**2. Latest genetic discoveries**

Even though Jean-Martin Charcot initially described ALS in 1869, it took more than a centu‐ ry until the first disease-causing gene – the Cu/Zn superoxide dismutase (SOD1) - was iden‐ tified [16]. Mutations in this gene account for approximately 10-20% of the familial ALS (FALS) cases (Anderson 2006) and about 1% of the sporadic (SALS). Up to now, mutations in 21 different genes have been linked to ALS, although some of them present with atypical disease characteristics [17]. FALS is usually inherited in an autosomal-dominant manner, but in rarer cases, it appears also recessive or X-linked. Along with the rising number of genes and mutations involved, the differentiation between sporadic and familial ALS be‐ comes increasingly difficult and depends on the definition applied. With the most stringent classification, a patient is considered to suffer from the familial form if he/she has at least one first- or second-degree relative affected by ALS [18]. However, other studies define FALS when at least one relative is affected by motor neuron disease, i.e. ALS, primary later‐ al sclerosis (PLS) or progressive muscular atrophy (PMA) [19]. In addition, several indica‐ tions exist that mutations leading to ALS are also involved in the development of other neurodegenerative diseases such as different types of dementia or Parkinsons disease. This further broadens the range of possible familial linkage [19]. Missing family history data and the existence of mutations with incomplete penetrance, thus masking inherited genetic forms of the disease as sporadic, further contribute to complicate the discrimination be‐ tween FALS and SALS.

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

In the same years, from 2007 to present, *in vitro* technologies to model neurological disor‐

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://

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‐

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

Even though Jean-Martin Charcot initially described ALS in 1869, it took more than a centu‐ ry until the first disease-causing gene – the Cu/Zn superoxide dismutase (SOD1) - was iden‐ tified [16]. Mutations in this gene account for approximately 10-20% of the familial ALS (FALS) cases (Anderson 2006) and about 1% of the sporadic (SALS). Up to now, mutations in 21 different genes have been linked to ALS, although some of them present with atypical disease characteristics [17]. FALS is usually inherited in an autosomal-dominant manner, but in rarer cases, it appears also recessive or X-linked. Along with the rising number of genes and mutations involved, the differentiation between sporadic and familial ALS be‐ comes increasingly difficult and depends on the definition applied. With the most stringent classification, a patient is considered to suffer from the familial form if he/she has at least one first- or second-degree relative affected by ALS [18]. However, other studies define FALS when at least one relative is affected by motor neuron disease, i.e. ALS, primary later‐ al sclerosis (PLS) or progressive muscular atrophy (PMA) [19]. In addition, several indica‐ tions exist that mutations leading to ALS are also involved in the development of other neurodegenerative diseases such as different types of dementia or Parkinsons disease. This further broadens the range of possible familial linkage [19]. Missing family history data and

familial cases and 5-7% of sporadic cases [10-12].

160 Current Advances in Amyotrophic Lateral Sclerosis

www.coriell.org/stem-cells).

terations in the cells used.

**2. Latest genetic discoveries**

research.

ders have also undergone an impressive development.

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 similar manner as observed in patients.

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 ALS cases.

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‐ ting a broad patient population.

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‐ though TDP-43 depletion affects the expression and splicing of many different RNAs in the CNS, the vast majority of the more than 40 ALS causing mutations identified so far, lie with‐ in the C-terminal domain encoding a glycine rich stretch that is important for protein-pro‐ tein interaction. While in healthy individuals TDP-43 is mainly localized in the nucleus, it gets mislocalized and trapped in cytoplasmic aggregates in ALS patients, leading to reduced levels in the nucleus [22]. The trapped TDP-43 seems to be heavily modified displaying ubiquitination, phosphorylation and cleavage and in some cases, misfolding. The exact meaning of TDP-43 mislocalization in ALS and other neurodegenerative diseases remains to be elucidated, as so far, it is unclear whether the inclusions actively participate in the disease development and progression or rather represent a mere indicator of other dysregulated cel‐ lular mechanisms. The observed changes in RNA regulation could arise from both, the mis‐ localization itself, leading to a reduced abundance in the nucleus, or function altering mutations or modifications of the protein. In addition, it was reported that certain mutations increase the stability of the protein and therefore its overall abundance in the cell [28], which might lead to the visible accumulations and alterations in RNA metabolism.

