br Discussion Although animal models have provided valuable

Discussion Although animal models have provided valuable mechanistic insights into the pathophysiology of ALS, there has been a lack of success in translating these findings to therapies (Gladman et al., 2012). This is perhaps due to species-specific differences between humans and rodents, or because there are issues with the animal model itself where a phenotype is observed only when the mutant protein is overexpressed at non-physiological levels. The use of patient-derived iPSCs promises to circumvent some of these obstacles by providing human neurons expressing the mutant protein at physiological levels in the relevant genetic background. However, one of the caveats of iPSC-based disease modeling is the phenotypic variation observed due to the underlying genetic differences between different iPSC lines. In addition, since iPSC-derived neurons are considered to be fetal-like (Sances et al., 2016), it is important to establish that the in vitro model is capable of recapitulating disease phenotypes observed in ALS patients. To exclude the possibility of observing phenotypic differences due to genetic variation in the iPSC lines, we generated isogenic iPSC lines by correcting the point mutation in the SOD1 genomic locus using CRISPR-Cas9 genome editing technology. Next, we confirmed the maturity of our iPSC-derived neuronal cultures by assaying for ARQ 621 of CHAT and MAP2, bona fide markers of MN maturity. Further, by assaying for phenotypes that were observed in either ALS postmortem tissue or rodent models, we established that our in vitro model faithfully recapitulates specific aspects of the disease. Having established that we were able to capture ALS disease pathophysiology in our in vitro system, we sought to uncover additional pathways dysregulated in ALS MN by deep RNA-seq. Analysis of the RNA-seq data identified several pathways commonly dysregulated in ALS MNs. For instance, activation of cell-cycle genes and p53 was observed in mutant SOD1 MNs. Since neurons are post-mitotic, reactivation of the cell cycle in neurons results in activation of the apoptotic pathway, partly in a p53-dependent manner (Herrup and Yang, 2007). Hence, we expected that inhibition of either p53 or the cell-cycle program would result in increased survivability of ALS MNs. However, inhibition of the cell-cycle pathway by targeting the CDK proteins diminished cell survival. This suggests that CDKs may have cell-cycle-independent functions in MN essential for homeostasis. Inhibition of p53 resulted in a modest improvement in survival, indicating that p53 may not be a driver of neurodegeneration, an observation supported by rodent ALS models (Kuntz et al., 2000). How does mutant SOD1 activate MAPK signaling? Recently, it was shown that ER stress leads to activation of JNK signaling via phosphorylation of HIPK2 in the SOD1 G93A mouse model of ALS. Activation of HIPK2 and JNK closely correlated with SOD1 aggregation and cell death, suggesting a mechanism whereby increased SOD1 aggregation leads to a heightened ER stress that in turn causes cell death via activation of JNK (Lee et al., 2016). It is possible that the ER stress may result in activation of other members of the MAPK family, including ERK, via hitherto unknown intermediary kinases. This would make the ER stress pathway an attractive target to identify ALS therapeutics. However, it must be noted that inhibiting the ER stress pathway via RNAi or pharmacological inhibition in an iPSC model of SOD1 ALS led to only modest improvement in MN survival (Kiskinis et al., 2014). On the other hand, FUS-R521C transgenic mice did not display any increase in ER stress markers or JNK activation in spite of showing progressive neurodegeneration (Lee et al., 2016). Consistent with these observations, we find that MNs derived from mutant FUS iPSCs do not display activation of JNK signaling. However, these mutant FUS MNs show activation of other members of the MAPK family, namely p38 and ERK. Taken together, this suggests that the ER stress may contribute toward activating the MAPK pathway in ALS, although to a minor extent.