**3. The role of wild type SOD1 in ALS**

In 1993 for the first time mutations in SOD1 were identified as cause of familial ALS and were found to be responsible for about 20% of familial cases [16]. Since that discovery nearly twenty years ago, more than 160 mutations in SOD1 have been identified (http:// alsod.iop.kcl.ac.uk/) and cellular and animal models of the disease carrying different forms of mutant SOD1 have been generated [36, 37]. Experiments using animal models revealed that the toxicity of mutant SOD1 is not related to a loss of function of the enzyme [38], but 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‐ ed FALS, as well as sporadic cases carrying SOD1 mutations.

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

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

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.

In 1993 for the first time mutations in SOD1 were identified as cause of familial ALS and were found to be responsible for about 20% of familial cases [16]. Since that discovery nearly twenty years ago, more than 160 mutations in SOD1 have been identified (http:// alsod.iop.kcl.ac.uk/) and cellular and animal models of the disease carrying different forms of mutant SOD1 have been generated [36, 37]. Experiments using animal models revealed that the toxicity of mutant SOD1 is not related to a loss of function of the enzyme [38], but

link [19, 34].

widely devoid of TDP-43 and contain p62.

164 Current Advances in Amyotrophic Lateral Sclerosis

**3. The role of wild type SOD1 in ALS**

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

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 could be a link between familial and sporadic ALS.

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.

In order to determine whether *in vivo* wild-type SOD1 can undergo misfolding and can be detected without altering the original sample, recent studies have focused on the production and investigation of new antibodies able to distinguish mutant/misfolded/monomeric SOD1 as opposed to its wild-type form. Although some of these antibodies have been tested only on limited samples and their ability to discriminate between aberrant conformations of SOD1 is debatable, their use has led to potentially interesting findings. One of the first anti‐ bodies produced to detect abnormalities in SOD1 post-translational processing was the SOD1-exposed dimer interface (SEDI). This antibody was prepared with the peptide at the dimer interface of SOD1. When SOD1 is folded as a homodimer in its active state, this site is inaccessible, while it is exposed upon monomerization [48]. This antibody successfully stained inclusions in motor neurons from SALS samples, however it did not detect positive inclusions in SALS spinal cords where no SOD1 mutations were detected [49]. Similarly, an antibody developed against the region Leu42-His48, which specifically recognizes SOD1 in which the beta barrel is unfolded, failed to detect misfolded SOD1 in SALS spinal cord sam‐ ples, but succeeded in recognizing aggregates in the FALS samples [50]. Despite these re‐ sults, it could not be concluded that wild-type SOD1 does not contribute to the pathogenic mechanisms occurring in SALS. Indeed, Forsberg et al. have produced a series of polyclonal antibodies against several SOD1 peptides that react with the denaturated enzyme, but not with the wild-type form. Using these antibodies, small inclusions were detected in the motor neurons of SALS patients [51] as well as in the nuclei of astrocytes, microglia and oligoden‐ drocytes [52]. These studies supported the hypothesis that wild-type SOD1, although not in‐ volved in the formation of Lewy body-like inclusions in SALS, is likely to undergo conformational changes, thus contributing to the pathologic mechanism.

normal SOD1 is in the range of protein stability that can cause disease and suggests that mu‐ tant forms of SOD1 with high stability might be related to low penetrance and be therefore categorized as sporadic forms of ALS. The authors suggest that, similarly, other complex ge‐ netic, environmental and lifestyle factors can influence the stability of normal SOD1 causing its misfolding in SALS cases. However, this does not exclude that the toxicity related to L117V mutation could derive by the interaction with other proteins and not by its stability. The importance of finding common pathways or players between sporadic and familial ALS is crucial for therapeutic approaches. One recent study explored this possibility [15]. The au‐ thors assessed the toxicity of astrocytes derived from neural progenitors isolated from the spinal cord of sporadic and familial ALS patients. This study showed for the first time that astrocytes from sporadic cases of disease are as toxic to motor neurons as astrocytes carrying mutations in SOD1. As expected, the shRNA mediated reduction of mutated SOD1 led to a complete rescue of motor neurons in this co-culture system. Of particular interest however, was the finding that even the knock down of wild type SOD1 in astrocytes from sporadic

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

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

167

The data summarized in this section provide strong evidence for a pathologic role of wildtype SOD1 in sporadic disease. This hypothesis opens new frontiers for future therapeutic

