**1. Introduction**

[68] Wang, L, Deng, H. X, Grisotti, G, Zhai, H, Siddique, T, & Roos, R. P. (2009). Wildtype SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse. *Hum*

[69] Wang, L, Sharma, K, Deng, H. X, Siddique, T, Grisotti, G, Liu, E, & Roos, R. P. (2008). Restricted expression of mutant SOD1 in spinal motor neurons and interneurons in‐

[70] Watanabe, M, Dykes-hoberg, M, Culotta, V. C, Price, D. L, Wong, P. C, & Rothstein, J. D. (2001). Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues. *Neurobiol Dis* , 8, 933-941. [71] Watanabe, S, Nagano, S, Duce, J, Kiaei, M, Li, Q. -X, Tucker, S. M, Tiwari, A, Brown, J. R. H, Beal, M. F, Hayward, L. J, Culotta, V. C, Yoshihara, S, Sakoda, S, & Bush, A. I. (2007). Increased affinity for copper mediated by cysteine 111 in forms of mutant su‐ peroxide dismutase 1 linked to amyotrophic lateral sclerosis. *Free Radical Biology and*

[72] Wiedau-pazos, M, Gato, J. J, Rabizadeh, S, Gralla, E. B, Roe, B, Lee, C. K, Valentine, J. S, & Bredesen, D. E. (1996). Alterd reactivity of superoxide dismutase in familial

[73] Witan, H, Gorlovoy, P, Kaya, A. M, Koziollek-drechsler, I, Neumann, H, Behl, C, & Clement, A. M. (2009). Wild-type Cu/Zn superoxide dismutase (SOD1) does not facil‐ itate, but impedes the formation of protein aggregates of amyotrophic lateral sclero‐

[74] Yim, M. B, Chock, P. B, & Stadtman, E. R. (1990). Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. *Proceedings of the Na‐*

[75] Yim, M. B, Kang, J. H, Yim, H. S, Kwak, H. S, Chock, P. B, & Stadtman, E. R. function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen per‐ oxide. *Proceedings of the National Academy of Sciences of the United States of America* , 93,

*tional Academy of Sciences of the United States of America* , 87, 5006-5010.

duces motor neuron pathology. *Neurobiol Dis* , 29, 400-408.

amyotrophic lateral sclerosis. *Science* , 271, 515-518.

sis causing mutant SOD1. *Neurobiol Dis* , 36, 331-342.

*Mol Genet* , 18, 1642-1651.

158 Current Advances in Amyotrophic Lateral Sclerosis

*Medicine* , 42, 1534-1542.

5709-5714.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by death of upper and lower motor neurons, which results in muscle wasting and death from respiratory failure typically within 2-5 years from diagnosis.

ALS is a multifactorial disease [1] where different cell types, i.e. astrocytes, microglia and oligodendrocytes, contribute to the pathologic mechanism [2, 3]. For a long time ALS was thought to be a pure motor neuron disease, however, thorough pathological investigations and recent findings linking mutations in transactive response DNA-binding protein gene (TARDBP) to familial and sporadic cases of ALS have relocated this disease within a spec‐ trum of neurological disorders, ranging from pure motor neuron disease to frontotemporal dementia [4, 5].

Since 1993, when the first mutation in the Cu/Zn superoxide dismutase (SOD1) enzyme was linked to familial forms of ALS, researchers have tried to unravel the mechanisms underly‐ ing this disease by interrogating *in vivo* and *in vitro* models overexpressing human SOD1. Although these models have highly contributed to understanding the pathogenic mecha‐ nisms involved in motor neuron degeneration, they only account for less than 2% of all cas‐ es. Hence, the ALS field is still lacking effective therapies and a deep understanding of the etiology of the sporadic disease.

