**1. Introduction**

436 Amyotrophic Lateral Sclerosis

Tavolato BF. (1975). Immunoglobulin G distribution in multiple sclerosis brain. An

Tobinick E., H. Gross, A. Weinberger, H. Cohen. (2006). TNF-alpha modulation for treatment of Alzheimer disease: a 6-month pilot study. MedGenMed, 8, 25. Tobinick E., H. Gross. (2008). Rapid improvement in verbal fluency and aplasia following

Tracey D., L. Klareskog, E.H. Sasso, J.G. Salfeld, P.P. Tak. (2008). TNF antagonist mechanisms of action : a comprehensive review. Pharmacol Ther, 117, 244-279. Troost D, Van den Oord JJ, Vianney de Jong JM. (1990). Immunohistochemical

Van oosten B.W., F. Barkhof, L. Truyen, J.B. Boringa, F.W. Bertelsmann, B.M. Von Blomberg,

Veglianese P, Lo Coco D, Bao Cutrona M, Magnoni R, Pennacchini D, Pozzi B, Gowing G,

Wengenack TM. (2004). Activation of programmed cell death markers in ventral horn motor

Westall FC, Rubin R, Gospodarowicz D. (1983). Brain-derived fibroblast growth factor: a

Weydt, P., Weiss, M.D., Möller, T., Carter, G.T. (2002). Neuro-inflammation as a therapeutic target amyotrophic lateral sclerosis. Curr. Opin. Investig. Drugs 12, 1720–1724. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. (1997). Effects of a

Vlahopoulos S, Boldogh I, Casola A, Brasier AR. (1999). Nuclear factor-kappaB-dependent

Wong GHW and Goeddel DV. (1988). Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science 242: 941-944. Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH. (2000).

Yoshihara T., Ishigaki S., Yamamoto M., Liang Y., Niwa J., Takeuchi H., Doyu M., Sobue G.

Zhao X, Bausano B, Pike BR, Newcomb-Fernandez JK, Wang KK, Shohami E, Ringger NC,

characterization of the inflammatory infiltrate in amyotrophic lateral sclerosis.

J.N. Woody, H.P. Hartung, C.H. Polman (1996). Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal

Julien JP, Tortarolo M, Bendotti C. (2006). Activation of the p38MAPK cascade is associated with upregulation of TNF alpha receptors in the spinal motor neurons of

neurons during early presyntomatic stages of amyotrophic lateral sclerosis in a

polymorphism in the human tumor necrosis factor alpha pronoter on

induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation.

Tumor necrosis factor-alpha generates reactive oxygen species via acytosolic

(2002). Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic

DeFord SM, Anderson DK, Hayes RL. (2001). TNF-alpha stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures. J

immunofluorescence study. J Neurol Sci. Jan;24(1):1-11.

Neuropathol Appl Neurobiol. Oct;16(5):401-10.

anti-TNF antibody cA2. Neurology, 47, 1531-1534.

transgenic mouse model. Brain Res. 1027:73-86

lateral sclerosis. J.Neurochem. 80:158-167

Blood 94: 1878-1889.

Neurosci Res 64:121–131

study of its inactivation. Life Sci. Dec 12;33(24):2425-9.

perispinal etanercept in Alzheimer disease. BMC Neurol, 8, 27.

mouse models of familial ALS. Mol Cell Neurosci. 31(2):218-31

transcriptional activation. Proc Natl Acad Sci USA; 94:3195-3199.

phospholipase A2-linked cascade. J. Biol. Chem. 275: 32357-32362.

### **1.1 ALS and the SOD1 rodent models**

Amyotrophic lateral sclerosis (ALS) is a progressive disorder that leads to degeneration of upper and lower motor neurons, muscular atrophy, and (ultimately) death. A clinical diagnosis of ALS requires signs of progressive degeneration in both upper and lower motor neurons, with no evidence that suggest that the signs can be explained by other disease processes (Brooks et al., 1994, 2000). The incidence rate of the disease is around 2 in 100,000 people (Hirtz et al., 2007). The onset age of sporadic and most familial form of ALS is between 50-60 years, and is generally fatal within 1-5 years of onset (Cleveland & Rothstein, 2001). Riluzile is the only drug that demonstrates a beneficial effect on ALS patients, but only increases survival by a matter of months (Zoccolella et al., 2009).

Motor neuron cell death in ALS probably involves multiple pathways. Most ALS cases are sporadic in nature, while ~10% arise from a dominantly inherited trait (familial ALS or FALS) (Brown, 1995). The cause for sporadic ALS remains unclear, while 20% of FALS patients have a point mutation in the cytosolic Cu2+/Zn2+ superoxide dismutase 1 (SOD1) gene (Rosen et al., 1993). Recent reports suggested that other causes of FALS also include mutations in TDP-43 (the 43-KDa TAR DNA binding protein) and FUS (Fused in sarcoma/translocated in liposarcoma) genes (Ticozzi et al, 2011). From various lines of transgenic mice, we can observe that motor neuron disease is developed in mutants with elevated SOD1 levels (ex. hSOD1-G93A line), while no symptoms are observed in SOD1 knockout mice. The combined effect shows that SOD1 acts through a toxic gain of function rather than loss of dismutase activity (Julien et al., 2001). Both mouse and rat models overexpressing SOD1 genes show similar disease phenotypes and disease progression to those observed in human ALS patients (Gurney, 1994; Nagai et al., 2001; Howland et al., 2002).

