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

176 Amyotrophic Lateral Sclerosis

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

In ALS, it is difficult to assess which of all the processes that have been found to be altered are causal to neurodegeneration and which are homeostatic, adaptive mechanisms that are actually allowing the maintenance function. Despite this, it is useful to map out the timing of the various altered properties collected from the mouse models, as presented in Figure 1. Depending on the particular SOD1 mouse model studied, the magnitude and timing of alterations observed does vary (reviewed in Elbasiouny et al, previous chapter). However, for this chapter, the deficits in these mice will be considered in their entirety and not

Long before the onset of overt symptoms, within the first week after birth, electrical properties are altered. These properties include an increase in excitability (as measured by both the Na+- and Ca2+– mediated persistent inward current; PIC) and an increased neuronal size (including increased dendritic branching and increased specific input conductance). Significantly larger PICs first appear in cultured embryonic spinal and cortical motoneurons (Kuo et al., 2005, Pieri et al., 2009), persist at an age of about one week in spinal and hypoglossal motoneurons (van Zundert et al., 2008, Quinlan et al., 2011) and are likely still present in the spinal and cortical motoneurons of adults (Carunchio et al., 2010, Meehan et al., 2010). Interestingly, although the PIC is upregulated very early, what might otherwise be the beginning of motoneuron hyperexcitability is instead moderated by changes in size and specific input conductance (Amendola and Durand, 2008, Elbasiouny et al., 2010, Quinlan et al., 2011). In adulthood, but still well before the onset of symptoms, there are signs of defective protein degradation, endoplasmic reticulum (ER) stress, impaired axon transport, and deficiencies in mitochondrial function. Signs of aberrant protein clearance include increased expression of genes related to ubiquitination, UPR, and ER stress (Saxena et al., 2009). As these changes might suggest, there is a buildup of insoluble SOD1 proteins at this time (Johnston et al., 2000, Turner et al., 2003a), followed shortly by fragmentation of the Golgi (Mourelatos et al., 1996). The next signs of impairment appear in the mitochondria and in the cellular transport system (Zhang et al., 1997, Warita et al., 1999, Williamson and Cleveland, 1999, Mattiazzi et al., 2002, Kieran et al., 2005, Damiano et al., 2006, De Vos et al., 2007, Bilsland et al., 2008, Jaiswal et al., 2009, Nguyen et al., 2009, Bilsland et al., 2010, Li et al., 2010). The immune response is initiated next (Alexianu et al., 2001, Chiu et al., 2008, Gowing et al., 2008, Chiu et al., 2009). After this, denervation of the motor units and loss of maximal force begins (Kennel et al., 1996, Frey et al., 2000, Fischer et al., 2004, Hegedus et al., 2007, Hegedus et al., 2008), but the impairment of normal function in the mouse is subtle and onset of overt symptoms is several weeks off, even in the most severe models. Just before the impending functional loss, several of the

separated based on the particular model from which the results were obtained.

malfunction in the neurons that are most vulnerable to the disease.

**3. Timeline of deficits** 

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.

Molecular and Electrical Abnormalities in the Mouse Model of Amyotrophic Lateral Sclerosis 179

While Ca2+ currents are increased in SOD1 motoneurons, large spinal and hypoglossal motoneurons do not have Ca2+-binding proteins calbindin and parvalbumin and thus cannot quickly neutralize large influxes of Ca2+. Instead, they depend heavily on mitochondrial uptake of Ca2+ (Ren and Ruda, 1994, Lips and Keller, 1998, Palecek et al., 1999, Bergmann and Keller, 2004). Small ocular motoneurons which have calbindin, parvalbumin and high Ca2+-buffering capacities are unaffected by ALS (Vanselow and Keller, 2000, von Lewinski and Keller, 2005). Ca2+-binding ratio, Ks, depends on Ca2+ binding proteins, the intracellular [Ca2+]i, and the size and geometry of the cell (Neher, 1995). Although Ca2+ buffering at the soma of neonatal SOD1 and WT motoneurons was similar (von Lewinski et al., 2008), buffering has not been measured in adult motoneurons or in the processes, where Ca2+ channels are located (Sukiasyan et al., 2009). Ca2+ buffering could also change postnatally in motoneurons, as in rat Purkinje cells in which the Ca2+-binding ratio more than doubles between P6 and P15 (Fierro and Llano, 1996). The increasing Ca2+ entry with postnatal maturation combined with the lack of Ca2+-buffering proteins seems likely to contribute to motoneuronal vulnerability

The lack of Ca2+-buffering proteins in vulnerable motoneurons make the mitochondria even more critical to their function. Mitochondria are normally highly mobile both in axons and dendrites (MacAskill et al., 2010). Mitochondrial movement can be halted by increased concentrations of ADP, so they tend to remain in compartments which are highly metabolically-active (Mironov, 2007). Mitochondrial movements are also regulated through Ca2+ signaling and synaptic activity (Rintoul et al., 2003, Yi et al., 2004, Macaskill et al., 2009). When glutamate binds NMDA- or certain AMPA-type receptor-channels, it allows the influx of Na+ and Ca2+ into the cell. The Ca2+-sensitive domain of Miro, the mitochondrial trafficking protein, then interacts with Ca2+ and the transport factors TRAK and KIF5, and pauses in its movement at active synapses (Rintoul et al., 2003, MacAskill et al., 2009). Postsynaptic NMDA receptors are also associated with PSD95 and with nitric oxide synthase (NOS) which, through nitric oxide (NO), also pauses mitochondrial movement (Rintoul et al., 2006). Once at a synapse, the mitochondria are probably tethered by neurofilaments, a process that depends both on the state of phosphorylation of the neurofilaments and a high mitochondrial membrane potential which indicates a high level