In ALS the cells mainly affected by the disease, the motor neurons and the glia, are located in the motor cortex and the spinal cord, which are accessible only post-mortem. The scarce availability of CNS samples, along with post-mortem delay and different preservation tech‐ niques that can affect the quality of the tissue and limit its use, are great challenges when studying this disease. Moreover, post-mortem material is only representative of the end stage of disease and, although used in microarray studies to unravel the mechanisms of neu‐ rodegeneration, it is unlikely to help identify early biomarkers. For these reasons, peripheral tissues, i.e. blood, fibroblasts and cerebrospinal fluid (CSF) have been preferentially used in high-throughput screening assays for biomarkers identification as well as gene expression

Multiple research groups have used post-mortem samples to identify the pathways in‐ volved in the neurodegenerative process of ALS. The studies utilizing complex tissues, rep‐ resentative of a mixed cell population, i.e. motor cortex [56] or spinal cord [57], have mainly recorded gene expression changes indicating the presence of an aggressive inflammatory re‐ action and active astrogliosis. These processes are prevalent in the spinal cord of ALS pa‐ tients and have masked the transcriptional changes occurring in motor neurons. However, the motor cortex seems to be affected to a lesser extent by astrogliosis and this enabled Le‐ derer and colleagues to identify important changes in transcripts involved in the cytoskele‐

patients markedly attenuated toxicity towards motor neurons.

approaches in the treatment of ALS.

profiling.

**4.1. Gene expression profiling**

**4. Human samples to study ALS**

Another antibody that has been used to detect misfolded SOD1 is C4F6. This peptide was raised against metal depleted (apo) SOD1 with G93A mutation [53]. Although this antibody was raised against a specific mutant form of SOD1, it successfully recognised skein-like in‐ clusions in FALS spinal cord samples, as well as inclusions in SALS [54].

Recently, a monoclonal antibody, called 3H1, was used to detect misfolded SOD1 in a subset of SALS cases displaying TDP-43/FUS-positive inclusions. This antibody recognizes a pep‐ tide corresponding to a structurally disrupted SOD1 electrostatic loop, detectable only when the protein is misfolded. Spinal cord immunocytochemistry showed that, in some SALS samples, TDP-43/FUS-positive inclusions were also positive for 3H1 antibody, suggesting that the pathologic mechanisms involved in ALS might trigger SOD1 misfolding, thus trig‐ gering toxic pathways common to both sporadic and familial ALS [55].

Besides the efforts to generate antibodies able to detect misfolded SOD1, no consensus has been reached on which antibodies, if any, can reliably and consistently detect the different forms of misfolded SOD1. Other studies have, therefore, used a different approach, trying to understand whether normal SOD1 shares common characteristics with the mutant form of the enzyme. Recently, a novel rare mutation in SOD1 (L117V) was identified in two Syrian ALS families [39]. Unusually, the disease showed uncommon low penetrance and slow pro‐ gression. Biochemical analysis of L117V SOD1 showed that its properties were indistin‐ guishable from the wild-type form and yet causing the disease. This study highlights that normal SOD1 is in the range of protein stability that can cause disease and suggests that mu‐ tant forms of SOD1 with high stability might be related to low penetrance and be therefore categorized as sporadic forms of ALS. The authors suggest that, similarly, other complex ge‐ netic, environmental and lifestyle factors can influence the stability of normal SOD1 causing its misfolding in SALS cases. However, this does not exclude that the toxicity related to L117V mutation could derive by the interaction with other proteins and not by its stability.

The importance of finding common pathways or players between sporadic and familial ALS is crucial for therapeutic approaches. One recent study explored this possibility [15]. The au‐ thors assessed the toxicity of astrocytes derived from neural progenitors isolated from the spinal cord of sporadic and familial ALS patients. This study showed for the first time that astrocytes from sporadic cases of disease are as toxic to motor neurons as astrocytes carrying mutations in SOD1. As expected, the shRNA mediated reduction of mutated SOD1 led to a complete rescue of motor neurons in this co-culture system. Of particular interest however, was the finding that even the knock down of wild type SOD1 in astrocytes from sporadic patients markedly attenuated toxicity towards motor neurons.

The data summarized in this section provide strong evidence for a pathologic role of wildtype SOD1 in sporadic disease. This hypothesis opens new frontiers for future therapeutic approaches in the treatment of ALS.
