**2. ALS animal models**

Most cases of ALS are spontaneous (sALS), while the heritable form, familial ALS (fALS), represents about 5% of total ALS cases (Byrne et al., 2011). Of fALS patients, 20% have a mutation in the gene that encodes for the superoxide dismutase 1 (SOD1) copper/zinc enzyme (Rosen et al., 1993), 5% have a mutation in the TARDBP gene which encodes DNAbinding protein 43 (TDP-43), another 5% have a mutation in the FUS gene which encodes for the fused in sarcoma FUS/TLS protein (Mackenzie et al., 2010), some possess a mutation in the gene encoding vesicle-associated membrane protein (VAPB) (Nishimura et al., 2004), and a new study shows that some of those remaining have a mutation in the gene coding for the ubiquitin-like protein ubiquilin-2 (Deng et al., 2011). Transgenic mice expressing one of the various mutations of human SOD1, hereafter referred to as SOD1 mice; (Gurney et al., 1994, Bruijn et al., 1997, Zhang et al., 1997) are very common animal models of ALS; numerous other models of fALS are reviewed by Van Den Bosch (2011). It is not known how the SOD1 mutation leads to the degeneration of motoneurons, though it is probably not due to loss of its normal function converting superoxide into hydrogen peroxide. The mutant, misfolded protein likely possesses a toxic gain-of-function, as some mouse lines retain nearly normal levels of SOD1 enzymatic activity and still develop the disease, while SOD1

Molecular and Electrical Abnormalities in the Mouse Model of Amyotrophic Lateral Sclerosis 177

last changes before overt onset of symptoms involve the glia: activation of astrocytes, expression of different splice variants of EAAT2, decreased expression of the GluR2 subunit, and decreased number of glial K+ channels (Bruijn et al., 1997, Bendotti et al., 2001, Sasaki et al., 2001, Munch et al., 2002, Warita et al., 2002, Fischer et al., 2004, Ignacio

It is tempting to assume that the order of appearance of the altered parameters represents a chain reaction of events, but this is not necessarily the case. There is considerable interplay between these components within the neurons, such that one pathway cannot be altered without affecting any other aspect of cellular or synaptic function. These interactions will be

Entry of Ca2+ occurs through voltage-gated Ca2+ channels and through ligand-gated channels activated by glutamate, particularly the NMDA-type glutamate receptors and those AMPA-type glutamate receptors which lack the Ca2+-impermeable GluR2 subunit. Most voltage-gated Ca2+ channels open only when the cell depolarizes; however, the L-type Cav1.3 channels, which contribute to the PIC, open near the resting membrane potential (- 40mV) and allow some Ca2+ influx even when the neuron is at rest (Xu and Lipscombe, 2001). There is very little expression of Cav1.3 channels in spinal motoneurons at birth, but Cav1.3 channels are increasingly present as the motoneurons mature, reaching adult levels by postnatal day 18 (P18) in mice, (Jiang et al., 1999, Quinlan et al., 2011). The PIC sets the level of excitability in neurons: PICs allow neurons to repetitively fire action potentials, and with large PICs, neurons can sustain firing long after the depolarizing stimulus is removed (Heckman et al., 2008). In addition, motoneurons from SOD1G93A-high-expressor mice show a significantly larger PIC during postnatal development, including significantly larger amplitudes of both Ca2+ and Na+ currents (Quinlan et al., 2011). Larger PICs can increase the overall excitability of a neuron (though other factors, like size, can mitigate this), and the influx of Ca2+ could have many other consequences in cell-signaling. An increased PIC is found in cultured, embryonic, SOD1G93A-high motoneurons (both spinal and cortical), though at this point the PIC is completely Na+-based (Kuo et al., 2005, Pieri et al., 2009). Postnatally, both spinal and brainstem SOD1 motoneurons show an increased PIC (van Zundert et al., 2008, Quinlan et al., 2011), and indirect evidence suggests larger PICs persist into adulthood in SOD1 cortical and spinal motoneurons (Carunchio et al., 2010, Meehan et al., 2010). In addition to the maturation of the PIC, there is an increase in AMPA-type glutamate receptors on motoneurons (Vinay et al., 2000). These receptors normally would not contribute to Ca2+ influx since, due to a single amino acid in the pore-forming GluR2 subunit they are impermeable to Ca2+. However, in presymptomatic SOD1 motoneurons, there are fewer Ca2+-impermeable GluR2 subunits; and more Ca2+-permeable GluR3 subunits (Tortarolo et al., 2006). In sALS patients, AMPA receptors also are more Ca2+ permeable, but through a different mechanism. Spinal motoneurons of symptomatic sALS patients, but not SOD1 rats, showed inefficient editing of the mRNA, resulting in mutant, GluR2Q subunits that are Ca2+-permeable (Kawahara et al., 2004, Kwak and Kawahara, 2005, Kawahara et al., 2006). As motoneurons mature they must cope with an everincreasing burden of Ca2+ influx through voltage-gated Ca2+ channels (as the Ca2+ PIC increases with age) and SOD1 motoneurons have a heavier burden due to potentiation of

the Ca2+PIC and altered AMPA receptors which are more Ca2+-permeable.

et al., 2005, Kaiser et al., 2006).

**4. Calcium: No buffer for increased currents** 

considered next.

knockout mice, which do not possess any SOD1 enzymatic activity, do not develop the disease (Gurney et al., 1994, Reaume et al., 1996, Wong et al., 1995). Whatever the mechanism(s) leading to neurodegeneration, it is not immediate. The SOD1 enzyme is present throughout the nervous system (Pardo et al., 1995) starting embryonically, but does not lead to onset of overt symptoms until well into adulthood, even in mice that express high levels of the protein (Gurney et al., 1994). And within the nervous system, only certain neurons show susceptibility to the disease. This chapter will explore the earliest signs of malfunction in the neurons that are most vulnerable to the disease.