and FUS might directly interact with each other as they were detected in the same com‐ plex in cultured cells [28, 30]. Even though further data from patients and animal models are needed to confirm this finding, it is an interesting observation, as TDP-43 does not seem to be mislocalized in ALS cases that have accumulation of FUS containing aggre‐

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Following TDP-43 and FUS, the thorough analysis of the cytoplasmic aggregates found in ALS patients led to the identification of additional common components such as optineurin and ubiquilin-2. Several of these proteins were afterwards identified to be mutated in a

In 2011, the identification of a new ALS gene harboring a different type of mutation was achieved. The association of the chromosomal locus 9p21.2 with ALS and FTD had al‐ ready been described in 2006 [32]. The improvement of sequencing techniques and contin‐ uous research finally led to the identification of the disease causing gene: C9ORF72. While the function of this widely expressed protein is unknown, the type of mutation dif‐ fers from other ALS related genes. It consists of a massive GGGGCC-hexanucleotide re‐ peat expansion in intron 1 between two non-coding exons. Whereas healthy individuals carry up to about 23 repeats, affected patients have at least 30, but in some cases, many

The first reports about the mutation in this gene came from two independent studies analyz‐ ing relatively small cohorts respectively of sporadic and familial ALS cases in Finland and Europe and ALS/FTD cases in the USA. Both studies started off with re-sequencing of the 9p21.2 locus from well defined families, in which the linkage of the disease to this chromo‐ some 9 location had been previously demonstrated [11, 12]. After the detection of the repeat,

The most striking discovery of these studies is the high frequency of mutations in this gene in the analysed cohorts. Between 9 and 20% of American patients suffering from familial FTD and up to 38% of familial ALS cases from different European countries resulted posi‐ tive for the new mutation. For the sporadic cases, the percentage lies around 7% for the American FTD patient population and 21% of sporadic ALS patients in the genetically ho‐ mogenous Finnish population. With these initially published percentages, C9ORF72 muta‐ tions appeared to be the so far most frequent known cause of ALS and FTD. However, the cohorts were recruited through only few institutions and were rather small. Recently, a cross-sectional study including more patients from various different countries and with dif‐ fering genetic background has been published. In this study, a total of 588 familial ALS cases and 403 familial FTD cases were screened for the mutation. For FALS, 37.6% of patients were identified to carry the pathological repeat, for FTD the percentage was 25.1% [33]. Al‐ though further studies are needed to confirm these findings, they are truly exciting, consid‐ ering the fact that together with SOD1, which accounts for approximately 10-20% of FALS, almost 50% of the familial disease cases can now be explained by mutations in one of these

gates in the cytoplasm [28].

hundred copies of it [11, 12].

two genes.

smaller portion of ALS patients as well [31].

the analyses were expanded to larger cohorts.

Until now, a plethora of different animal models including Drosophila, mouse and rats has been generated. Unfortunately at present, TDP-43 models have originated controversial re‐ sults. In fact, overexpressing wild type TDP-43 in the CNS appears to be toxic by itself, while the effects of the mutant protein vary broadly ranging from no symptoms to severe neurodegeneration in different regions of the mouse brain [20]. Mostly, it is unclear whether the toxicity is due to the mutation or the simple presence of the transgene.

