**7. Protein degradation and endoplasmic reticulum stress**

180 Amyotrophic Lateral Sclerosis

the motoneurons that are vulnerable are the largest: the fast, fatiguable alpha motoneurons (Pun et al., 2006, Hegedus et al., 2007, Hegedus et al., 2008). Evidence for further increases size in SOD1 motoneurons is reviewed in the previous chapter by Elbasiouny et al. Perhaps the size of the motoneuron and deficits in transport go hand in hand to produce

Axon transport has been extensively studied and is likely to contribute to ALS and to several neurodegenerative diseases, reviewed by (De Vos et al., 2008). In ALS, both slow and fast axon transport appear to be altered (Zhang et al., 1997, Warita et al., 1999, Williamson and Cleveland, 1999, Kieran et al., 2005, De Vos et al., 2007, Bilsland et al., 2010). Excessive glutamate could cause these deficiencies: high levels of glutamate activate a family of mitogen-activated protein kinases that phosphorylate neurofilaments, thereby decreasing transport (Ackerley et al., 2000, Hiruma et al., 2003, Stevenson et al., 2009). This process can be induced by NMDA or AMPA, blocked by removal of extracellular Ca2+, or reduced by application of riluzole (Hiruma et al., 2003, Stevenson et al., 2009). The protein kinases JNKs, cdk/p35 and p38, which phosphorylate heavy and light chains of kinesin and medium and heavy neurofilament sidearms, may link glutamate neurotransmission and axon transport deficits (Kawasaki et al., 1997, Schwarzschild et al., 1997, Ackerley et al., 2000, Brownlees et al., 2000, Lee et al., 2000). Further suggesting this, p38 has been found to be activated in SOD1 mice and ALS patients (Raoul et al., 2002, Tortarolo et al., 2003, Ackerley et al., 2004). Axon transport deficiencies occur early, with reports of impaired axonal integrity and dieback from the neuromuscular junction occurring weeks in advance of onset of symptoms in SOD1 mice, and appearing in cultured embryonic neurons (Kennel et al., 1996, Zhang et al., 1997, Williamson and Cleveland, 1999, Frey et al., 2000, Fischer et al., 2004, Pun et al., 2006, De Vos et al., 2007, Hegedus et al., 2007, Hegedus et al., 2008, Bilsland et al., 2010). Strengthening these results, transgenic TDP-43 mice show significantly lower levels of expression of heavy and light neurofilaments, though axon transport itself has not yet been

In motoneurons under normal conditions, the mitochondrial membrane potential powers both the Ca2+ uniporter and ATP synthase, so in periods of heavy Ca2+ influx, ATP production could be impaired (Mattson et al., 2008, Nguyen et al., 2009). The increased Ca2+ influx in SOD1 motoneurons is likely to further impair the function of mitochondria under these conditions. In addition, SOD1 mitochondria appear to be impaired in function under basal conditions (Mattiazzi et al., 2002, Nguyen et al., 2009, Li et al., 2010). Before the onset of symptoms, SOD1 mitochondria show decreased protein import, altered Ca2+ sequestering, and an exaggerated response of the electrical gradient of the inner membrane to stimulation-induced Ca2+ influx (Damiano et al., 2006, Bilsland et al., 2008, Jaiswal et al., 2009, Nguyen et al., 2009, Li et al., 2010). By the time symptoms appear there is severe damage to mitochondrial membrane potentials, respiration, the electron transport chain and ATP synthesis (Mattiazzi et al., 2002, Jaiswal and Keller, 2009). Another impairment is misfolded SOD1 binding to VDAC1, the general diffusion pore for anions and cations, including Ca2+. Both mitochondrial conductance and the uptake of ADP are thereby reduced, however, this is not observed until after the onset of symptoms (Israelson et al., 2010). Early alterations in SOD1 mitochondria must take place though

vulnerability.

assessed (Swarup et al., 2011).

another mechanism.

