**4.** *In vitro* **models**

Despite *in vivo* models are extremely helpful for studying ALS, when the aim consists in studying the molecular mechanisms of MN death and the role and the involvement of the different cells in the pathological mechanisms, simplified *in vitro* models such as MN and organotypic cultures can be useful, allowing to control the experimental conditions.

### **4.1 Motoneuron and glia culture**

Since the major feature of ALS consists in the progressive loss of upper and lower MNs, cultures of spinal MNs are a valuable tool for studying ALS pathomechanisms. Many authors have developed protocols to isolate MNs from newborn or embryonic murine spinal cord (Berg & Fischbach, 1978; Gingras et al., 2007; Schnaar & Schaffner, 1981), allowing to identify MNs for their size and their Choline Acetyl Transferase (ChAT) activity. However, more recently, Wiese and coll. (2010) have isolated embryonic spinal MNs from rodents using p75 neurotrophin receptor (NTR)-antibody panning step, a technique with low toxicity and high efficiency; in fact, at the embryonic stage, MNs are the only neurons expressing the p75 low-affinity nerve growth factor receptor (Camu & Henderson, 1992). Unlike most studies performed on cultured embryonic spinal MNs, those of cultures of spinal MNs from newborn rodents is less common. In 2004, Anderson and coworkers described a protocol for isolating and culturing neonatal spinal MNs positive to p75NTR and ChAT, in presence of a growth factor cocktail containing glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) (Anderson et al., 2004).

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

In conclusion, the pathological features observed in both models do not properly correlate with hALS characteristics. Dynein mutation models will need further investigation, they

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

Despite *in vivo* models are extremely helpful for studying ALS, when the aim consists in studying the molecular mechanisms of MN death and the role and the involvement of the different cells in the pathological mechanisms, simplified *in vitro* models such as MN and

Since the major feature of ALS consists in the progressive loss of upper and lower MNs, cultures of spinal MNs are a valuable tool for studying ALS pathomechanisms. Many authors have developed protocols to isolate MNs from newborn or embryonic murine spinal cord (Berg & Fischbach, 1978; Gingras et al., 2007; Schnaar & Schaffner, 1981), allowing to identify MNs for their size and their Choline Acetyl Transferase (ChAT) activity. However, more recently, Wiese and coll. (2010) have isolated embryonic spinal MNs from rodents using p75 neurotrophin receptor (NTR)-antibody panning step, a technique with low toxicity and high efficiency; in fact, at the embryonic stage, MNs are the only neurons expressing the p75 low-affinity nerve growth factor receptor (Camu & Henderson, 1992). Unlike most studies performed on cultured embryonic spinal MNs, those of cultures of spinal MNs from newborn rodents is less common. In 2004, Anderson and coworkers described a protocol for isolating and culturing neonatal spinal MNs positive to p75NTR and ChAT, in presence of a growth factor cocktail containing glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and ciliary

organotypic cultures can be useful, allowing to control the experimental conditions.

better represent a model for sensory neuropathy rather than for MN disease.

**3.6 Hereditary canine spinal muscular atrophy** 

neurodegeneration (Green et al., 2005).

**4.1 Motoneuron and glia culture** 

neurotrophic factor (CNTF) (Anderson et al., 2004).

(Dupuis et al., 2009).

**4.** *In vitro* **models** 

*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 antagonists, consequently used as neuroprotective agents.

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

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

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., 2007).

#### **4.2 Cell line culture**

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 (Cashman et al., 1992).

Advantages and Pitfalls in Experimental Models Of ALS 139

This *in vitro* model is helpful either for studying specific pathways/organelles or for testing treatments. Particularly, Tolosa and coll. (2008) studied the effects of VEGF on the survival and vulnerability to excitotoxicity of spinal cord MNs treated with THA, demonstrating that this growth factor plays an important role in neuroprotection by activating the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Similarly Kosuge and coworkers (2009) tested the effect of GDNF, showing that threo-hydroxyaspartate -induced MN death was significantly inhibited in G93A mice slice. The pathway of caspase-12 was among the causes of MNs death. Also the anti-convulsant drugs topiramate (Maragakis et al., 2003), valproic acid (Sugai et al., 2004) and lithium (Caldero et al., 2010) were found to

ALS is a devastating neurodegenerative disease the etiology of which is still unclear. Even though the clinical hallmarks are similar, several different genetic and sporadic causes have been found or hypothesized. Experimental models of ALS have been created in transgenic animals, and spontaneously mutated animals affected by MN disease have been identified. Due to the multifactorial causes of the disease, all these models, even though they show motor impairment and neuropathological aspects typical of the human disease, cannot fully address the complexity of the human disease. Nevertheless, they represent useful tools to study the early and late development of the disease from a neuropathological point of view, and the molecular mechanisms involved. The major pitfall of the use of animals models thus far consists in the failure to translate the positive therapeutic outcome in human patients. This might be ascribed to mistakes in both preclinical and clinical research strategy: on one hand, animals, unlike patients, tend to be treated in the early phases of the disease and in any case represent a restricted, uniform population in terms of age, sex and genetic characteristics; on the other hand, patients represent a much more variable population. Therefore, an increasing collaboration between preclinical researchers and clinicians is

Andersen, P.M. (2000). Genetic factors in the early diagnosis of ALS. *Amyotroph Lateral Scler* 

Andersen, P.M., Borasio, G.D., Dengler, R., Hardiman, O., Kollewe, K., Leigh, P.N., Pradat,

Anderson, K.N., Potter, A.C., Piccenna, L.G., Quah, A.K., Davies, K.E. & Cheema, S.S. (2004).

