**3. Astrocytes in excitotoxicity**

In addition to the clear inflammatory role that astrocytes play in ALS, they also contribute to the mechanism of excitotoxicity. Their effect in the latter mechanism is two-fold: firstly, astrocytes facilitate the removal of excessive glutamate at the synaptic cleft and secondly, they affect the calcium permeability of AMPA receptors of motor neurons.

### **3.1 Excitotoxicity in ALS explained**

Glutamate is the initiator of excitotoxicity in ALS. This neurotransmitter is the most abundant excitatory neurotransmitter in the brain and binds to NMDA, AMPA, and kainate (ionotropic) and metabotropic glutamate receptors (mGluR). Packaged into vesicles by the pre-synaptic neuron, glutamate is released into the synaptic cleft by the fusion of vesicles to the membrane of the neuron to excite the post synaptic neuron. This process is inhibited by riluzole (Siniscalhi et al., 1999). Increased levels of glutamate are detected in fALS, sALS (Fiszman et al., 2010; Spreux-Varoquaux et al., 2002) and is confirmed in spinal cords of ALS mice and rats. The detrimental role of glumate in the disease is demonstrated by the pronounced cell death that occurs to neurons in vitro when exposed to low levels of glutamate, even as low as physiologically detected in CSF (Cid et al., 2003). To further illustrate the detrimental role of glutamate in ALS, administration of compounds that block the formation of glutamate increase cell survival, both in vivo and in vitro (Cid et al., 2003).

### **3.2 Astrocytes in excitotoxicity in ALS: EAAT2/GLT-1**

After glutamate release from the pre-synaptic neuron and binding of glutamate to ionotropic or metabotropic receptors on the post-synapse (increasing the concentration of

Fig. 1. Mutant SOD1 in astrocytes affecting (motor) neuron survival.

they affect the calcium permeability of AMPA receptors of motor neurons.

In addition to the clear inflammatory role that astrocytes play in ALS, they also contribute to the mechanism of excitotoxicity. Their effect in the latter mechanism is two-fold: firstly, astrocytes facilitate the removal of excessive glutamate at the synaptic cleft and secondly,

Glutamate is the initiator of excitotoxicity in ALS. This neurotransmitter is the most abundant excitatory neurotransmitter in the brain and binds to NMDA, AMPA, and kainate (ionotropic) and metabotropic glutamate receptors (mGluR). Packaged into vesicles by the pre-synaptic neuron, glutamate is released into the synaptic cleft by the fusion of vesicles to the membrane of the neuron to excite the post synaptic neuron. This process is inhibited by riluzole (Siniscalhi et al., 1999). Increased levels of glutamate are detected in fALS, sALS (Fiszman et al., 2010; Spreux-Varoquaux et al., 2002) and is confirmed in spinal cords of ALS mice and rats. The detrimental role of glumate in the disease is demonstrated by the pronounced cell death that occurs to neurons in vitro when exposed to low levels of glutamate, even as low as physiologically detected in CSF (Cid et al., 2003). To further illustrate the detrimental role of glutamate in ALS, administration of compounds that block the formation of glutamate increase cell survival, both in vivo and in vitro (Cid et al., 2003).

After glutamate release from the pre-synaptic neuron and binding of glutamate to ionotropic or metabotropic receptors on the post-synapse (increasing the concentration of

**3. Astrocytes in excitotoxicity** 

**3.1 Excitotoxicity in ALS explained** 

**3.2 Astrocytes in excitotoxicity in ALS: EAAT2/GLT-1** 

intracellular calcium), glutamate is recycled for further use by the glial and endothelial cells, including astrocytes. Astrocytic glutamate re-uptake occurs by the glutamate transporters excitatory amino acid transporter 1 (EAAT1) and excitatory amino acid transporter 2 (EAAT2; also known as glutamate aspartate transporter (GLAST1) and glutamate transporter 1 (GLT-1), respectively). These transporters internalise glutamate, eg. into the astrocyte, for conversion to glutamine that is returned to the pre-synaptic neuron to be release again as glutamate (Laake et al., 1995).

