**4. Human samples to study ALS**

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

166 Current Advances in Amyotrophic Lateral Sclerosis

conformational changes, thus contributing to the pathologic mechanism.

clusions in FALS spinal cord samples, as well as inclusions in SALS [54].

gering toxic pathways common to both sporadic and familial ALS [55].

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‐

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‐

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

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 profiling.

#### **4.1. Gene expression profiling**

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‐ tal, mitochondrial and proteasomal functions, as well as ion homeostasis and glycolysis [56], in agreement with other lines of research in ALS [1].

transcripts involved in RNA processing as well as upregulation of inflammatory genes. This suggests that the mechanisms affecting motor neurons, also strike other cell type. However, because of their post-mitotic characteristics and their unique function, motor neurons are the

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

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

169

A more recent study performed gene expression profiling on peripheral blood mononuclear cells (PBMCs) from patients with SALS [62]. The results show upregulation of LPS/TLR4 signaling associated genes in response to elevated LPS plasma levels. A similar transcription pattern was obtained by culturing PBMCs from normal controls with LPS for a short time *in*

Similarly, Mougeot et al. found that peripheral blood lymphocytes (PBLs) display dysregu‐ lation of the ubiquitin/proteasome system (UPS) [63]. In particular, microarray analysis re‐ vealed upregulation of the ubiquitin-protein ligase E3-alpha-2 (UBR2) expression. UBR2 is known to act in synergy with UBR1 in a quality control mechanism for degradation of un‐ folded proteins. UBR2 upregulation correlated inversely with time since onset of disease and directly with the ALS functional rating scale (ALSFRS-R), suggesting that UBR2 is in‐ creased early in the disease course and decreases as disease progresses. The authors con‐ firmed with *in vitro* experiments that cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner, as

Very recently, human samples have been used to interrogate micro RNA (miRNA) expres‐ sion [64, 65]. Two studies successfully identified dysregulation of miRNAs in peripheral leu‐ kocytes [64] and monocytes [65] from SALS samples compared to controls. These miRNA are involved in pathways relevant to the CNS and, in particular, Butovsky et al. identified changes relevant for the inflammatory response, similarly to the expression pattern dis‐ played by monocytes isolated from multiple sclerosis (MS) patients. These results suggest that miRNAs profiles found in the peripheral blood cells can be relevant to understand the

ALS is fatal rapidly progressive neurodegenerative disorder, characterized by the activation of an intricate network of pathways and still lacking an effective treatment beside Riluzole [1]. The diagnosis of ALS is still mainly based on clinical assessment of progression of symp‐ toms, which results in a delay of about a year from symptom onset to diagnosis. Although the clinical course of disease can considerably vary from case to case, in the majority of cases

In this scenario, significant effort has been spent trying to identify molecules that could help classify different forms of ALS and lead to early diagnosis, as well as monitor disease pro‐

The tissues mainly utilized for biomarker screening in the past five years have been periph‐ eral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF). PBMCs have already been shown to display some of the traits of the disease, such as increase in inflammatory

most susceptible.

expected from the transcription data.

**4.2. Biomarkers in ALS**

gression.

death occurs within 2-5 years [66].

pathogenesis of ALS and/or used as biomarkers of the disease.

*vitro*.

In order to determine those genes differentially expressed in the cell type most affected in ALS, i.e. spinal motor neurons, laser capture microdissection (LCM) has been used to isolate single cells from human post mortem spinal cord samples.

Gene expression profiling of motor neurons has been performed on sporadic ALS cases [58], as well as ALS cases carrying mutations in the SOD1 and chromatin modifying protein 2B (*CHMP2B*) genes [59, 60]. The three studies highlighted the activation of different pathogen‐ ic pathways. The motor neurons isolated from sporadic cases showed decreased expression of genes associated with the cytoskeleton and transcription, whilst cell death-associated transcripts were increased. Moreover, genes involved in cell cycle activation and progres‐ sion were found to be upregulated, supporting the theory that inappropriate activation of the cell cycle in these post-mitotic cells can lead to cell death [58].