Aoki, M., Abe, K., Houi, K., Ogasawara, M., Matsubara, Y., Kobayashi, T., Mochio, S.,

*Other Motor Neuron Disord*, 1 Suppl 1, (Mar), pp. (S31-42), 1466-0822 (Print) 1466-

P.F., Silani, V. & Tomik, B. (2007). Good practice in the management of amyotrophic lateral sclerosis: clinical guidelines. An evidence-based review with good practice points. EALSC Working Group. *Amyotroph Lateral Scler*, 8, 4, (Aug),

Isolation and culture of motor neurons from the newborn mouse spinal cord. *Brain Res Brain Res Protoc*, 12, 3, (Feb), pp. (132-136), 1385-299X (Print) 1385-299X

Narisawa, K. & Itoyama, Y. (1995). Variance of age at onset in a Japanese family

significantly prevent MN degeneration in this model.

needed to favor translation from benchside to clinics back and forth.

pp. (195-213), 1748-2968 (Print) 1471-180X (Linking)

**5. Conclusion** 

**6. References** 

0822 (Linking)

(Linking)

NSC-34 can be transfected with vectors containing mutant forms of hSOD1, allowing to study specific neurodegenerative aspects such as mitochondrial degeneration, NF accumulation or Golgi apparatus disruption (Gomes et al., 2008; Menzies et al., 2002a; Menzies et al., 2002b). This model can also enlighten on the implicated cell death pathways in ALS, i.e. the involvement of MAP kinases (Guo & Bhat, 2007), the interaction between bcl2-A1 and pro-caspase-3 (Iaccarino et al., 2011) or the involvement of kynurenine pathway implicated in the regulatory mechanisms of the immune response (Chen et al., 2011).

In addition, different therapeutic agents have been tested in culture, such as the vascular endothelial growth factor (VEGF) (Kulshreshtha et al., 2011) or the semi-synthetic tetracycline called minocycline (Guo & Bhat, 2007), known for their neuroprotective effects in neurodegeneration models (Orsucci et al., 2009; Storkebaum et al., 2004).

Similarly, other cell lines have been transfected with WT or mutant (G93A) hSOD1 and used for the same purpose: N18TG2 neuroblastoma, the non-neuronal Madin–Darby Canine Kidney (MDCK) (Raimondi et al., 2006) or the mouse embryonic fibroblast NIH3T3 cell lines (Shinder et al., 2001).

The recent finding of human induced pluripotent stem cells (iPSCs) present a novel opportunity for *in vitro* disease modeling. iPSCs can be generated from readily accessible tissue from patients. iPSCs show similar capacity for directed MN differentiation as ESCs (Boulting et al., 2011). iPSCs can be generated from patients, thus carrying the actual mutations associated with the disease and allowing to correlate, any cellular phenotype with the patient's clinical characteristics such as onset, duration, and severity of disease at the time of tissue collection. In 2008, iPSCs were generated from a skin sample from an elderly patient with fALS displaying a SOD1 mutation (Dimos et al., 2008)*.*

### **4.3 Organotypic cultures**

Since ALS is a complex disease originating from an important crosstalk among MNs, glia and muscles (Strong, 2003), a step forward is represented by using organotypic cultures, where the *in vivo* architecture is maintained and MNs as well as neighboring cells (interneurons and glial cells) are preserved. However, this system presents an important limitation, due to the lack of correlation between morphological results and behavioral impairment (as well as for all the *in vitro* preparations) and the isolated slices undergo a series of little traumas (axotomy, deafferentation…) that can unintentionally originate a neuron selection.

Preparations of spinal cord organotypic culture from newborn animals is actually done according the Stoppini's procedure (1991): MNs survive more than 2 months, maintaining their structural and metabolic characteristics and allowing both morphological and electrophysiological experiments. Moreover, these experimental conditions stimulate glial proliferation, reproducing the glial activation observed *in vivo* in ALS patients/animal models (Hall et al., 1998; Vercelli et al., 2008).

As already mentioned, such model requires tissue obtained from embryos or pups, when ALS alterations are not full-blown (Avossa et al., 2006). Therefore, it is possible to use slice cultures obtained from normal animals and to add a glutamate transporter inhibitor (as threohydroxyaspartate (THA) or D,L-threo-B-benzyloxyaspartate), able to induce a chronic glutamate neurotoxicity. This treatment causes selective MNs death, simulating the human ALS neurodegeneration (Bilak et al., 2004; Rothstein et al., 1993). Some authors prepare organotypic spinal slice cultures directly from G93A SOD1 mice and expose them to THA in order to induce loss of MNs (Kosuge et al., 2009).

This *in vitro* model is helpful either for studying specific pathways/organelles or for testing treatments. Particularly, Tolosa and coll. (2008) studied the effects of VEGF on the survival and vulnerability to excitotoxicity of spinal cord MNs treated with THA, demonstrating that this growth factor plays an important role in neuroprotection by activating the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Similarly Kosuge and coworkers (2009) tested the effect of GDNF, showing that threo-hydroxyaspartate -induced MN death was significantly inhibited in G93A mice slice. The pathway of caspase-12 was among the causes of MNs death. Also the anti-convulsant drugs topiramate (Maragakis et al., 2003), valproic acid (Sugai et al., 2004) and lithium (Caldero et al., 2010) were found to significantly prevent MN degeneration in this model.