**6. Mitochondrial deficiency and energy balance** 

Misfolded proteins are degraded through autophagy (Yang and Klionsky, 2010). When the capacity of the cellular machinery in the ER to properly fold proteins is exceeded, cells react with the unfolded protein response (UPR) and signs of ER stress (reviewed by Ron and Walter, 2007). The UPR decreases most protein synthesis in the cell while upregulating synthesis of some ER proteins that assist in proper folding and processing of proteins. Another pathway, known as ER-associated protein degradation (ERAD), helps to clear the ER of misfolded proteins by exporting them to proteasomes where they are broken down (Bernasconi and Molinari, 2011). Proteins to be exported and degraded are marked by ubiquitination, a process in which ubiquitin molecules bind to the protein, tagging it for destruction (Bingol and Sheng, 2011). Normal ER function can be disrupted by blocking the ER-resident proteins from folding properly, inadequate functioning of the ubiquitinproteosome system, or failure to maintain a high level of Ca2+ inside the lumen of the ER (Paschen, 2003).

It is known that mice with the highest expression levels of mutant SOD1 protein have the earliest disease onset (Wong et al., 1995), and that markers for ER stress have been found in the spinal cords of sALS patients (Ilieva et al., 2007, Atkin et al., 2008, Ito et al., 2009). However, recent studies have shed more light on the role of protein degradation and ER stress in the pathology of ALS. In the first study, gene expression patterns from 3 different SOD1 mouse lines all showed an early increase in protein ubiquitination only in those motoneurons that are vulnerable to the disease. This is followed shortly by the UPR and signs of ER stress by P30 in SOD1G93A-high expressor mice (see Fig 1) (Saxena et al., 2009). In another study, cortical motoneurons from SOD1 mutant mice were compared to those from wild type mice that were fed a diet high in branched-chain amino acids (Carunchio et al., 2010). These branched chain amino acids are part of protein supplements that some athletes consume. Like mutant SOD1 neurons, cortical neurons from mice fed the highprotein diet were hyperexcitable compared with neurons from wild type mice on a normal diet. A return to normal levels of excitability after treatment with rapamycin was achieved for both the SOD1 and the amino- acid-supplement-treated cortical neurons (Carunchio et al., 2010). The protein kinase known as the mammalian target of rapamycin (mTOR) serves as an integration point for several cell signaling pathways. As its name suggests, mTOR is inhibited by rapamycin; it also inhibits protein degradation, and promotes increased cell size in some neurons (Lee et al., 2007). These results indicate that promoting autophagy with rapamycin can reduce abnormal excitability and could be beneficial for treatment of the disease (Carunchio et al., 2010). The third, most recent study described a mutation found in 5 different families, located in the gene encoding ubiquilin-2 as a novel genetic cause of fALS (Deng et al., 2011). The function of ubiquilin is to clear certain misfolded proteins during ERAD by shuttling ubiquitinated proteins from the ER to the proteasome, such that loss of ubiquilin leads to ER stress (Kim et al., 2008, Lim et al., 2009). The mutations in ubiquilin-2 found in ALS patients were also found to impair proteosome- mediated protein degradation *in vitro,* suggesting these mutations could be causing similar impairments in the

Molecular and Electrical Abnormalities in the Mouse Model of Amyotrophic Lateral Sclerosis 183

patients, there is inefficient editing of AMPRA receptor GluR2Q subunit mRNA which also causes a shift from Ca2+-impermeability of the receptors to Ca2+-permeability (Kawahara et al., 2004, Kwak and Kawahara, 2005, Kawahara et al., 2006). Glutamatergic signaling is probably a significant factor in the onset of symptoms since reducing excitatory sensory input delayed the onset of disease in SOD1 mice (Ilieva et al., 2008), and intrathecal administration of the glutamate agonist kainic acid in normal rats produced slow, selective motoneuron death similar to ALS (Sun et al., 2006). If changes in the transmission of glutamate are taking place early enough, it could alter the activity of spinal networks during normal development (Blankenship and Feller, 2010, Landmesser and O'Donovan, 1984, Marder and Rehm, 2005, Gonzalez-Islas and Wenner, 2006). Some evidence for alterations in network activity has been shown in SOD1 hypoglossal motoneurons (van Zundert et al., 2008) and spinal motoneurons (Amendola et al., 2004, Bories et al., 2007) in juvenile mice. After symptom onset, increased network activity has also been shown in the spinal cord (Jiang et al., 2009). However, considering all the documented changes in glutamatemediated neurotransmission, there has been surprisingly little research into the overall effects on cortical, brainstem and spinal network activity throughout the lifespan of the

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

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

SOD1 mouse.

**9. Future directions** 

**10. Conclusions** 

found to increase autophagy (Williams et al., 2008).

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

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.
