**2. Genetic animal models**

To date, at least 13 genes and loci of major effect in fALS have been identified. The most frequent mutations found in fALS consist in mutations in the gene encoding copper/zinc superoxide dismutase 1 (Cu/Zn SOD1), a ubiquitously expressed enzyme that plays a key role in oxygen free radical scavenging. SOD1 catalyzes the dismutation of superoxide (O2 -) to hydrogen peroxide (Scozzafava & Viezzoli, 1993; Tainer et al., 1982). On average, SOD1 mutations are responsible for about 20% of fALS cases (Deng et al., 1993; Rosen et al., 1993) and 5% of apparently sALS (Andersen et al., 2007). Over 70 different mutations at more than 60 residues throughout SOD1 (153 amino acids) have been linked to fALS (Andersen, 2000). The vast majority of mutations are amino acid substitutions, but a few cause C-terminal truncations of the protein. A spontaneous mutation in SOD1 has also been found in a canine degenerative myelopathy which resembles ALS (Awano et al., 2009).

Advantages and Pitfalls in Experimental Models Of ALS 127

transgenic mice tissues, suggesting that also G86R mutation has no effects on SOD activity. The early symptoms are variable, consisting in either a flaccid or a spastic paralysis of forelimbs/hindlimbs, whereas the time of the onset is very predictable and the progression of the disease extremely rapid: in fact, the disorder progresses from a mild gait abnormality to total paralysis within a 5-days period: animals are healthy at the age of 3 months and dye

The original G85R mutation observed in fALS was introduced in a mouse background by Bruijn and coll. (1997). Low levels of accumulated human G85R are sufficient to cause severe MN disease without altering the protein and activity levels of endogenous SOD1. After an 8 month-latency period, MN loss proceeds nearly synchronously with 40% of large spinal MN axons degenerating in 2 weeks. Moreover, prominent SOD1-containing inclusions in astrocytes appear prior to clinical signs and increase markedly during disease progression, indicating astrocytes as first players in mutant-SOD1 mediated damage. Furthermore, in this mouse model the presence of glial glutamate transporter, the major glutamate transporter in spinal cord, decreases in end-stage G85R mice. This finding is strikingly similar to that observed in patients with sALS (Rothstein et al., 1995), implicating glutamate mediated excitotoxicity as a mechanism to account for the nearly synchronous degeneration of MNs. Wong and coll. (1995) produced multiple lines of transgenic mice expressing the human G37R SOD1 mutant. These mice, expressing 5 to 14 times normal SOD1 activity levels, develop the clinical phenotype of MN disease. At lower levels of mutant protein accumulation, the pathology is restricted to lower MNs, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations (striatum, thalamus, hypothalamus, pyriform cortex), with conspicuous vacuolar degeneration of axons and dendrites even in pre-symptomatic mice. However, vacuolar degeneration is not a well recognized component of MN pathology in human G37R fALS patients. Furthermore, in these mice neurofilamentous accumulations in cell bodies and proximal axons are rarely present, although they are a constant feature of the pathology in SOD1-linked fALS and in

Wang and coll. (2002) engineered the human SOD1 gene, to encode mutations at the first two of the four histidine residues (46, 48, 63 and 120) that coordinately bind Cu2+ in the active site of the enzyme (Parge et al., 1992), as found in a Japanese and an English families, respectively (Aoki et al., 1995; Enayat et al., 1995). The experimental juxtaposition of diseaselinked mutations at histidine 46 and 48 (H46R/H48Q) in transgenic mice creates a mutant enzyme with little or no superoxide scavenging ability that induced MN disease (Wang et al., 2003). These mice develop MN disease before 1 year of age, proportionally to the expression level of the transgene. The most prominent pathological feature in the spinal cord from paralyzed mice consists in the accumulation of high molecular weight SOD1 aggregates, and non-native, detergent-insoluble species of mutant protein. Combining the two disease-causing mutations at histidines 46 and 48 with two experimental mutations at histidines 63 and 120 (H63G and H120G), a stable but inactive protein (H46G/H48Q/H63G/H120G or Quad) is obtained: its expression in mice results in a MN disease clinically and pathologically similar to the H46R/H48Q mouse model (Wang et al.,

Among the reported mutations, the hSOD1-D90A is the milder. Homozygous mice develop a fatal MN disease, but the progression is slow. Mice display bladder disturbances similar to those found in human fALS homozygous for this mutation (Jonsson et al., 2006). Transgenic

by 4 months.

2003).

sALS (Hirano et al., 1984; Kato et al., 1991).

Recently, mutations in the genes encoding the TAR DNA/RNA-binding protein 43 (TDP-43) (Corrado et al., 2009; Gitcho et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008; Yokoseki et al., 2008) and Fused in sarcoma/Translocated in liposarcoma (FUS/TLS) (Corrado et al., 2010; Kwiatkowski et al., 2009; Vance et al., 2009) have also been identified in approximately 5%-10% typical fALS.

Other genetic causes of rare and/or atypical fALS include mutations in the genes encoding alsin (ALS2; Hadano et al., 2001) and senataxin (ALS4; Chen et al., 2004) in juvenile ALS, spatacsin (ALS5; Orlacchio et al., 2010), vescicle-associated membrane protein B (VAPB; ALS8; Nishimura et al., 2004), angiogenin (ALS9; Greenway et al., 2006), optineurin (ALS12; Maruyama et al., 2010) and dynactin (Puls et al., 2003). Looking for a genotype-phenotype correlation for the different genes, a significant correlation have been demonstrated only for single mutations and considerable intrafamilial phenotypic differences were observed in some families carrying various mutations in the SOD1, TARDBP and FUS genes (Millecamps et al., 2010).

Aiming to unravel the pathogenic mechanisms leading to ALS, several animal models based on genetic mutations in fALS have been created.
