br Materials and methods br Results br Discussion Both anima

Materials and methods
Results
Discussion Both animal models and in vitro cell systems can contribute to the dissection of molecular pathways and cellular phenotypes underlying ALS. The SOD1 mouse model has been extensively used to study the non-cell autonomous effects of the mutations in different cell types. Muscle-restricted expression of mutant SOD1 elicited in this district toxic effects, due to increased oxidative stress, associated to signs of ALS and motoneuron degeneration in old mice (Dobrowolny et al., 2008; Wong and Martin, 2010). However, reduction of mutant SOD1 in muscles did not affect disease onset or survival of transgenic mice (Miller et al., 2006). A better understanding of the role of muscle is crucial to design effective therapeutic strategies for this multi-systemic disease. The genetic etiology of ALS also contributes to its complexity. Despite the list of genes underlying inherited ALS has been greatly expanded in the last decade, effects of non-SOD1 mutations in muscle cells, and their possible contribution to ALS onset or progression, have not been elucidated. In a recent report, Wachter et al. suggested that overexpression of FUS and TDP-43 in muscle order PS341 affects motoneuron neurite length in an in vitro murine co-culture system (Wächter et al., 2015). This study relied on the ectopic expression of human mutant proteins in a mouse system. So far, a proper human model system in which these evidences could be confirmed and implemented is still missing. In this paper we show that both normal and ALS patient-derived human iPSCs can be converted to functionally mature myotubes in vitro. One important point of strength of our system is the expression of physiological levels of mutant proteins by human iPSC-derived cells (Lenzi et al., 2015). Notably, ectopic expression of human WT FUS in murine systems (in vivo or in vitro) produced the same detrimental effects observed when mutant proteins are used (McGoldrick et al., 2013; Mitchell et al., 2013; Wächter et al., 2015). Such dose-dependent toxicity should be taken into consideration when analyzing the results of experiments relying on transfection of exogenous ALS-related genes. Human iPSCs and their differentiated derivatives, such as those described here, are not flawed by this concern. Our approach is based on the ectopic expression of the myogenic factor MyoD in an inducible way, in cells that had stably integrated the transgene. Transduced iPSCs represent a stable and expandable population, in which doxycycline treatment and medium switch are sufficient to induce myogenic differentiation in few days. Even if we do not provide comparative analysis, a survey in the literature suggests similar timing and efficiency of differentiation with our system compared to analogous methods (Tanaka et al., 2013; Shoji et al., 2016). In our previous work, we have reported comparable efficiency of motoneuron differentiation between WT and FUS or TDP-43 mutant iPSCs (Lenzi et al., 2015). Here we have extended this analysis to muscle differentiation showing that ALS mutations did not impair myogenesis, as assessed by muscle markers expression. Functionally, both control and ALS mutant iPSC-derived muscles expressed AChRs and responded to ACh application with transient intracellular calcium increase, suggesting that these cells could have reached a mature phenotype. Interestingly, we detected significantly smaller currents in the TDP-43 mutant compared to the control. Moreover, one of the FUS mutants did not respond to ACh. Despite the fact that the fraction of cells expressing the muscle marker MYH1 was not significantly different among the lines, we cannot exclude that these differences were due to suboptimal maturation. It should be also taken into consideration that iPSC lines used in the present work were derived by somatic cell reprogramming and therefore are not isogenic. In order to ascribe any functional impairment to the genetic defect it would be necessary, in the future, to extend this analysis to other mutants and/or compare cell lines that differ only with respect to disease mutations, i.e. otherwise isogenic, generated by gene editing.