**The Astrocytic Contribution in ALS: Inflammation and Excitotoxicity**

Kim Staats1,2 and Ludo Van Den Bosch1,2 *1University of Leuven, 2VIB Vesalius Research Center, Belgium* 

### **1. Introduction**

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, due to the loss of motor neurons and denervation of muscle fibres, resulting in increasing muscle weakness and paralysis. The disease has an incidence of 2.7 cases per 100,000 people in Europe (Longroscino et al., 2010). It is diagnosed from teen years onward, but is more prevalent in the later years of life. In lack of a medical cure, average life expectancy post diagnosis is between 2 and 5 years, though 10% of all patients live longer than 10 years. Patients mainly succumb to the disease by respiratory insufficiency or may opt for euthanasia where legislature permits (Maessen et al., 2010). Although ALS is characterised by degeneration of central nervous system tissue, mental functions remain largely unaffected resulting in a locked-in state (Kotchoubey et al., 2003). At current, there is but one medicine to treat the disease, riluzole, slowing disease progression moderately (Miller et al., 2007).

### **1.1 Basic genetics of ALS**

Mutations in the ubiquitously expressed Cu/Zn superoxide dismutase 1 (SOD1) gene can cause ALS. SOD1 detoxifies cell damaging free radicals and its mutations account for 20% of the ALS patients suffering from the disease by familial origin (fALS) worldwide. The remaining 90% of ALS patients suffer from the disease by unknown sporadic causes (sALS), though a common mechanism is predicted as fALS and sALS patients display indistinguishable clinical phenotypes. Overexpression of mutant forms of human SOD1 causes the ALS phenotype of transgenic SOD1 mice, accounting for an invaluable contribution to ALS research (Gurney et al., 1994). Many hallmarks of the disease are shared between patients and this rodent model, including specific motor neuron loss, aggregate formation, astrogliosis, microgliosis and progressive paralysis. As the genetic ablation of SOD1 does not produce an ALS-like phenotype in mice (Reaume et al., 1996; Shefner et al., 1999) the pathogenic mechanism of mutant SOD1 is a toxic gain of function. This gain of function may be exerted by protein misfolding, aggregation, impaired proteasome functioning, impaired retrograde transport, excitotoxic cell death or other mechanisms (reviewed in Bruijn et al., 2004). Mutations in other genes also cause familial ALS, including mutations in vesicle-associated membrane proteinassociated protein B (VAPB), TAR DNA binding protein (TDP-43), fused in

The Astrocytic Contribution in ALS: Inflammation and Excitotoxicity 379

An additional detrimental mechanism in ALS is excitotoxicity; an overstimulation of neurons causing neurodegeneration. Glutamate binds to the N-methyl D-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, allowing extracellular sodium and calcium to enter motor neurons. Increased levels of intracellular calcium consequently cause neuronal cell death. The importance of excitotoxicity in ALS is demonstrated by the beneficial effects obtained by treating patients with riluzole. Although the precise mechanism of this drug is not yet known, it blocks NMDA receptors, enhances re-uptake of glutamate from the synaptic cleft and inhibits glutamate release by blocking voltage-gated sodium channels (Siniscalhi et al., 1999), thus preventing motor neuron cell death. Riluzole treatment increases predicted lifespan with a significant 12% in ALS mice (Bensimon et al., 1994; Lacomblez et al., 1996) and increases the probability of one year survival in patients by 9% (Miller et al., 2007). Unfortunately, it does not halt disease

An overview of the current knowledge of the astrocytic contribution in ALS will be addressed in this chapter separately for the mechanisms inflammation and excitotoxicity.

Despite that microglia are the main immune cells of the central nervous system (reviewed in Ransohoff, 2010), astrocytes can also become reactive and contribute to neuroinflammation and are the focus of this chapter, with microglial inflammatory effects residing beyond the scope of this chapter. During neuroinjury or neurodegeneration the production of cytokines induce astrogliosis in which astrocytes increase glial fibrillary acidic protein (GFAP) and vimentin expression as well as an array of other genes. This response increases neuronal survival and includes both supportive factors (e.g. growth factors and glutamate transporters) and cytokines to sustain/promote neuroinflammation. Interestingly, during neuroinflammation the number of astrocytes increases by the differentiation of chondroitin sulfate proteoglycan, NG2, positive cells to astrocytes and not by astrocytic proliferation

To assess the effect of inflammation in ALS and thus to discover whether boosting the inherent inflammation would be beneficial, lipopolysaccharide (LPS) was daily administered to ALS mice (Nguyen et al., 2004). The effect of this treatment was a clear decrease in lifespan, implying that an increase of inflammation is detrimental in ALS (Nguyen et al., 2004). Another study, initially intended to decrease inflammation, administered macrophage colony stimulating factor (M-CSF) to ALS mice and observed an unexpected increase of microgliosis also leading to a decreased survival (Gowing et al., 2009). Although not directed specifically at astrocytes, this work has led to the

Reactive astrocytes alter gene expression including an upregulation of the intermediate filaments GFAP and vimentin that allow for visualisation of astrogliosis by increased immunoreactivity of these filaments in patient and ALS model tissue. Post mortem spinal

understanding of the hazardous character of neuroinflammation in ALS.

