Fig. 3: mutSOD1 astrocytes release β-secretase sensitive N-APP fragments that cause toxicity in ALS in vitro models.

a NTg (blue) or mutSOD1 (red) astrocytes infected with empty vector (EV) (n = 4 NTg and n = 7 mutSOD1), sh-SOD1 (n = 4 NTg and n = 7 mutSOD1) or sh-APP (n = 2 NTg and n = 4 mutSOD1) selected with puromycin for 4.5 days, replaced with regular astrocyte media and then were co-cultured with mouse ES-MNs expressing EGFP under the control of the MN-specific Hb9 promoter. Viability was measured at 7 DIV. MNs were counted using Metamorph software. Data for EV and sh-SOD1 are means ± SEM of independent experiments (n) and were analyzed by a two-way ANOVA (Interaction F_(2,22) = 7.824; P = 0.0027) followed by a Sidak post hoc test: ****P ≤ 0.0001 EV NTg vs mutSOD1 (CI: 32.34–88.86%; d = 7.29). Data for sh-APP in NTg (n = 2) are means only and were not analyzed by statistics. b Inhibitors of α-secretase (n = 3), β-secretase (n = 4), γ-secretase (n = 2), or vehicle control (n =4) were applied twice (1 DIV and 4 DIV) at 5 μM, 250 nM, and 500 nM, respectively to primary MN and GFP+ MNs were counted on DIV 7. Data for control, α-secretase and β-secretase are means ± SEM of independent experiments (n) and were analyzed by a two-way ANOVA (Interaction F_(3,18) = 13.97; P < 0.0001) followed by a Sidak post hoc test: ****P ≤ 0.0001 Control NTg vs mutSOD1 (CI: 24.9–49.69%; d = 12.27) and α-secretase NTg vs mutSOD1 (CI: 21.67–50.29%; d = 4.83). Data for γ-secretase in NTg (n = 2) are mean only and were not analyzed by statistics. c Schematic figure of protein domains of APP and E1 and E2 segments. See also Supplementary Figs. 2 and 3. Source data provided as source data file.