In contrast, microarray analysis of motor neurons isolated from *SOD1*-related ALS cases highlighted the activation of a cell survival pathway in the motor neurons that were spared by the disease. The study, in fact, revealed differential expression of genes involved in the protein kinase B/phosphatidylinositol-3 kinase (AKT/PI3K) pathway, along with decrease in phophastase and tensin homologue (*PTEN*) gene, a negative regulator of AKT [60]. The au‐ thors also showed that inhibition of PTEN led to increased activation of the AKT/PI3K path‐ way, with beneficial effects on primary motor neuron survival. Thus, activation of the AKT/ PI3K pathway is a potential candidate for future therapeutic strategies.

Finally, the transcriptional profiles from motor neurons isolated from the *CHMP2B*-related ALS cases showed dysregulation of genes involved in p38 MAPK signalling pathway, re‐ duced autophagy and repression of translation [59]. The significant impairment of the au‐ tophagy pathway reflects the function of *CHMP2B*, the gene mutated in these patient samples.

In spite of the differences between the pathways described above, dysregulation of calcium handling and cell cycle, as well as transcription, cytoskeleton assembly and metabolism, were common between the different genetic subtypes and SALS. Taking into account that these results derive from end-stage tissues, they support the evidence that etiologically di‐ verse forms of ALS converge into common mechanisms involved in motor neuron death.

#### *4.1.1. Results from use of human peripheral tissue*

Gene expression profiling has also been conducted on blood cells from ALS patients in order to identify biomarkers and/or achieve a better classification of disease subtypes through the identification of common transcriptional patterns [61-63]. In the study conducted by Saris and colleagues, microarray analysis of SALS and control whole blood samples was followed by hierarchical clustering of all differentially expressed transcripts [61]. This approach suc‐ cessfully identified different clusters that were able to differentiate between ALS and control samples. Interestingly, this study showed that peripheral blood can be used to investigate the pathways activated during disease, as the blood from ALS patients reveals decrease in transcripts involved in RNA processing as well as upregulation of inflammatory genes. This suggests that the mechanisms affecting motor neurons, also strike other cell type. However, because of their post-mitotic characteristics and their unique function, motor neurons are the most susceptible.

A more recent study performed gene expression profiling on peripheral blood mononuclear cells (PBMCs) from patients with SALS [62]. The results show upregulation of LPS/TLR4 signaling associated genes in response to elevated LPS plasma levels. A similar transcription pattern was obtained by culturing PBMCs from normal controls with LPS for a short time *in vitro*.

Similarly, Mougeot et al. found that peripheral blood lymphocytes (PBLs) display dysregu‐ lation of the ubiquitin/proteasome system (UPS) [63]. In particular, microarray analysis re‐ vealed upregulation of the ubiquitin-protein ligase E3-alpha-2 (UBR2) expression. UBR2 is known to act in synergy with UBR1 in a quality control mechanism for degradation of un‐ folded proteins. UBR2 upregulation correlated inversely with time since onset of disease and directly with the ALS functional rating scale (ALSFRS-R), suggesting that UBR2 is in‐ creased early in the disease course and decreases as disease progresses. The authors con‐ firmed with *in vitro* experiments that cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner, as expected from the transcription data.

Very recently, human samples have been used to interrogate micro RNA (miRNA) expres‐ sion [64, 65]. Two studies successfully identified dysregulation of miRNAs in peripheral leu‐ kocytes [64] and monocytes [65] from SALS samples compared to controls. These miRNA are involved in pathways relevant to the CNS and, in particular, Butovsky et al. identified changes relevant for the inflammatory response, similarly to the expression pattern dis‐ played by monocytes isolated from multiple sclerosis (MS) patients. These results suggest that miRNAs profiles found in the peripheral blood cells can be relevant to understand the pathogenesis of ALS and/or used as biomarkers of the disease.

