Fig. 4

Terazosin protects against NaArO₂-induced cell death, increases glycolysis and decreases mitochondrial respiration in mESC-MNs. (a) TDP-43^M337V mESC-MNs show a decreased survival in response to sodium arsenite (NaArO₂) stress compared to unstressed controls. Survival following terazosin treatment at all concentrations is maintained at 100%. (n = 3 differentiations; One-way ANOVA P < 0.001 with Dunnett's multiple comparisons). (b) Traces from the Seahorse analyser glycolytic rate assay showing glycolytic proton efflux rate (GlycoPER) following mitochondrial inhibition (oligomycin) and inhibition of the glycolysis pathway (2-deoxy-D-glucose; 2-DG). (c) TDP-43^M337V mESC-MNs demonstrate a significantly lower rate of basal glycolysis compared to TDP-43^WT controls, which is rescued by treatment of 2.5 µM terazosin (Tz) (One-way ANOVA P = 0.017 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P = 0.034, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P > 0.99). (d) TDP-43^M337V mESC-MNs show significantly reduced rates of compensatory glycolysis compared to TDP-43^WT controls, which is rescued by treatment of 2.5 µM terazosin (Tz) (One-way ANOVA P = 0.055 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P = 0.038, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P = 0.22). (e) Traces from the Seahorse analyser showing oxygen consumption rate (OCR) following mitochondrial inhibition (oligomycin), mitochondrial uncoupling (FCCP) and electron transport chain inhibition (antimycin A/rotenone). (f) Treatment with 2.5 µM terazosin significantly decreases basal respiration in TDP-43^M337V mESC-MNs compared to TDP-43^WT controls (One-way ANOVA P = 0.001 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P > 0.99, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P = 0.003). (g) Treatment with 2.5 µM terazosin significantly decreases maximal respiration in TDP-43^M337V mESC-MNs compared to TDP-43^WT controls (One-way ANOVA P = 0.003 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P = 0.091, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P = 0.002). (h) Treatment with 2.5 µM terazosin significantly decreases ATP production in TDP-43^M337V mESC-MNs compared to TDP-43^WT controls (One-way ANOVA P = 0.004 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P = 0.87, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P = 0.018). (i) Treatment with 2.5 µM terazosin does not change the spare respiratory capacity in TDP-43^M337V mESC-MNs compared to TDP-43^WT controls (One-way ANOVA P = 0.19 with Bonferroni's multiple comparison test: TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂P = 0.16, TDP-43^WT + NaArO₂ vs TDP-43^M337V + NaArO₂ + 2.5 µM Tz P = 0.38). Each data point represents a separate differentiation; n = 7 per glycolysis analysis; n = 6 per respiration analysis. Error bars represent s.e.m., ns=non-significant, * = P < 0.05, ** = P < 0.01.