The gene encoding FUS lies on chromosome 16 in a region that was already linked to familial ALS before the first mutations were identified. After the discovery of TDP-43 mu‐ tations, the focus on genes encoding RNA/DNA-binding proteins increased leading to the fast discovery of mutations in FALS patients by two independent groups [8, 9]. Similar to TDP-43, more than 40 different FUS mutations have been identified in FALS patients or patients suffering from FTD. The FUS protein consists of 526 amino acids and like TDP-43, it is widely expressed amongst different tissues. Knockout of FUS in different mouse strains led to differing results, indicating that the genetic background of the used mouse strain plays an important role as disease modifier. In the inbred strains (C57BL/6 and 129), the knockout causes death at birth, whereas outbred strains survive until adult‐ hood. In all cases, FUS depletion seems not to induce classical neurodegeneration as a primary effect. Interestingly, unlike TDP-43, the ALS related mutations in the FUS gene do not cluster in the glycine-rich region of the protein, but rather at the very end of the highly conserved C-terminus of the protein that contains the nuclear localization signal [22]. FUS is also mainly localized in the nucleus of cells in healthy individuals, but its mislocalization and aggregation in cytoplasmic granules in ALS patients leads to a less se‐ vere reduction in the nucleus than TDP-43 [22]. Up to now, only few binding partners of FUS (RNA or proteins) are known, making it further challenging to speculate about the function of the protein, which remains mostly unknown. In general, FUS is thought to be involved in regulation of gene expression, transcription, RNA splicing, RNA transport, translation, miRNA processing as well as DNA damage repair [29]. Interestingly, TDP-43 and FUS might directly interact with each other as they were detected in the same com‐ plex in cultured cells [28, 30]. Even though further data from patients and animal models are needed to confirm this finding, it is an interesting observation, as TDP-43 does not seem to be mislocalized in ALS cases that have accumulation of FUS containing aggre‐ gates in the cytoplasm [28].

though TDP-43 depletion affects the expression and splicing of many different RNAs in the CNS, the vast majority of the more than 40 ALS causing mutations identified so far, lie with‐ in the C-terminal domain encoding a glycine rich stretch that is important for protein-pro‐ tein interaction. While in healthy individuals TDP-43 is mainly localized in the nucleus, it gets mislocalized and trapped in cytoplasmic aggregates in ALS patients, leading to reduced levels in the nucleus [22]. The trapped TDP-43 seems to be heavily modified displaying ubiquitination, phosphorylation and cleavage and in some cases, misfolding. The exact meaning of TDP-43 mislocalization in ALS and other neurodegenerative diseases remains to be elucidated, as so far, it is unclear whether the inclusions actively participate in the disease development and progression or rather represent a mere indicator of other dysregulated cel‐ lular mechanisms. The observed changes in RNA regulation could arise from both, the mis‐ localization itself, leading to a reduced abundance in the nucleus, or function altering mutations or modifications of the protein. In addition, it was reported that certain mutations increase the stability of the protein and therefore its overall abundance in the cell [28], which

162 Current Advances in Amyotrophic Lateral Sclerosis

might lead to the visible accumulations and alterations in RNA metabolism.

the toxicity is due to the mutation or the simple presence of the transgene.

Until now, a plethora of different animal models including Drosophila, mouse and rats has been generated. Unfortunately at present, TDP-43 models have originated controversial re‐ sults. In fact, overexpressing wild type TDP-43 in the CNS appears to be toxic by itself, while the effects of the mutant protein vary broadly ranging from no symptoms to severe neurodegeneration in different regions of the mouse brain [20]. Mostly, it is unclear whether

The gene encoding FUS lies on chromosome 16 in a region that was already linked to familial ALS before the first mutations were identified. After the discovery of TDP-43 mu‐ tations, the focus on genes encoding RNA/DNA-binding proteins increased leading to the fast discovery of mutations in FALS patients by two independent groups [8, 9]. Similar to TDP-43, more than 40 different FUS mutations have been identified in FALS patients or patients suffering from FTD. The FUS protein consists of 526 amino acids and like TDP-43, it is widely expressed amongst different tissues. Knockout of FUS in different mouse strains led to differing results, indicating that the genetic background of the used mouse strain plays an important role as disease modifier. In the inbred strains (C57BL/6 and 129), the knockout causes death at birth, whereas outbred strains survive until adult‐ hood. In all cases, FUS depletion seems not to induce classical neurodegeneration as a primary effect. Interestingly, unlike TDP-43, the ALS related mutations in the FUS gene do not cluster in the glycine-rich region of the protein, but rather at the very end of the highly conserved C-terminus of the protein that contains the nuclear localization signal [22]. FUS is also mainly localized in the nucleus of cells in healthy individuals, but its mislocalization and aggregation in cytoplasmic granules in ALS patients leads to a less se‐ vere reduction in the nucleus than TDP-43 [22]. Up to now, only few binding partners of FUS (RNA or proteins) are known, making it further challenging to speculate about the function of the protein, which remains mostly unknown. In general, FUS is thought to be involved in regulation of gene expression, transcription, RNA splicing, RNA transport, translation, miRNA processing as well as DNA damage repair [29]. Interestingly, TDP-43 Following TDP-43 and FUS, the thorough analysis of the cytoplasmic aggregates found in ALS patients led to the identification of additional common components such as optineurin and ubiquilin-2. Several of these proteins were afterwards identified to be mutated in a smaller portion of ALS patients as well [31].