In axons, but not in the soma of cultured SOD1 motoneurons, mitochondria are more sparsely distributed (De Vos et al., 2007), and *in vivo* mitochondria show more frequent pauses in their movements in pre-symptomatic SOD1 mice (Bilsland et al., 2010). Unfortunately, movement of mitochondria and other membrane-bound organelles has not yet been well studied in the dendrites of SOD1 motoneurons. If the mitochondria are similarly sparse in the dendrites, where most Ca2+ channels are located, this could have serious consequences for Ca2+ buffering. Spinal motoneurons of SOD1 mice show a significant proliferation in dendritic branches (Amendola and Durand, 2008) and an increased Ca2+ PIC (Quinlan et al., 2011), which could make mitochondrial motility in the dendrites more challenging. Without mitochondria to take up Ca2+ at the synapses, this would further exacerbate the low Ca2+ buffering in vulnerable motoneurons and any increased Ca2+ entry with synaptic inputs (Tortarolo et al., 2006). It is also worth noting that

in adulthood.

**5. Impaired transport, more places to go** 

of activity (Wagner et al., 2003).

 1(Quinlan et al., 2011),2(Kuo et al., 2005), 3(van Zundert et al., 2008), 4(Meehan et al., 2010), 5(Carunchio et al., 2010), 6(Pieri et al., 2009), 7(Bories et al., 2007), 8(Amendola and Durand, 2008), 9(Saxena et al., 2009), 10(Johnston et al., 2000), 11(Turner et al., 2003b), 12(Mourelatos et al., 1996), 13(Li et al., 2010), 14(Jaiswal and Keller, 2009),15(Mattiazzi et al., 2002), 16(Nguyen et al., 2009), 17(Jaiswal et al., 2009), 18(Bilsland et al., 2008), 19(Damiano et al., 2006), 20(Bilsland et al., 2010), 21(De Vos et al., 2007), 22(Williamson and Cleveland, 1999), 23(Zhang et al., 1997), 24(Kieran et al., 2005), 25(Warita et al., 1999), 26(Alexianu et al., 2001), 27(Gowing et al., 2008), 28(Chiu et al., 2008), 29(Chiu et al., 2009), 30(Fischer et al., 2004), 31(Frey et al., 2000), 32(Pun et al., 2006), 33(Hegedus et al., 2007), 34(Hegedus et al., 2008), 35(Kennel et al., 1996), 36(Bruijn et al., 1997), 37(Munch et al., 2002), 38(Tortarolo et al., 2006), 39(Bendotti et al., 2001), 40(Dal Canto and Gurney, 1995), 41(Dal Canto and Gurney, 1994), 42(Kaiser et al., 2006).

Fig. 1. Timeline of deficits in mutant SOD1 mice. Earliest reported deficits in the above properties are used. Different SOD1 mutants were normalized to dates of overt symptom onset. When differences in timing between mouse lines were large (as it was for protein ubiquitination, stress of the ER, and activation of astrocytes), the range is indicated in the timeline with (///). † Also found in embryonic cultured motoneurons. **\*** Different aspects of mitochondrial function were impaired at different time points. The first alteration in function is decreased Ca2+ storage capacity19. Another property, mitochondrial membrane potential, is not altered until just before symptom onset17, while the function or regulation of the electron transport chain is impaired slightly before membrane potential16.

While Ca2+ currents are increased in SOD1 motoneurons, large spinal and hypoglossal motoneurons do not have Ca2+-binding proteins calbindin and parvalbumin and thus cannot quickly neutralize large influxes of Ca2+. Instead, they depend heavily on mitochondrial uptake of Ca2+ (Ren and Ruda, 1994, Lips and Keller, 1998, Palecek et al., 1999, Bergmann and Keller, 2004). Small ocular motoneurons which have calbindin, parvalbumin and high Ca2+-buffering capacities are unaffected by ALS (Vanselow and Keller, 2000, von Lewinski and Keller, 2005). Ca2+-binding ratio, Ks, depends on Ca2+ binding proteins, the intracellular [Ca2+]i, and the size and geometry of the cell (Neher, 1995). Although Ca2+ buffering at the soma of neonatal SOD1 and WT motoneurons was similar (von Lewinski et al., 2008), buffering has not been measured in adult motoneurons or in the processes, where Ca2+ channels are located (Sukiasyan et al., 2009). Ca2+ buffering could also change postnatally in motoneurons, as in rat Purkinje cells in which the Ca2+-binding ratio more than doubles between P6 and P15 (Fierro and Llano, 1996). The increasing Ca2+ entry with postnatal maturation combined with the lack of Ca2+-buffering proteins seems likely to contribute to motoneuronal vulnerability in adulthood.
