Fig 7. Overexpression of LSM12 or EPAC1 rescues NCT-relevant pathologies in C9-ALS patient-derived neurons.
(A) C9-ALS iPSNs express low levels of LSM12 and EPAC1 proteins. C9-ALS iPSNs (CS28, CS29, and CS52) and control iPSNs (CS0, CS29-ISO, CS4) were harvested 21 days after neuronal differentiation from NPCs. Total cell extracts from 3-week-old iPSNs were resolved by SDS-PAGE and immunoblotted with anti-LSM12, anti-EPAC1, and anti-tubulin (loading control) antibodies. The abundance of each protein was quantified as in Fig 1C. Error bars indicate SEM (n = 3 independent differentiation experiments). *P < 0.05, **P < 0.01, as determined by 1-way ANOVA with Dunnett post hoc test. (B) C9-ALS iPSNs express low levels of LSM12 and EPAC1 transcripts. Total RNA was prepared from 3-week-old iPSNs, and the abundance of each transcript was quantified as in Fig 5B. Data represent means ± SEM (n = 3 independent differentiation experiments). n.s., not significant; *P < 0.05, as determined by 1-way ANOVA with Dunnett post hoc test. (C, D) Overexpression of LSM12 or EPAC1 rescues the RAN gradient in C9-ALS iPSNs. NPCs from C9-ALS iPSCs (CS29) and their isogenic control cells (CS29-ISO) were transduced with individual recombinant lentiviruses that express the indicated FLAG-tagged proteins along with a GFP reporter. iPSNs were fixed 21 days after neuronal differentiation from NPCs and co-stained with anti-RAN antibody (red), anti-MAP2 antibody (magenta), and Hoechst 33258 (blue). The nucleocytoplasmic RAN gradient was quantified as in Fig 2D. Two-way ANOVA detected significant interaction effects of C9-ALS and lentiviral overexpression on the RAN gradient (P = 0.0051 for LSM12; P = 0.0066 for LSM12V135I; P = 0.0041 for EPAC1). Data represent means ± SEM (n = 100–105 GFP–positive cells from 4 independent differentiation experiments). n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, as determined by Tukey post hoc test. (E, F) Overexpression of LSM12 or EPAC1 suppresses the pathogenic mislocalization of TDP-43 in the cytoplasm of C9-ALS iPSNs. Three-week-old iPSNs (CS29-ISO and C9-ALS CS29) were co-stained with anti-TDP-43 antibody (red), anti-MAP2 antibody (magenta), and Hoechst 33258 (blue). The relative distribution of endogenous TDP-43 proteins was quantified similarly as above. Two-way ANOVA detected significant interaction effects of C9-ALS and lentiviral overexpression on the RAN gradient (P = 0.0154 for LSM12; P = 0.0120 for EPAC1). Data represent means ± SEM (n = 102–104 GFP–positive cells from 4 independent differentiation experiments). n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, as determined by 2-way ANOVA with Tukey post hoc test. (G, H) Overexpression of LSM12 or EPAC1 suppresses caspase-3 activation in C9-ALS iPSNs. Three-week-old iPSNs (CS29-ISO and C9-ALS CS29) were co-stained with anti-cleaved caspase-3 antibody (red), anti-MAP2 antibody, and Hoechst 33258 (blue). The relative percentages of iPSNs expressing cleaved caspase-3 were averaged from 15 confocal images of random fields of interest per condition (n = 771–1,129 GFP-positive cells from 3 independent differentiation experiments). Data represent means ± SEM. n.s., not significant; ***P < 0.001, as determined by 2-way ANOVA with Tukey post hoc test. All underlying numerical values are available in S1 Data. ANOVA, analysis of variance; ATXN2, ataxin-2; C9-ALS, C9ORF72-associated amyotrophic lateral sclerosis; EPAC1, exchange protein directly activated by cyclic AMP 1; GFP, green fluorescent protein; LSM12, like-Sm protein 12; N/C, nuclear to cytoplasmic; NCT, nucleocytoplasmic transport; NPC, neural progenitor cell; SEM, standard error of the mean.