The mechanism underlying motor neuron death in ALS is still unknown. However, SOD1 mutant induces non-cell-autonomous motor neuron killing by an unknown gain of toxicity, which means the gain of toxicity arises from damage to cells other than motor neurons (Boillée et al., 2006a). Multiple mechanisms account for the selective vulnerability of motor neurons including protein misfolding, mitochondrial dysfunction, oxidative damage, defective axonal transport, excitoxicity, insufficient growth factor signaling, and inflammation (Boillée et al., 2006a). Of course there are a lot of shortcomings for using

Stem Cell Application for Amyotrophic Lateral Sclerosis: Growth Factor Delivery and Cell Therapy 439

barrier; (ii) unwanted side effects in non-targeted sites, and (iii) a relative short half-life of the protein. The significance of these issues is amplified in the human nervous system because of greater cross-sectional area when compared to rodents. Further penetration is needed for the injected growth factor to reach the deep structure in the brain or spinal cord to give its desired effect. Similar issues are found in clinical trials for patients with

The second method is to deliver the chemical of interest by implanting a catheter directly into the site of the brain that needs the growth factor, as seen in a couple Parkinson's disease studies (Gill et al., 2003; Slevin et al., 2005). It is better than the previous method as it overcomes the distance problem seen in large animals. However, there are a couple of drawbacks if this is applied to ALS patients to deliver the growth factor into the spinal cord instead of the brain for Parkinson's disease. The implanted catheter might interrupt the ascending and/or descending white matter track, and the natural movement of the spinal cord in patients increase the shearing forces may cause further damage. Therefore catheter

The last approach uses viral vectors to circumvent all those issues. Those viruses include lentivirus (Cisterni et al., 2000; Hottinger et al., 2000; Azzouz et al., 2004), adenovirus (Acsadi et al., 2002; Hasse et al., 2007), and adeno-associated virus (AAV) (Kasper et al., 2003; Wang et al., 2002). They are used because of the ability to deliver genes to non-dividing cells, which includes mature neurons. Thus they are ready to be engineered to encode the therapeutic protein. Extensive studies of AAV delivery of potential drugs to specific brain regions have

Studies have been done to inject vectors encoding the growth factor of interest into two distinctive types of tissues: (i) limb/respiratory muscles and (ii) the connecting motor neurons. In most ALS studies the vectors are injected in the muscle. Although positive results are shown in studies with GDNF and IGF-1, researchers believes that motor neurons may detach from the muscle at early stages of the disease (Fischer et al., 2004), or the cellular transport mechanism is heavily impaired (Williamson & Cleveland, 1999; De Vos et al., 2007). Again due to their large cross-sectional area, retrograde transport is more severely affected in larger mammals when compared to mice, and thus requires a longer distance of transport. This factor may slow the translation of this successful strategy to clinical trials. To overcome the potential problems of retrograde transport that may be encountered in muscle injections in humans, studies that inject vectors directly to motor neurons within spinal cord has been performed. Surprisingly, only a few studies have been published on this approach and the effect is less significant than the muscle injection studies. In a GDNF study on ALS mice, neuroprotection is only seen on facial but not lumbar motor neurons (Guillot, 2004). Another study supports the above idea by showing that GDNF is neuroprotective when it is overexpressed in skeletal muscles, but has no effect when the growth factor is overexpressed in motor neurons (Li et al., 2007). Disease progression is only slowed when GDNF is expressed in skeletal muscles, but not when it is expressed in the motor neurons.

**2.3 Insights from growth factor studies to understand ALS disease progression**  Although the ultimate goal of growth factor therapy for ALS is to alleviate symptoms, prolong survival, delay onset, and slow disease progression, during the course of

Parkinson's disease using the same strategy to deliver growth factors.

delivery would not be a desirable method of ALS growth factor delivery.

been published, suggesting viral vector delivery is a practical method.

**2.2.2 Sites of delivery** 

G93A and other SOD1 transgenic rodent models as SOD1 mutation is only found in a small proportion of human ALS patients. However, it is still an excellent tool for ALS researchers as transgenic mice have proven to be one of the most useful tools to understand the complexity of neurodegenerative diseases because of their usefulness to unveil underlying mechanisms of the disease and evaluating potential treatments (Rothstein, 2004). In this review we will overview the extensive use of SOD1 transgenic rodent models in ALS research and how those findings can be transferred to treat human ALS patients.