In 2011, the identification of a new ALS gene harboring a different type of mutation was achieved. The association of the chromosomal locus 9p21.2 with ALS and FTD had al‐ ready been described in 2006 [32]. The improvement of sequencing techniques and contin‐ uous research finally led to the identification of the disease causing gene: C9ORF72. While the function of this widely expressed protein is unknown, the type of mutation dif‐ fers from other ALS related genes. It consists of a massive GGGGCC-hexanucleotide re‐ peat expansion in intron 1 between two non-coding exons. Whereas healthy individuals carry up to about 23 repeats, affected patients have at least 30, but in some cases, many hundred copies of it [11, 12].

The first reports about the mutation in this gene came from two independent studies analyz‐ ing relatively small cohorts respectively of sporadic and familial ALS cases in Finland and Europe and ALS/FTD cases in the USA. Both studies started off with re-sequencing of the 9p21.2 locus from well defined families, in which the linkage of the disease to this chromo‐ some 9 location had been previously demonstrated [11, 12]. After the detection of the repeat, the analyses were expanded to larger cohorts.

The most striking discovery of these studies is the high frequency of mutations in this gene in the analysed cohorts. Between 9 and 20% of American patients suffering from familial FTD and up to 38% of familial ALS cases from different European countries resulted posi‐ tive for the new mutation. For the sporadic cases, the percentage lies around 7% for the American FTD patient population and 21% of sporadic ALS patients in the genetically ho‐ mogenous Finnish population. With these initially published percentages, C9ORF72 muta‐ tions appeared to be the so far most frequent known cause of ALS and FTD. However, the cohorts were recruited through only few institutions and were rather small. Recently, a cross-sectional study including more patients from various different countries and with dif‐ fering genetic background has been published. In this study, a total of 588 familial ALS cases and 403 familial FTD cases were screened for the mutation. For FALS, 37.6% of patients were identified to carry the pathological repeat, for FTD the percentage was 25.1% [33]. Al‐ though further studies are needed to confirm these findings, they are truly exciting, consid‐ ering the fact that together with SOD1, which accounts for approximately 10-20% of FALS, almost 50% of the familial disease cases can now be explained by mutations in one of these two genes.

The observation that C9ORF72 mutations not only cause ALS, but also FTD and clinically mixed syndromes such as ALS-FTD, is in line with the clinical spectrum caused by TDP-43 and FUS mutations as well as wild type aggregations in both diseases. This further strength‐ ens the indications that these two clinically distinct syndromes share a common pathogenic link [19, 34].

rather a gain of toxic function. In the past twenty years different forms of mutant SOD1 have been characterized for their biochemical properties, however no common characteristics be‐ tween mutations have been found. In fact, different mutations seem to cause different changes in enzymatic function or no change at all [39-41], leading to the conclusion that SOD1 dismutase activity is not responsible for protein toxicity. Although the nature of the toxic function gained by mutant SOD1 is still obscure, there is clear evidence that the mutant enzyme undergoes conformational changes leading to its misfolding and subsequent aggre‐ gation [41, 42]. SOD1 aggregates are, in fact, one of the histological hallmarks of SOD1-relat‐

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Although SOD1 mutations are responsible for less than 2% of ALS cases and this disease is mainly of sporadic origin, sporadic and familial cases are clinically undistinguishable. More‐ over, with the exception of patients carrying C9ORF72 mutation, which seem to define a specific clinical subgroup [35], other genetic mutations do not determine different clinical characteristics. This observation has led to the conclusion that familial and sporadic ALS must share common pathogenic mechanisms [1]. Consequently, in recent years, efforts have been made in understanding whether the genes causing familial ALS can be responsible or can be involved in the pathophysiology of sporadic cases. Recently, strong evidence has

