Fig. 7
Edaravone induces the GDNF/RET neurotrophic signaling pathway in miMNs and the mouse spinal cord. (A and B) Control and ALS miMNs were subjected to edaravone treatment (10 μM, 24 h) or DMSO as the vehicle control. Total cellular proteins were subjected to western blotting of genes for validation (A, left) and quantification from three blotting results (B, right). Total cellular RNAs were subjected to qPCR analysis of three TF genes (B). (*: p < 0.05, t test with bonferroni correction for multiple comparisons). (C) Control and ALS miMNs with +/− edaravone treatment (10 μM, 24 h) were subjected to western blotting (left) and quantification from three blotting results (right) to measure the levels of GDNF/RET signaling components (*: p < 0.05, t test with the Bonferroni correction for multiple comparisons). (D and E) Control and ALS miMNs (day 7 of differentiation) were pre-treated with +/− edaravone (10 μM) and GDNF (1 ng/ml) for 16 h in neurotrophic factor-free medium and were treated with H2O2 (50 μM) or PBS as the vehicle control for 24 h. Neurite length was quantified from 6 wells for each condition as technical replicates, after Calcium AM staining (Bar = 10 μm). Edaravone+GDNF more effectively alleviated H2O2-induced neurite damage than single treatment. (F) Control miMNs and astrocytes differentiated from human NSCs were subjected to edaravone treatment (10 μM) for 48 h. GDNF release in the culture medium was measured by ELISA with 3 technical replicates. (G) Control miMNs were cultured in neuron maturation medium with +/− edaravone and GDNF/BDNF for 15 days. Brightfield (BF) and Calcium AM staining images were shown (Bar = 10 μm). Neurite length from 6 wells for each condition as technical replicates was compared among different conditions. (H and I) Mice (n = 6 for each group) received intraperitoneal injection of edaravone (15 mg/kg daily) or vehicle (saline) for 5 days. Total proteins from spinal cord tissues were analyzed by western blotting (G) Representative western blotting and quantification of protein fold expression normalized to Actin (*: p < 0.05, t test with the Bonferroni correction for multiple comparisons). Total proteins from spinal cord tissues from 6 mice were also analyzed by GDNF ELISA (H, *: p < 0.01, t test). Data represents Mean ± SEM