Decreased glutamate uptake and EAAT2 protein levels are a common feature in both fALS and sALS and both in vitro and in vivo model systems (Staats and Van Den Bosch, 2009). In vitro transfection of primary cultured astrocytes with either mutant SOD1 or wild type human SOD1 down-regulates EAAT2 post transcriptionally (Tortarolo et al., 2004) and decreases EAAT2 transcription (Yang et al., 2009). Accordingly, glutamate transport is decreased in a neuronal cell line by mutant SOD1 transfection (Sala et al., 2005). Interestingly, this down-regulation also occurs in ALS model rats at pre-symptomatic stages through to end stage (Howland et al., 2002), at end stage only (Warita et al., 2002), in ALS model mice at end stage (Bendotti et al., 2001; Guo et al., 2010) and in post mortem patient spinal cords (Sasaki et al., 2001). In addition, in patient material the loss of EAAT2 and decreased tissue glutamate transport does not coincide with decreased levels of gene expression (Bristol and Rothstein, 1996), indicating that the loss is induced post transcriptionally, also in humans. Interestingly, a decrease of EAAT2 protein levels is not only in mutant SOD1 ALS models, but also a model of ALS/PDC (Wilson et al., 2003). In this model wild-type mice are fed with washed cycad flour containing β-methylaminoalanine (BMAA), which causes an ALS-like phenotype (Wilson et al., 2002). Although the loss of EAAT2 in ALS is apparent, it remains unclear whether this post transcriptional loss of EAAT2 proceeds or follows the loss of motor neurons.

### **3.3 Targeting (astrocytic) EAAT2**

To assess whether the loss of glutamate transport or the loss of EAAT2 specifically results in motor neuron loss, pharmacological and genetic tools have been used. To begin, research conducted by pharmacologically inhibiting glutamate transport in the rat spinal cord, failed to show any motor neuron loss despite the increased levels of glutamate (Tovar et al., 2009). In contrast, a similar experiment has been performed to address whether EAAT2 loss specifically induces motor neuron loss. EAAT2 null mice live for approximately 6 weeks before they succumb to epileptic seizures and are vulnerability to acute brain injury (Tanaka et al., 1997). To this end, heterozygous mice demonstrated the effect of approximately 40% knockdown of EAAT2 in the spinal cord in ALS mice (Pardo et al., 2006). This knockdown resulted in a non-significant decrease of symptom onset and significant, but moderate, decrease of lifespan in ALS mice (Pardo et al., 2006).

To assess the expected beneficial role of EAAT2 in ALS, transgenic mice overexpressing human EAAT2 in specifically astrocytes were crossbred with mutant SOD1 mice. Although glutamate uptake is increased in vivo and an overexpression of human EAAT2 is protective on cortical neurons in vitro, an effect on symptom onset or lifespan was absent (Guo et al., 2003). Possibly, the expression levels were insufficient to induce an effect or human EAAT2 is not as efficient as murine EAAT2 in mouse, as administration of ceftriaxone (a β-lactam antibiotic) and GPI-1046 (a synthetic, non-immunosuppressive derivative of FK506) increase EAAT2 protein levels and extend lifespan of ALS mice (Rothstein et al., 2005; Ganel et al.,

The Astrocytic Contribution in ALS: Inflammation and Excitotoxicity 385

Interestingly, the surrounding astrocytes influence the expression level of the GluR2 subunit in motor neurons, as soluble factor(s) released from astrocytes effect GluR2 gene expression and neuronal vulnerability to excitotoxic insults, both in vitro and in vivo (Van Damme et al., 2007). Moreover, the presence of mutant SOD1 interferes with the production and/or secretion of this factor(s) to increase GluR2 expression and thus decreases motor neuronal

Astrocytes clearly contribute to ALS decrease progression in both neuroinflammation and excitotoxicity. An intriguing aspect of astrocytes in ALS disease pathology is whether the mutant SOD1 astrocytic properties, of LIGHT dependent cell death and diminished GluR2 editing for example, are also important in other ALS causing mutations and in sporadic cases of ALS. Initial work performed implies that these characteristics are not solely dependent on mutant SOD1 in patients. In addition, both mechanisms in which astrocytes function seem successfully targetable in mice. Future research may benefit from further assessing the role of also non-SOD1 ALS causing mutations in astrocytes on ALS and optimizing therapeutic strategies against

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**4. Conclusions and future directions** 

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**5. References** 

2006) by enhancing EAAT2 transcription (Lee et al., 2008). In addition, EAAT2 is also expressed by other cells types than astrocytes alone (Anderson and Swanson, 2000), which are not targeted in this genetic experimental design. Interestingly, removal of mutant SOD1 from astrocytes leads to prolonged survival without affecting astrogliosis, but does preserve EAAT2 levels potentially explaining the extended lifespan (Wang et al., 2011a).