For 15 years the SOD1 models have been the only available, until, in 2008, mutations in TARDBP were found to be responsible for familial and sporadic forms of ALS [6, 7]. This led to the discovery that mutations in a second RNA/DNA-binding protein called fused in sar‐ coma (FUS) or translocated in liposarcoma (TLS) were also cause of the disease [8, 9]. More

recently, the field of ALS has seen a breakthrough with the association of GGGGCC-hexanu‐ cleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) to 35-40% of familial cases and 5-7% of sporadic cases [10-12].

the existence of mutations with incomplete penetrance, thus masking inherited genetic forms of the disease as sporadic, further contribute to complicate the discrimination be‐

The Use of Human Samples to Study Familial and Sporadic Amyotrophic Lateral Sclerosis: New Frontiers…

http://dx.doi.org/10.5772/56487

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In the last 20 years, most efforts were concentrated on studying the effect of SOD1 muta‐ tions, resulting in the generation of over 30 different animal models including *Drosophila, C. elegans, D. rerio, mice, rats* and *dogs* [20]. In most cases, expression of human mutant SOD1 in the animal models led to astrogliosis, inflammation and degeneration of motor neurons in a

The generated SOD1 animal models highly contributed to the understanding of SOD1 func‐ tions in the central nervous system (CNS) leading to the development of potential therapeu‐ tic strategies targeting these pathways. Unfortunately, most of the therapeutics that show an effect in rodent models, fail in human clinical trials. Overall, SOD1 only accounts for about 2% of all ALS cases, therefore the question arose how applicable the findings from these models really are for other familial cases and especially for the huge majority of sporadic

In 2006, the transactive response DNA-binding protein (TDP-43) was identified as a major component of intraneuronal inclusions, a form of protein aggregates representing a hall‐ mark of SALS and non-SOD1-FALS cases [21]. Soon after, researchers found ALS causing mutations in this gene [6, 7]. One year later, mutations in a second RNA/DNA-binding pro‐ tein called fused in sarcoma (FUS) or translocated in liposarcoma (TLS) were published [8, 9]. While TDP-43 mutations account for 4% of FALS, FUS mutations are less frequent and account for approximately 1-2% [22]. The discovery of the involvement of these two genes can be considered a milestone in ALS research, not necessarily because of the mutation fre‐ quency, but rather because of the wide presence of these proteins in the aggregates charac‐ terizing tissues from sporadic ALS cases. Mutations in TDP-43 and FUS can also be found in some forms of frontotemporal dementia (FTD), while aggregates of the non-mutated protein seem to be an even more common feature for neurodegenerative diseases including Hun‐ tington's, Alzheimer's and Parkinson's [23]. As both proteins are involved in RNA metabo‐ lism, a common disease mechanism underlying sporadic and familial forms of ALS might exist. This link rises hope that a common therapeutic strategy could be developed benefit‐

The TARDBP gene encoding TDP-43 lies on chromosome 1p36.2. The TDP-43 protein con‐ sists of 414 amino acids and is highly conserved among species [7]. The expression pattern is almost ubiquitous with high levels during development. Loss of TDP-43 is detrimental in rodents as knockouts in mice are lethal in both cases, either when performed during embry‐ onic stages, or also as conditional knockouts in the adult mouse [24-26]. As mentioned above, the protein is involved in RNA metabolism, but therein, various functions including regulation of alternative splicing, transcription, miRNA levels, RNA stabilization, as well as formation of stress and RNA granules have been described. TDP-43 seems to preferentially bind RNAs with unusually long introns and/or such that are involved in neuronal function like synaptic activity and neuronal development. Some of these RNAs encode proteins which have previously been shown to be involved in neurodegenerative diseases [27]. Al‐

tween FALS and SALS.

ALS cases.

similar manner as observed in patients.

ting a broad patient population.

In the same years, from 2007 to present, *in vitro* technologies to model neurological disor‐ ders have also undergone an impressive development.

With the discovery that adult human fibroblasts could be reprogrammed to induced pluri‐ potent stem (iPS) cells with the use of selected transcription factors [13], the field of ALS saw the opportunity to finally model not only the familial, but especially the sporadic disease *in vitro*. In fact, in 2008, the first human iPS-derived motor neurons from patients were cul‐ tured in a petri dish [14]. Since then, several iPS lines have been produced from patients and healthy individuals and they have been made commercially available (http:// www.coriell.org/stem-cells).

Moreover, in 2011, neural progenitors cells (NPCs) were isolated from post-mortem spinal cord samples of ALS patients and successfully cultured and differentiated into motor neu‐ rons, astrocytes and oligodendrocytes *in vitro* [15]. This technology provided for the first time the possibility to model all forms of ALS *in vitro* without inducing major epigenetic al‐ terations in the cells used.

In this chapter we will give an overview of how human tissues have been used so far, what discoveries they have led to since 2007, and how the recent advances in technology com‐ bined with the recent genetic discoveries, have tremendously widened the horizon of ALS research.
