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  • To investigate whether the activated pathways identified in

    2018-10-24

    To investigate whether the activated pathways identified in ALS MNs were causal or merely associated with the disease phenotype, we assayed MN survival in vitro after pharmacological inhibition of the identified pathways. MNs differentiated from mutant SOD1 iPSCs were treated with small-molecule inhibitors targeting buy D-Luciferin the p38, ERK, JNK, and CDK kinases, as well as p53 and WNT pathways, at the indicated concentrations starting at day 30 and continued until day 44 (Figure 4A, Table S1). Untreated (vehicle only control) SOD1 MNs showed a significant loss of survival with ∼50% of the MNs lost at day 44 compared with day 30 (Figure 4A). Strikingly, inhibition of the AP1 complex, especially via targeting ERK signaling, reduced MN loss in a dose-dependent manner such that only 16% of the MNs were lost at day 44 at the highest concentration used (Figure 4A). Inhibition of p38/MAPK signaling similarly restricted mutant SOD1 MN loss to 25% (Figure 4A). Treatment of SOD1 MNs with a JNK inhibitor or an inhibitor of the WNT pathway had a significant but modest effect on SOD1 MN survival compared with the control treatment (Figure 4A). Surprisingly, inhibition of the p53 pathway displayed only a modest improvement on the survival of ALS MNs, suggesting that p53 activation may play a minor role in driving neurodegeneration in ALS (Figure 4A). The CDK inhibitor did not promote survival of ALS MNs and, unexpectedly, was toxic to MNs at the higher concentrations used (Figure 4A). In concordance with our phenotypic screen, we observed activation of ERK1/2 and JNK1/2 in the SOD1 mutant MNs (Figure 4B). Surprisingly, contrary to our expectation, we did not observe activation of p38 in mutant SOD1 MNs compared with isogenic control MNs (Figure 4B). We performed immunofluorescence assays in ALS and isogenic control MNs to confirm activation of the AP1 and WNT pathways. For a given target protein (i.e., JUN), we measured the median fluorescent signal in the nuclei of diseased and isogenic control MNs and plotted the distribution of intensities (Figure 4C). Immunofluorescence assays confirmed increased nuclear localization of the AP1 complex member JUN as well as β-catenin in mutant SOD1 MNs compared with the isogenic control MNs (Figures 4C and S3A), confirming activation of the AP1 and WNT pathways in ALS MNs. ALS is characterized by selective loss of MNs, while other buy D-Luciferin remain viable even though they harbor the same germline mutations. Using our in vitro model, we asked if there was differential activation of signaling pathways in MNs and non-MNs that might confer disease-specific loss of MNs. We analyzed nuclear localization of JUN and β-catenin in MNs (ISL1+) and non-MNs (ISL1−). Strikingly, we found that the levels of nuclear JUN were almost five times higher in MNs compared with non-MNs (Figure 4D). This difference between MNs and non-MNs was evident even in the isogenic controls and was heightened in the SOD1 cultures (Figure 4D). This observation suggests a mechanism where MNs require an active but tightly regulated AP1 pathway for normal homeostasis that is not required by non-MNs. Mutant SOD1 might cause hyperactivation of the AP1 pathway via ERK and JNK activation, perturbing the homeostasis and thereby driving neurodegeneration (Figure 4E). Although we observed a modest increase in nuclear JUN in the non-MN population, the absolute levels of the protein might not be sufficient to perturb homeostasis. This might explain the selective loss of MNs due to over-activation of the AP1 pathway. We observed a similar trend for WNT activation, where MNs displayed higher WNT activity compared with non-MNs, although the difference was less striking compared with that observed for JUN (Figure S3B). Next, we asked whether pathways driving neurodegeneration in SOD1 MNs were also activated by other ALS-associated mutations. To answer this question, we sought to analyze the MAPK pathway in MNs bearing mutations in the FUS gene. We obtained iPSCs from a patient homozygous for the recessive H517Q mutation in the FUS gene (Kwiatkowski et al., 2009) and corrected this recessive mutation to heterozygous wild-type using the CRISPR-Cas9 system. Correction of the homozygous point mutation was confirmed by PCR amplification of the targeted genomic region followed by DNA sequencing (Figure 5A). We further confirmed that the corrected FUS iPSCs displayed a normal karyotype (Figure 5B). We were able to differentiate both the mutant and corrected FUS iPSCs into ISL1+/TUJ1+ spinal MNs with efficiencies similar to those obtained with the SOD1 iPSCs (H517Q/H517Q, 73% ± 1.7%; +/H517Q, 70% ± 0.74%; mean ± SEM) (Figure 5C). In addition, greater than 85% of ISL1+ MNs (day 30) expressed the mature MN marker CHAT (Figure 5C), similar to efficiencies obtained using SOD1 and control iPSCs. To investigate activation of the MAPK pathway, we assayed expression of individual MAPK family members in protein lysates derived from the mutant and corrected FUS MNs. Strikingly, we found activation of the p38 kinase and ERK kinase in the FUS mutant MNs, while the JNK kinase was not activated (Figure 5D). These observations indicate that MAPK activation is a common feature related to both SOD1 and FUS mutations, although these mutations activate distinct members of the MAPK family.