD1G93A transgenic mice motor neuron death [72]. In a different in vitro model, motor [151]. Jung et al. [152] and Mattiazzi et al. [153] reported that the neurons in mixed spinal cord cultures from SOD1G93A mice activity of several mitochondrial respiratory chain complexes were shown to be more sensitive to glutamate, which resulted in was reduced and that ATP synthesis was diminished in mutant a higher free radical production [42]. In contrast, we and others SOD1G93A mice. These deficits in mitochondrial function could not find an enhanced sensitivity of cultured motor became more pronounced with progression of the disease and neurons overexpressing mutant SOD1 to excitotoxicity affected the spinal cord more than other tissues. This [145,146]. mitochondrial dysfunction caused by the presence of mutant In vivo, treatment protocols that interfered with excitotoxi- SOD1 may have many consequences. First, it is possible that the city delayed selective motor neuron death and prolonged localization of mutant SOD1 in the mitochondria on itself is survival in the SOD1G93A mice. The AMPA receptor antago- pathogenic (independent of excitotoxicity). When mutant SOD1 nists RPR119990 [147], 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- was targeted to different subcellular compartments, targeting it

Fig. 5. Mechanisms of slow excitotoxicity in ALS. (A) Factors and growth factors released from the astroglial cells surrounding the synapse activate the expression of the GluR2 subunit in the motor neuron. As a consequence, the number of Ca2+-permeable AMPA receptors in the postsynaptic neuron is controlled and stimulation of the AMPA receptors only leads to a moderate influx of Ca2+ through this type of receptors. (B) If the release of the astroglial factor is diminished, the number of Ca2+- permeable AMPA receptors is increased and the influx of Ca2+ into the postsynaptic neuron increases. Moreover, reduced editing of the mRNAs encoding the GluR2 subunit can also be responsible for an increase in the Ca2+ permeability of the AMPA receptors. As the presence of mutant (mt) SOD1 compromises the Ca2+-buffering capacity of the mitochondria, the intracellular Ca2+ concentration in the cytoplasm of the postsynaptic neuron raises. (C) A dramatic increase in the Ca2+ permeability of the AMPA receptors in combination with a drastic decrease in the Ca2+-buffering capacity of the mitochondria leads to an excessive activation of different types of Ca2+-dependent enzymes. As in classical excitotoxicity, this interferes with the normal neuronal function and ultimately leads to neuronal death. 1076 L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 to the mitochondria was sufficient to induce cell death in a motor neuron degeneration induced by ischemia was also neuronal cell line [154]. It was shown that the presence of clearly different in the two different rat strains. This difference mutant SOD1 in the mitochondria triggered the release of in the sensitivity to excitotoxicity was preserved in cultured cytochrome c followed by the activation of the caspase cascade spinal motor neurons on top of a astrocytic feeder layer isolated [154]. from these two different rat strains [160,161]. Motor neurons Second, mitochondrial dysfunction makes it difficult for the cultured from the rat strain with the lowest sensitivity to affected neuron to meet the demands of its many ATP- transient ischemia expressed AMPA receptors with a low Ca2+ dependent processes. The delicate ion balance thus becomes permeability and a high GluR2 expression [160]. Remarkably, disturbed and may give rise to slow excitotoxicity and lead to the astrocytic feeder layer on which these motor neurons were cell death of neurons with a high glutamatergic input, such as cultured determined these characteristics. When motor neurons motor neurons. Moreover, the damage caused by mutant SOD1 were shifted from one astrocytic feeder layer to the other, they on mitochondria may also be more pronounced in these motor changed their characteristics according to the origin of the neurons as these cells have a high metabolic demand. astrocytes. [161]. We conclude from these experiments that Last but not least, dysfunction of mitochondria will also factors released from astrocytes can influence the sensitivity of have a dramatic effect on the mitochondrial Ca2+-buffering motor neurons to excitotoxicity by influencing the expression capacity of these organelles [155]. This defect will have more level of GluR2 and the relative amount of Ca2+-permeable pronounced consequences in the motor neurons as these AMPA receptors. Moreover, it seems that the presence of organelles play a crucial role in the Ca2+ buffering of these mutant SOD1 in the astrocytes can affect the secretion of these cells (cfr supra). As a consequence, the cytoplasmic Ca2+ factors, leading to a lower expression of GluR2, more Ca2+ concentration rises and over-activates a number of Ca2+- permeable AMPA receptors and a higher sensitivity to dependent enzymes and this could lead to neuronal death excitotoxicity (unpublished results). (Fig. 5). Further evidence for an effect of glial cells on the characteristics of the AMPA