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  • br Materials and methods br Results br

    2018-11-12


    Materials and methods
    Results
    Discussion It is known that mutation types of SOD1 affect not only enzymatic activity and protein half-life of SOD1, but also age at onset and the survival time of SOD1-ALS patients (Hayward et al., 2002) (Borchelt et al., 1994) (Regal et al., 2006). However, there are very few studies focusing on comparative study of SOD1 mutations. Here, we generated SOD1-ALS models derived from mutant SOD1-overexpressing hESCs with identical genetic background and we found mutation-specific morphological alterations. Furthermore, our models revealed that mutation types affect drug responses. Recently, two papers reported that mutant SOD1s influenced not only cell death but also the morphologies such as soma size, neurite length and abnormal organelles in MNs derived from the patient-specific iPSCs (Chen et al., 2014; Kiskinis et al., 2014). Although cell death was not detected in our ALS models, the reduction of neurite length, which could be a cellular disease phenotype, was observed in our ALS models except in G85R-SOD1. Morphological changes, primarily the dpp-iv inhibitors of MN neurite length, were detected following treatment with the compounds we investigated. Atrophic changes or axonopathy of MNs is observed in ALS (Ferraiuolo et al., 2011; Fischer et al., 2004; Vinsant et al., 2013); hence, the MN neurite length change of our ALS models might be a good predictor of drug efficacy in ALS. Our ALS models have potential as cell materials for cell-based high-throughput drug screening, similar to previous studies using patient-iPSCs (Egawa et al., 2012) (Yang et al., 2013). Riluzole, which is widely used and the approved drug for ALS, showed effects on A4V- and G93A-MNs at a single concentration of 10 and 100nM, respectively, in our dpp-iv inhibitors models. It is unclear why the drug was effective at a single dose but not in a dose-dependent manner in this study. This drug was reported to provide protective effects to primary rat MNs treated with media conditioned from primary astrocytes derived from mouse ALS models (including G93A and G86R SOD1 variants) (Rojas et al., 2014). Similarly, MCI-186, which is known as a neuroprotective drug (Shichinohe et al., 2004), only affected the elongation of neurites in MNs expressing G93A-SOD1. These results suggest that neuroprotective drugs might be effective at treating ALS phenotypes with respect to non-cell-autonomous toxicity or specific types of SOD1 mutations. Mutant SOD1 variants are divided into two classes on the basis of the metal ion content and the position of the mutation: WT-like mutants and metal-binding region (MBR) mutants (Rodriguez et al., 2002; Tiwari and Hayward, 2005). A4V and G93A are WT-like mutants, whereas G85R is a MBR mutant. We found that dbcAMP and Y27632 showed efficacy in non-MNs from all mutant-SOD1, WT-SOD1, and parental cell lines examined. However, they were only effective in MNs from cell lines expressing SOD1 variants in the WT-like mutant group. In contrast, kenpaullone was not effective at increasing the longest neurite length in non-MNs, but was widely effective in MNs, regardless of mutation type (although the effects on MNs expressing G93A were not statistically significant). TRO19622 showed effectiveness similar to kenpaullone despite the fact that the targets of these two compounds are different: TRO19622 inhibits the mitochondrial permeability transition pore complex (Bordet et al., 2007), while kenpaullone inhibits glycogen synthase kinase 3 and some cyclin-dependent kinases (Martinez et al., 2002; Senderowicz and Sausville, 2000). In this study, out of over 100 SOD1 mutants, only three SOD1 mutants were examined. By using SOD1-ALS models with a large number of other mutation types, SOD1 mutations could be potentially classified into drug response-based categories. Astrocytes are additionally implicated in MN death by secretion of neurotoxic factors (Wada et al., 2012) (Yamanaka et al., 2008) (Re et al., 2014) (Rojas et al., 2014) (Nagai et al., 2007). To the best of our knowledge, there are no reports that astrocytes show SOD1 mutation-dependent responses to drug in a manner similar to MNs. Future research should address questions regarding the classification of ALS into treatment-responsive categories and the associated role of astrocytes.