**3.3 PMN mouse**

Bommel and colleagues (2002) identified animals suffering of a progressive motor neuronopathy (pmn), caused by an autosomal recessive mutation in the tubulin-specific chaperone e gene (tbce). The mutation results from a tryptophan to glycine substitution at position 524 (T524G; Martin et al., 2002). This protein is one of the main cofactors involved in MT stability or polymerization dynamics. These mice between P14 and P20 display a progressive caudal-cranial degeneration of motor axons leading to muscular atrophy, hindlimb paralysis and death in 4 to 6 weeks after birth (Schmalbruch et al., 1991).

Although the pathophysiology of MN death in pmn mice is mainly due to loss of neuromuscular integrity by distal axonopathy rather than alteration of neuron soma (which is instead a common feature in ALS), this model can be considered a good one for MN neurodegeneration.

### **3.4 WASTED mouse**

Wasted (wst) mice bear a spontaneous autosomal mutation that results in the loss of gene encoding the translation factor eEF1A2. From the 3rd week of age they exhibit immunodeficiency and neurological disorders in terms of tremors and disturbance of gait. Subsequently these mice present MN degeneration and hindlimb paralysis.

Moreover, Newbery and coll. (2005) observed reactive gliosis at P19 in the cervical spinal cord with a rostrocaudal gradient, and retraction of MN fibers. Mice develop a rapid neurodegeneration and die around P28. Finally, several studies demonstrated that this model bears increased chromosomal breakage and DNA damage (Shultz et al., 1982).

### **3.5 LOA and CRA mice**

"Legs at odd angles" (Loa) and "Cramping 1" (Cra) mice arose as the result of two independent N-ethyl-N-nitrosourea-(ENU) induced mutagenenesis experiments (Hrabe de Angelis et al., 2000; Rogers et al., 2001).

Loa mouse presents a point mutation that changes phenylalanine to tyrosine at position 580 (F580Y) in the gene encoding the heavy chain of dynein. On the other hand, Cra mice have a substitution from tyrosine to cytosine at position 1055 (Y1055C) in the same gene. Dynein is

Advantages and Pitfalls in Experimental Models Of ALS 137

*In vitro* preparations are age-dependent, since cells or tissues are generally obtained from animals at late embryonic or neonatal stages, but ALS related-alterations or neurodegenerative defects are not yet evident at this early stage. Indeed Avossa and coll. (2006) characterized the alterations in WT and G93A embryonic spinal cord cells analyzing MNs, glial cells, interneurons, distribution of hSOD1, mitochondria, MTs, NFs and synapses. They did not find any substantial difference in all these parameters, excepted for a significant increase in inhibitory synapses compared to excitatory ones in mutant cells. Therefore, it is necessary to use specific molecules or drugs to induce cell death mechanisms in these apparently normal cells. Since in ALS an imbalance in the glutamatergic system has been described, leading to an excitotoxic environment around MNs (Boillee et al., 2006), an *in vitro* approach can be represented by NMDA exposure. Rothstein and coll. (1993) were the first to use this model, demonstrating that MN degeneration is prevented by non-NMDA glutamate receptor

Similarly, some authors have proposed that spinal MNs are more vulnerable to AMPA receptor agonists than to NMDA, particularly in spinal cord cultures (Carriedo et al., 1996; Van Damme et al., 2002). In fact, microdialysis perfusion of AMPA agonists in spinal cord reveals a consistent loss of spinal MNs and the consequently paralysis of the hindlimbs,

Finally, it is well-known that also the glia (astrocytes and microglial cells) actively contributes to the death of MNs (Philips & Robberecht, 2011). Glia can be easily obtained and cultured both from embryos/newborns and adults; the first method has been described in 1972 by Booher and Sensenbrenner (1972). Now, concerning astroglia culture, "primary culture", "subculture" and "shaken culture (once or twice)" can be performed (Du et al., 2010), each one with advantages and pitfalls relative to purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin, a protein potentially involved in embryo implantation, which is markedly up-regulated in reactive astrocytes (Fang et al., 2008). Similarly, protocols to isolate microglial cells are easy and reproducible (Ni & Aschner, 2010; Yip et al., 2009), and they have been recently improved by the column-free magnetic separation technology: the cells can be labeled and isolated on the strength of their expression of CD11b, a specific microglial marker, thus allowing to isolate an high number

Cocultures of healthy hMNs with human astrocytes carrying either the WT or mutated SOD1 cDNA have demonstrated the role of astrocytes in ALS disease, since MN number decreased about 50% in the presence of mutant SOD1-expressing astrocytes (Marchetto et al., 2008). This neurotoxicity is probably mediated by the release of soluble factors from astrocytes, finally involving the Bax-dependent death machinery within MNs (Nagai et al.,

Due to the technical difficulties in establishing a MN culture for their poor proliferation ability when differentiated, an alternative is represented by the use of a neural hybrid cell line named NSC-34 (neuroblastoma spinal cord), derived from the fusion of neuroblastoma cells with MN enriched spinal cord. These cells perfectly show morphological and physiological properties of primary MNs: extending processes, contacts with cultured myotubes, synthesis and storage of acetylcholine, action potentials and NF proteins

probably due to an increased cytoplasmic Ca2+ concentration (Corona & Tapia, 2007).

of cells and significantly reducing the animals needed (Gordon et al., 2011).

2007).

**4.2 Cell line culture** 

(Cashman et al., 1992).

antagonists, consequently used as neuroprotective agents.

a motor protein which determines retrograde transport along MTs and this model has been partially used to evaluate the role of dynein in the neuronal migration. However, the disturbance in axonal transport leads to selective MN death, making these mice useful models in ALS studies. Particularly, Loa mice exhibit pathological features similar to human ones, as Lewy body-like inclusions containing SOD1, CDK5 and ubiquitin. However, Ilieva et al., (2008) found no -MN loss at any age, but a sensory axon deficit of the large proprioceptive (type Ia) axons and the decrease of -motor axons innervating muscle spindles. Similarly, Cra mice do not show MN degeneration but rather peripheral sensory neuropathy due to loss of axons in dorsal roots and decrement of large proprioceptive axons (Dupuis et al., 2009).

In conclusion, the pathological features observed in both models do not properly correlate with hALS characteristics. Dynein mutation models will need further investigation, they better represent a model for sensory neuropathy rather than for MN disease.

### **3.6 Hereditary canine spinal muscular atrophy**

Hereditary canine spinal muscular atrophy (HCSMA) is a lower motor neuron disease found in Brittany Spaniels. It shares clinical and pathological features with human ALS (Green et al., 2002). These animals show signs of oxidative stress (Green et al., 2001), but do not have mutations in the SOD1 gene (Green et al., 2002). From the histopathological point of view these animals are characterized by aberrant accumulation of extensively phosphorylated heavy (high molecular weight) neurofilament (NFH) and neurodegeneration (Green et al., 2005).
