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. Author manuscript; available in PMC: 2015 Nov 19.
Published in final edited form as: Cell Stem Cell. 2014 Apr 3;14(6):781–795. doi: 10.1016/j.stem.2014.03.004

Figure 5. SOD1+/A4V Motor Neurons Exhibit Signatures of an Unfolded Protein Response and are Selectively Vulnerable to ER Stress Induction.

Figure 5

(A) Gene set enrichment analysis of transcriptional changes in SOD1+/A4V MNs shows strong downregulation (normalized enrichment score: -3.31) for genes involved in translational capacity. Horizontal black bars represent individual genes with representative examples indicated. Blue or red represents genes which were up or downregulated respectively in SOD1+/A4V relative to isogenic control MNs. Out of 139 genes detected that are annotated as involved in regulation of translation, 130 were downregulated, consistent with activation of the UPR pathway. (B) Diagram illustrating the canonical unfolded protein response. (C) WB analysis demonstrates increased levels of phosphorylated EIF2α, a marker for activation of the UPR pathway, in SOD1+/A4V MN cultures. Percentage relative to control samples for each time point is shown. (D) SOD1+/A4V MNs exhibit increased levels of XBP1 splicing, a marker of ER stress. RNA was isolated from purified Hb9∷RFP MNs after 15 days in culture (n=3, +/-SEM, P<0.05). U: unspliced, S: spliced. (E) Experimental strategy used to assess the contribution of XBP1 and ATF4 in the survival of SOD1+/A4V MNs. (F) Representative images of untreated and treated MN cultures are shown. (G) SOD1+/A4V MN numbers selectively increase after XBP1 knockdown, while ATF4 knockdown significantly decreases numbers in both control and SOD1+/A4V cases (n=3, m>9800 and m>8500, P<0.05), +/-SEM, P<0.05). (H) Salubrinal has a modest but positive effect on survival of SOD 1+/A4V MNs (n=2, m>10000, +/-SEM, P<0.05).