**1.4 Excitotoxicity in ALS** 

progression.

(Gowing et al., 2008).

**2.2 Astrogliosis in ALS** 

**2. Astrocytes in inflammation** 

**2.1 Increasing inflammation in ALS** 

sarcoma/translocated in liposarcoma (FUS/TLS), optineurin and valsolin containing protein (VCP) (Johnson et al.; Maruyama et al.; Rutherford et al., 2008; Van Deerlin et al., 2008; Del Bo et al., 2009; Kwiatkowski et al., 2009; Vance et al., 2009). Unfortunately, the discovery of these mutant genes has not yet progressed into useful ALS model organisms, so most of the work described below was conducted with mutant SOD1 based ALS models.

### **1.2 Non-cell autonomous ALS**

Multiple cell types contribute to the pathology making ALS a non-cell autonomous disease (Boillee et al., 2006a). By addition or deletion of mutant SOD1 in specific cell types, it is known that mutant SOD1 influences the disease depending on the cell type, including astrocytes (Yamanaka et al., 2008; Wang et al., 2011a), microglia (Boillee et al., 2006b), Schwann cells (Lobsiger et al., 2009) and motor neurons (Jaarsma et al., 2008). Additionally, ablation of T-cells (Beers et al., 2008; Chiu et al., 2008), B-cells (Naor et al., 2009), CD4+ and CD8+ cells (Beers et al., 2008) decrease survival of ALS mice, demonstrating the role of immune cells in disease progression. Although ALS is a non-cell autonomous disease, mutant SOD1 expressed solely in motor neurons is sufficient to initiate the disease, albeit with a slower disease progression (Jaarsma et al., 2008). Motor neurons in the motor cortex, brainstem and spinal cord undergo cell death selectively in patients. A number of hypotheses attempt to explain this cell type selectivity, including the long axons of the motor neurons (Fischer and Glass, 2007), their poor intracellular calcium buffering capacity (Grosskreutz et al., 2010) and motor neuron specific cell death pathways (Raoul et al., 2002; Raoul et al., 2006; Genestine et al., 2011).

The contribution of mutant SOD1 expressing astrocytes in the non-cell autonomous character of ALS has been studied by excising mutant SOD1 from astrocytes which increases survival in two different mutant SOD1 mouse models (Yamanaka et al., 2008; Wang et al., 2011a). These results denote the toxic character of mutant SOD1 in astrocytes that accelerate disease progression significantly by mechanisms such as, but not exclusively, the below described mechanisms of neuroinflammation and excitotoxicity. This is schematically presented in figure 1.

### **1.3 Neuroinflammation observed in ALS**

Neuroinflammation occurs in a number of neurodegenative diseases, including ALS (reviewed in Papadimitriou et al., 2010 and Philips and Robberecht, 2011), and entails the reactive state of astrocytes (astrogliosis) and microglia (microgliosis) and the infiltration of lymphocytes. Initially perceived as a bystander effect, neuroinflammation is currently seen as beneficial at first, removing damaged cells and secreting supportive factors, and potentially detrimental thereafter by excessive release of cytokines (Beers et al., 2011a). Evidence of inflammation is detected in post mortem tissue (Schiffer et al., 1996; Anneser et al., 2004; Casula et al., 2011; Sta et al., 2011; Wang et al., 2011b), in cerebrospinal fluid (CSF) (Baron et al., 2005; Tateishi et al., 2010) and in blood samples of ALS patients (Poloni et al., 2000). In accordance, similar parameters of neuroinflammation are detected in ALS rodent models (among many others in Kiaei et al., 2006; Keller et al., 2009; Beers et al., 2011b). Inflammation is generally perceived as hazardous in ALS, as increasing inflammation in ALS models exacerbates disease progression and diminishes survival (Nguyen et al., 2004; Gowing et al., 2009). Fittingly, therapeutic strategies targeting inflammation are often advantageous in ALS rodent models (see below).