### **4.2. Biomarkers in ALS**

tal, mitochondrial and proteasomal functions, as well as ion homeostasis and glycolysis [56],

In order to determine those genes differentially expressed in the cell type most affected in ALS, i.e. spinal motor neurons, laser capture microdissection (LCM) has been used to isolate

Gene expression profiling of motor neurons has been performed on sporadic ALS cases [58], as well as ALS cases carrying mutations in the SOD1 and chromatin modifying protein 2B (*CHMP2B*) genes [59, 60]. The three studies highlighted the activation of different pathogen‐ ic pathways. The motor neurons isolated from sporadic cases showed decreased expression of genes associated with the cytoskeleton and transcription, whilst cell death-associated transcripts were increased. Moreover, genes involved in cell cycle activation and progres‐ sion were found to be upregulated, supporting the theory that inappropriate activation of

In contrast, microarray analysis of motor neurons isolated from *SOD1*-related ALS cases highlighted the activation of a cell survival pathway in the motor neurons that were spared by the disease. The study, in fact, revealed differential expression of genes involved in the protein kinase B/phosphatidylinositol-3 kinase (AKT/PI3K) pathway, along with decrease in phophastase and tensin homologue (*PTEN*) gene, a negative regulator of AKT [60]. The au‐ thors also showed that inhibition of PTEN led to increased activation of the AKT/PI3K path‐ way, with beneficial effects on primary motor neuron survival. Thus, activation of the AKT/

Finally, the transcriptional profiles from motor neurons isolated from the *CHMP2B*-related ALS cases showed dysregulation of genes involved in p38 MAPK signalling pathway, re‐ duced autophagy and repression of translation [59]. The significant impairment of the au‐ tophagy pathway reflects the function of *CHMP2B*, the gene mutated in these patient

In spite of the differences between the pathways described above, dysregulation of calcium handling and cell cycle, as well as transcription, cytoskeleton assembly and metabolism, were common between the different genetic subtypes and SALS. Taking into account that these results derive from end-stage tissues, they support the evidence that etiologically di‐ verse forms of ALS converge into common mechanisms involved in motor neuron death.

Gene expression profiling has also been conducted on blood cells from ALS patients in order to identify biomarkers and/or achieve a better classification of disease subtypes through the identification of common transcriptional patterns [61-63]. In the study conducted by Saris and colleagues, microarray analysis of SALS and control whole blood samples was followed by hierarchical clustering of all differentially expressed transcripts [61]. This approach suc‐ cessfully identified different clusters that were able to differentiate between ALS and control samples. Interestingly, this study showed that peripheral blood can be used to investigate the pathways activated during disease, as the blood from ALS patients reveals decrease in

in agreement with other lines of research in ALS [1].

168 Current Advances in Amyotrophic Lateral Sclerosis

single cells from human post mortem spinal cord samples.

the cell cycle in these post-mitotic cells can lead to cell death [58].

PI3K pathway is a potential candidate for future therapeutic strategies.

*4.1.1. Results from use of human peripheral tissue*

samples.

ALS is fatal rapidly progressive neurodegenerative disorder, characterized by the activation of an intricate network of pathways and still lacking an effective treatment beside Riluzole [1]. The diagnosis of ALS is still mainly based on clinical assessment of progression of symp‐ toms, which results in a delay of about a year from symptom onset to diagnosis. Although the clinical course of disease can considerably vary from case to case, in the majority of cases death occurs within 2-5 years [66].

In this scenario, significant effort has been spent trying to identify molecules that could help classify different forms of ALS and lead to early diagnosis, as well as monitor disease pro‐ gression.

The tissues mainly utilized for biomarker screening in the past five years have been periph‐ eral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF). PBMCs have already been shown to display some of the traits of the disease, such as increase in inflammatory genes [65] and downregulation of Bcl-2 [67] and might, therefore, be used to investigate the disease during its progression as well as provide unique biomarkers.

drastically alter gene expression in the target cells until some of them eventually become pluripotent and can then be isolated and amplified. Initially, the transcription factors were introduced by retroviral or lentiviral constructs leading to the integration of the transgene into the target genome. As the random integration of additional genes can disrupt/alter the expression of endogenous genes, more recent approaches rely on less invasive techniques