receptors comes from the 2.3.3. Reduced editing of the GluR2 mRNA observation that both glial derived neurotrophic factor Under normal conditions, all the GluR2 mRNA is edited (GDNF) as well as brain derived neurotrophic factor (BDNF) resulting in the change of a CAG codon into a CGG codon. If enhance GluR2 expression. The GluR2 promoter contains a this edited GluR2 subunit is incorporated in the AMPA receptor, neuron-restrictive silencer element (NRSE). Binding of neuron- it is Ca2+ impermeable. However, it was demonstrated that the restrictive silencing factor (NRSF) to this sequence in non- editing efficiency of GluR2 mRNA was lower in the ventral neuronal cells results in silencing of genes with a NRSE in their spinal gray matter of ALS patients compared to controls [156]. promoter [162–164]. It was shown that the effect of GDNF on This deficiency in GluR2 editing was confirmed in single motor the GluR2 promotor was mediated by NRSE [165]. Delivery of neurons isolated with laser microdissection from five ALS GDNF to motor neurons with adeno-associated viral or patients [157]. The consequence of this editing deficiency is that adenoviral vectors resulted in an increase of the life span of AMPA receptors that contain GluR2 translated from unedited mutant SOD1G93A mice by 14% [166,167]. Whether GDNF- mRNA will have a neutral glutamine instead of the positively induced upregulation of GluR2 and/or interference with charged arginine and will be Ca2+ permeable (Fig. 5). excitotoxicity contributed to this protective effect was not Apparently, this phenomenon is limited to sporadic ALS investigated. patients as it was not found in mutant SOD1G93A mice (our unpublished results) or in mutant SOD1G93A rats [158]. 2.3.5. Interference with axonal transport As motor neurons have extremely long axons, interference 2.3.4. Astrocytes affect sensitivity of motor neurons to with axonal transport will have dramatic consequences on the excitotoxicity functioning of these cells. Moreover, a pathological hallmark of There is increasing evidence that the selective motor neuron ALS is the abnormal accumulation of neurofilaments in the death in mutant SOD1-dependent motor neuron death is a non- perikaryon and in the axons of motor neurons [168,169]. cell-autonomous process [159]. Cells surrounding the motor Although it is not yet clear how these accumulations contribute neuron that do not contain mutant SOD1 can protect mutant to the neurodegenerative process, their presence suggests that SOD1 containing motor neurons and vice versa. Moreover, it is axonal transport is disturbed in affected motor neurons. An generally accepted that astrocytes are crucial to keep the interesting link between excitotoxicity and axonal transport was synaptic glutamate concentration very low (cfr supra). We found in primary cortical neurons. After treatment of these recently discovered that astrocytes can also influence the neurons with glutamate, a dose-dependent slow down of the subunit composition of the AMPA receptor as they determine anterograde axonal transport was observed [170].This the GluR2 expression in motor neurons (Fig. 5). We found that phenomenon was accompanied by phosphorylation of the two rat strains differed in their sensitivity to transient spinal cord neurofilament side-arm domains by different members of the ischemia induced by clamping the aortic arch and left mitogen-activated protein kinase family [171]. Although, this subclavian artery. This process was mediated by AMPA phenomenon was dependent on the stimulation of NMDA receptor stimulation, as it was completely prevented by pre- receptors, a similar process involving (excessive) stimulation of treatment with the AMPA receptor antagonist NBQX. The the Ca2+-permeable AMPA receptors could have comparable L. Van Den Bosch et al. / Biochimica et Biophysica Acta 1762 (2006) 1068–1082 1077 effects in motor neurons, although experimental evidence for neurons to high intracellular Ca2+ concentrations by inducing this attractive hypothesis is lacking. defence mechanisms and/or to inhibit the downstream pathways activated by an increased intracellular Ca2+ concentration. 3. Conclusions Moreover, recent research indicates that therapeutic options do not have to focus on motor neurons alone, as ALS seems to be a Since its description by Charcot more than 130 years ago, the non-cell-autonomous disease. As a consequence, inhibiting pathogenesis of selective motor neuron degeneration in microglial activation which prevents the release of toxic amyotrophic lateral sclerosis (ALS) remains unresolved. Over substances by these cells could also be a valuable option. In the years, many pathogenic mechanisms have been proposed. addition, stimulating astrocytes to increase glutamate uptake Amongst others these include: oxidative stress, aggregate and/or to secrete (growth) factors that modify the properties of formation of mutant SOD1, inflammation, growth factor the AMPA receptors present on the motor neurons could also deficiency, neurofilament disorganisation and last but not least have a positive effect. In conclusion, a combined pharmaco- excitotoxicity. 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