Muscle fasciculations are commonly observed in ALS patients

Muscle fasciculations are commonly observed in ALS patients and are related to underlying axonal hyperexcitability (Kanai et al., 2006). MN hyperexcitability has also been observed in vitro in a SOD1 A4V iPSC model, and inhibiting neuronal excitability via retigabine enhanced MN survival (Wainger et al., 2014). On the other hand, MNs derived from iPSCs bearing the hexanucleotide repeat expansions in C9ORF72 showed diminished activity (Devlin et al., 2015; Sareen et al., 2013). A recent study elegantly reconciled these two contradictory findings, suggesting that ALS MNs display hyperexcitability in the early stages but become hypoexcitable as the disease progresses (Devlin et al., 2015). Interestingly, neuronal firing has been shown to activate the ERK pathway, while a hyper-active ERK pathway has been shown to result in epilepsy (Guo et al., 2014; Nateri et al., 2007). This suggests a positive feedback between neuronal firing and activation of the ERK signaling cascade. Hyperexcitability in early-stage ALS MNs could result in activation of ERK signaling, thereby triggering the auto-feedback loop leading to neurodegeneration. Accordingly, inhibiting this loop either by targeting the ERK pathway or neuronal excitability promotes MN survival. It would be interesting to see whether inhibiting the ERK pathway or other members of the MAPK family would result in decreased neuronal excitability or whether inhibiting neuronal firing via retigabine reduces MAPK activity. It must be noted that protein aggregation and an elevated ER stress have also been identified in other familial forms of ALS, including the most common ARQ 621 observed in C9ORF72 (Taylor et al., 2016; Dafinca et al., 2016; Kaus and Sareen, 2015). In addition, many sporadic ALS cases commonly display TDP-43 protein inclusions in spinal MNs (Mackenzie et al., 2007), and these have been shown to correlate with ER stress and JNK activation (Lee et al., 2016). Further, neuronal hyperexcitability has been established as a recurring theme across sporadic as well as familial forms of ALS (Geevasinga et al., 2016). Hence, it is reasonable to suppose that a combination of the ER stress mediated by TDP-43 inclusions and neuronal hyperexcitability might contribute toward activation of the MAPK pathway in other forms of ALS as well. Due to differences in iPSC generation and MN differentiation protocols, the range and extent of phenotypes observed in ALS iPSC models are expected to vary. However, comparison across our iPSC model and other published ALS iPSC models revealed shared features among MNs bearing different ALS mutations. MNs bearing mutant SOD1 and TDP43 display decreased survival and increased apoptosis in extended cultures (Bilican et al., 2012; Chen et al., 2014; Kiskinis et al., 2014; Figures 2B and 2D). MNs differentiated from C9ORF72 mutant iPSCs did not display survival deficits in vitro under standard culture conditions (Devlin et al., 2015) but displayed enhanced susceptibility to cellular stressors (Donnelly et al., 2013; Haeusler et al., 2014). Interestingly, a recent report using patient-derived iPSCs bearing C9ORF72 expansion of greater than 500 repeats displayed increased neuronal death and apoptosis in vitro (Dafinca et al., 2016). Apoptotic MNs also display defects in soma size and neurite length (Chen et al., 2014; Kiskinis et al., 2014). Although neurite defects have not been observed in C9ORF72 mutant MNs, interference with actin dynamics via interaction of C9ORF72 with cofilin can be expected to result in axonal defects (Sivadasan et al., 2016). An activated p53 response was observed in C9ORF72 mutant MNs (Lopez-Gonzalez et al., 2016) similar to our observation of an increased nuclear p53 in SOD1 MNs compared with the control and isogenic corrected MNs. ER stress has also been detected in MNs derived from mutant SOD1 and C9ORF72 iPSCs in parallel to the detection of SOD1 aggregation and RAN dipeptide accumulation, respectively (Dafinca et al., 2016; Kiskinis et al., 2014). On the other hand, defects in nucleocytoplasmic transport that have been observed in C9ORF72 ALS have yet to be detected in other familial forms of ALS (Freibaum et al., 2015; Zhang et al., 2015).