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

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

171

The use of stem cell technologies in ALS research started in 2007 when mouse embryonic stem cells from the most prominent SOD1 model carrying the G93A mutation were estab‐ lished [75]. When differentiated into motor neurons, mild phenotypic differences between motor neurons expressing human wild type SOD1 or the G93A mutation could be observed. After several weeks in culture, SOD1 containing inclusions as well as the overall level of ubiquitinated proteins were more frequent in the motor neurons expressing the mutant hu‐ man protein. In 2009, a human embryonic stem cell line was used to generate motor neurons that were then transfected with different SOD1 mutation containing constructs [76]. The re‐ searchers observed a reduction in neurite length and in line with the study of the mouse G93A embryonic stem cell derived motor neurons, reduced survival. However, in the hu‐ man study, it is unclear whether the observed phenotype arises from the mutations or the increase of SOD1 abundance itself, as a control overexpressing wild type SOD1, was not

Since the discovery of the iPS technology, huge efforts were put in the generation of patient specific iPS lines. Up to now, several hundred lines with various mutations have been gener‐ ated and some are now becoming commercially available thereby getting accessible to a

In 2008, Dimos et al reported the successful generation of motor neurons and glial cells from an ALS patient derived iPS line carrying a SOD1 mutation causing a mild disease phenotype [14]. Surprisingly, unlike the previous studies with mouse and human embryonic stem cells, no disease related phenotype was reported from these cells until now. This potential lack of phenotype could in part be explained by the patient's late onset and mild disease form. An‐ other report from 2011, where motor neurons were generated from a patient harboring a VAPB mutation, did also not mention any phenotype despite reduced levels of VAPB. As the levels of this protein were already reduced in the fibroblasts used for the reprogram‐ ming, the lower levels in the resulting motor neurons could either be due to mutation in‐ duced expression or translational changes or could also be explained by an incomplete reprogramming of this genomic locus in the generated iPS lines [77]. Recently, the first re‐ port of an iPS line harboring a TDP-43 mutation was published [78]. The motor neurons generated from this line showed elevated TDP-43 levels, but no change in localization or signs of aggregate formation. In addition, motor neurons from both, control and TDP-43 mutant were phenotypically and functionally similar despite an elevated sensitivity to PI3K signaling inhibition and elevated cell death. These data suggest that the toxicity of this TDP-43 mutation might arise from its increased stability leading to a higher overall protein

such as transposons or RNA transfection [74]

generated.

broad scientific community.

amount in the cell.

Recently, Nardo et al. performed proteomic analysis of PBMCs isolated from 60 sporadic ALS patients and 30 healthy controls [68]. The authors identified and validated in a second cohort 14 protein biomarkers, that could discriminate between ALS patients and controls re‐ gardless of age and gender. Remarkably, of these 14 biomarkers, 5 were able to discriminate between ALS patients and individuals with other neuropathies and 3, among which TDP43, were markers of disease severity. Notably, these results are consistent with a CSF biomarker study reporting that TDP43 levels were increased in ALS patients [69]. The value of this re‐ sult goes beyond the finding of a disease biomarker, as it supports the even more interesting hypothesis that TDP43 could be a common player in early disease in familial as well as sporadic ALS cases. This would confirm what is already suggested by the presence of TDP-43 positive aggregates in SALS biopsy samples.

Although there is still no consensus on valid biomarkers for ALS [70], proteome analysis has recently led to the identification of fetuin-A and transthyretin (TTR) as candidates to distin‐ guish ALS patients with rapid versus slow disease progression. The upregulation of TTR and fetuin-A, involved in immune regulatory functions, could be associated with the inflam‐ matory state of the CNS. At present, these markers were tested in two independent cohorts of 18 and 20 patients with a follow up of 2 years [71] and TTR had already been identified as a potential biomarker for ALS compared to controls in a previous study [72]. Although fur‐ ther validation is needed, these results are encouraging and would provide an invaluable tool to discriminate between patients with different disease progression rates. This would help clinician determine the timing for clinical intervention such as gastrostomy and noninvasive ventilation [73].