Wild-type human SOD1 is a 32KDa homodimer known to be one of the most stable proteins with a melting temperature around 90°C. However, its stability is highly dependent upon post-translational processes including binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Impairment or retardation of these post-translational processes can disrupt SOD1 stability, causing the formation of misfolded structures and ag‐ gregates. Indeed, in 2007, it was shown that oxidised wild-type SOD1 could acquire *in vitro* aberrant properties leading to association with poly-ubiquitin, Hsp70 and chromogranin B, similarly to the mutant enzyme [43]. The same year, another group used covalent chemical modification to show that spinal cord samples from both familial and sporadic ALS cases displayed a form of SOD1 that was absent in non-neurological controls as well as in spinal cord samples from patients affected by other neurodegenerative disorders [44]. Recently, Guareschi et al [45] managed to immunoprecipitate SOD1 from sporadic and familial ALS patients' lymphoblasts and then analysed the presence of oxidized carbonyl groups. A form of over-oxidized wild-type SOD1 was indeed found in a subset of SALS patients. This posttranslationally modified form of the wild-type enzyme recapitulates some of the toxic prop‐ erties attributed to mutant SOD1, i.e. the ability to cause mitochondrial damage through interaction with Bcl-2 [45]. Altogether, these findings supported the hypothesis that SOD1

On another front, the use of the SOD1 mouse model also provided important clues as to whether normal SOD1 can play a role in the disease. Surprisingly, overexpression of wildtype human SOD1 accelerated disease onset in several transgenic mouse models of ALS [46, 47], supporting the involvement of wild type SOD1 in the disease mechanism. However, these results have to be interpreted with caution, as they might derive by the toxicity of

transgene accumulation rather than a specific SOD1-related mechanism.

ed FALS, as well as sporadic cases carrying SOD1 mutations.

could be a link between familial and sporadic ALS.

been gathered suggesting that SOD1 might play a crucial role also in SALS.

Interestingly, the pathological features of C9ORF72 related ALS seem very unusual and dis‐ tinct up to the point that the mutation itself can actually be predicted from the observed pathology [35]. While the spinal cord shows the typical neuronal loss and TDP-43 positive cytoplasmic inclusions, other regions of the brain seem to accumulate aggregates that are widely devoid of TDP-43 and contain p62.

The mechanism through which this expansion repeat conveys toxicity to neurons still has to be elucidated. Two major hypotheses can be distinguished: 1) The expansion repeat alters or abolishes expression of all or certain C9ORF72 protein isoforms leading to reduced protein levels and a loss of functionality, 2) the expanded repeat itself conveys toxicity by sequestra‐ tion of other RNA binding proteins and aggregate formation inside the nucleus, thus inhib‐ iting proper functionality of the bound proteins. To date, it is not known which hypothesis applies in the case of C9ORF72 expansions, but the experience from various other toxic ex‐ pansion repeat diseases like Huntington's disease are favoring the second. While Renton et. al demonstrate the presence of aggregates in the nucleus of fibroblasts from affected pa‐ tients, the results from the second study by DeJesus et al are less clear concerning accumula‐ tion of RNA granules. The latter study also lies more emphasis on a change in the expression levels of different C9ORF72 mRNA isoforms, which would support the first hy‐ pothesis rather than the second. Currently, the tools for a detailed analysis of protein expres‐ sion and function are still lacking, but considering the importance of the mutation, huge efforts are put into the development of better antibodies, probes and assays.

Overall, the finding that mutations in TDP43, FUS and C9ORF72 might cause ALS by alter‐ ing the normal interaction of these proteins with RNA or might cause a toxic gain of func‐ tion leading to unexpected protein-RNA interaction opens new avenues for ALS research. These recent genetic discoveries have shifted the attention to cellular processes, i.e. RNA metabolism, transport and processing, that were not under investigation in the SOD1 mod‐ els. These pathways might represent a common mechanism for different forms of ALS, as well as creating a link between ALS and a wider spectrum of neurodegenerative conditions.
