**9. Future directions**

182 Amyotrophic Lateral Sclerosis

families from whom they were isolated (Deng et al., 2011). Even in sALS patients, ubiquilin-2 was found in abnormal protein aggregates in degenerating neurons, indicating it could play a broad role in both fALS and sALS pathology (Deng et al., 2011). These studies suggest

In healthy neurons, the resting [Ca2+] in the ER remains high. When ER [Ca2+] drops, the Ca2+-sensing STIM proteins promote Ca2+-channel formation (Luik et al., 2008). Blocking this ER-mediated Ca2+-entry affects neuronal activity and under conditions of chronic hyperexcitability, STIM proteins are upregulated (Steinbeck et al., 2011). Contributions to electrophysiological excitation-mediated Ca2+ transients from ER Ca2+ release have been documented in motoneurons (Scamps et al., 2004, Jahn et al., 2006). Supporting the possibility that neuronal excitability and neuronal protein processing and ER function could share common pathways, blocking L-type Ca2+ channels has been reported to increase autophagy (Williams et al., 2008). To summarize, due to the large role Ca2+ plays in cell signaling, (McCue et al., 2010, Pivovarova and Andrews, 2010), even small changes in

electrophysiological properties could have broad consequences in cellular function.

**8. Non-cell autonomous deficits: Astrocytes and glutamate excitotoxicity** 

Recent work has shown that the vulnerability of motoneurons is not cell autonomous, and that glia play critical roles in neurodegeneration in SOD1 mice. The involvement of astrocytes and microglia in the disease were elegantly demonstrated in a series of studies using mice with deletable mutant SOD1, mice with a selective knockdown of SOD1, and SOD1/WT chimera mice (Clement et al., 2003, Boillee et al., 2006, Yamanaka et al., 2008, Wang et al., 2009). Simply culturing WT motoneurons on mutant SOD1 astrocytes was sufficient to confer toxicity to motoneurons (Nagai et al., 2007). Glia have this effect on motoneurons through a variety of pathways, including activation of astrocytes, microglia, and T cells shortly after the first signs of pathology appear. The glial response is thought to influence the progression, but not the onset, of the disease (Beers et al., 2006, Boillee et al., 2006, Yamanaka et al., 2008, Wang et al., 2009, Philips and Robberecht, 2011). Presymptomatic involvement of the glia includes a reduction of glial K+ channel expression shortly before the onset of symptoms (Kaiser et al., 2006) and later in the course of the disease, a reduced expression of astroglial glutamate transporters, GLT1/EAAT2 which mediate glutamate reuptake at synapses and help prevent glutamate excitotoxicity (Bruijn et al., 1997, Bendotti et al., 2001, Warita et al., 2002). Earlier alterations in EAAT2 function are likely due to expression of different splice variants rather than decreased expressions levels (Sasaki et al., 2001, Munch et al., 2002, Ignacio et al., 2005). Some ALS patients also show abnormal splice variants of EAAT2, which could lead to decreased glutamate transport (Rothstein et al., 1992, Maragakis et al., 2004, Lauriat et al., 2007). Stimulation of the expression and transporter activity of EAAT2/GLT1 increases the lifespan of mutant SOD1 mice (Rothstein et al., 2005). An additional, critical function of the glia is regulation of the glutamate receptor's pore-forming GluR2 subunit (Van Damme et al., 2007). The challenges of Ca2+ buffering are exacerbated by alterations in the glutamate signaling across disease models of ALS. In SOD1 motoneurons, expression of subunits in the AMPA-type glutamate receptors is shifted from Ca2+-impermeable to Ca2+-permeable (Tortarolo et al., 2006). In TDP mice, levels of RNA that encode proteins involved in synaptic activity, including glutamate receptors, ion channels and voltage gated Ca2+ channels, are altered, with unknown consequences on synaptic transmission (Polymenidou et al., 2011). Lastly, in sALS

a key role for protein degradation and ER stress in ALS pathology.

There are many possibilities to explore for new treatments of ALS besides the nowstandard drug riluzole (Bellingham, 2011). The neuroinflammation response is a promising approach (Philips and Robberecht, 2011); another could be to manipulate neuromodulatory input to the spinal cord. Serotonin (5HT) and norepinephrine (NE) have potent effects on motoneurons, including increasing PIC amplitude, decreasing input conductance, hyperpolarizing spike threshold, and depolarizing resting potential (Hounsgaard and Kiehn, 1989, Lee and Heckman, 1999, Powers and Binder, 2001, Alaburda et al., 2002, Hultborn et al., 2004, Perrier and Delgado-Lezama, 2005, Heckman et al., 2008). Furthermore, neuromodulators are constantly scaling the level of activation of motoneurons as needed (Heckman et al., 2004). Activation of 5HT2 receptors strongly depresses high-voltage-activated Ca2+ channels while probably increasing basal [Ca2+]I by potentiating the Ca2+ PIC (Hounsgaard et al., 1988, Bayliss et al., 1995, Hsiao et al., 1998, Ladewig et al., 2004, Li et al., 2007). Both 5HT and dopamine (DA) modulate KIF-5 dependent cellular transport, including transport of mitochondria. Acting through the GSK3 regulator of KIF-5, 5HT is observed to increase transport, while DA decreases it (Chen et al., 2007, Chen et al., 2008). Other neuromodulators, such as nitric oxide, GABAB, and adenosine, could also be worth investigating as modulators of motoneuron synaptic strength, reduction of the Ca2+ PIC, and modulation of both high-voltage-activated Ca2+ channels and input conductance, respectively (Marks et al., 1993, Mynlieff and Beam, 1994, Li et al., 2004, Moreno-Lopez et al., 2011). Another useful target of neuromodulators that modify Ca2+ influx is protein clearance; inhibition of L-type Ca2+ channels has been found to increase autophagy (Williams et al., 2008).

### **10. Conclusions**

Factors causing neurodegeneration in ALS are present long before motor function is adversely affected. From research on the animal models of ALS, it is thought that excessive

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Ca2+ entry, increased motoneuronal size, altered glutamate neurotransmission, astrocyte dysfunction, mitochondrial deficits, failures in axon transport, and problems in protein degradation act in concert and gradually push motoneurons outside the parameters under which they can function properly. The fact that motoneurons are able to remain functioning for as long as they do under adverse conditions suggests that there is a large window of time and intrinsic conditions within which motoneurons can maintain normal function. Hopefully future treatments can target these altered pathways to extend the time motoneuron properties remain within these parameters.