### **3.4 Astrocytic replacement therapy in ALS mice**

The beneficial effect of EAAT2 is often used as an explanation of positive effects found by cell transfers in ALS model rodents. For instance, the systemic transplantation of c-kit positive cell from bone marrow in mutant SOD1 mice significantly increased the lifespan, which is, at least in part, attributed to increased EAAT2 expression induced by the transferred cells (Corti et al., 2010). The same holds true for the prolonged survival of ALS rats when treated with focal transplantation-based astrocyte replacement with wild type glial-restricted precursors (GRPs) (Lepore et al., 2008b). This study also focussed on EAAT2 by also transplanting EAAT2 overexpressing GRPs and EAAT2 null GRPs. The ALS mice treated with the EAAT2 overexpressing GRPs showed no additional increase of lifespan compared to wild type GRP treated ALS mice (already increased compared to controls). Intriguingly, this positive effect of transplantation of the wild type GRPs is diminished in mice transplanted with EAAT2 null GRPs (Lepore et al., 2008b). In addition, co-cultures of human adipose-derived stem cells with astrocytes induce higher levels of EAAT2 in astrocytes (Gu et al., 2010), though this treatment has not (yet) been shown to affect motor neuron survival in vitro or in vivo.

### **3.5 Astrocytes in excitotoxicity in ALS: AMPA receptor permeability**

After the release of glutamate from the pre-synaptic neuron into the synaptic cleft, glutamate binds to NMDA, AMPA receptors or the metabotropic receptors. High levels of calcium entering through AMPA receptors into the post-synaptic neuron can cause neuronal death. The AMPA receptor is formed as a tetramer combining, usually pairwise, a combination of its four different subunits (glutamate receptor unit 1-4 (GluR1-4)) (Shi et al., 1999). Each subunit can bind glutamate and the channel opens after occupation of at least 2 binding locations (Mayer, 2005). The importance of this receptor in ALS is demonstrated by the ablation of glutamate induced apoptosis in cortical neurons in vitro (Cid et al., 2003) and in vivo when administering an AMPA receptor antagonist (Van Damme et al., 2003; Tortarolo et al., 2006).

The AMPA receptor plays an imperative role in excitotoxicity by its calcium permeability that is determined by the incorporation of the GluR2 subunit in the receptor complex. In most conditions, the AMPA receptor complex contains at least one GluR2 subunit and it prevents the influx of extracellular calcium into the neuron (Seeburg et al., 2001). In contrast, receptors lacking the GluR2 subunit are highly calcium permeable (Seeburg et al., 2001). A general decrease of GluR2 is found in ALS model mice, portraying an increased vulnerability of these mice to excitotoxic insults (Tortarolo et al., 2006; Zhao et al., 2008). The role of GluR2 in ALS is investigated by genetically ablating GluR2 in ALS mice, which decreases survival in vivo and decreases cell survival in vitro (Van Damme et al., 2005). The opposite has been shown by up-regulating GluR2 expression in motor neurons of ALS mice, as hereby survival is increased (Tateno et al., 2002). In addition, pharmacological inhibition of the AMPA receptor prolonged survival in ALS model mice (Van Damme et al., 2003; Tortarolo et al., 2006).

Interestingly, the surrounding astrocytes influence the expression level of the GluR2 subunit in motor neurons, as soluble factor(s) released from astrocytes effect GluR2 gene expression and neuronal vulnerability to excitotoxic insults, both in vitro and in vivo (Van Damme et al., 2007). Moreover, the presence of mutant SOD1 interferes with the production and/or secretion of this factor(s) to increase GluR2 expression and thus decreases motor neuronal resistance to excitotoxicity (Van Damme et al., 2007).
