Fig. 7.

DITPA rescues oligodendroglial cell death mediated by SLC16A2 knock down, retaining its myelination capability.

(a) Lentivirus (LV) was generated to carry the SLC16A2-shRNA with a mCherry reporter (b) Immunostaining for mCherry and cleaved caspase-3 (cl caspase-3) on NKX2.1-GFP + sorted OPCs at stage V, day 12 (3 days post-transduction with non-targeting, SLC16A2-shRNA, or SLC16A2-shRNA + DITPA (10 ng/mL)) counterstained with DAPI. (b and c) The proportion of mCherry +/cl caspase-3 + apoptotic cells was increased upon SLC16A2-shRNA transduction. DITPA treatment (10 ng/mL) reduced the SLC16A2-shRNA-mediated apoptosis. Scale bar = 50 μm. (d–j) Western blot analysis of lysates from OPCs transduced either with non-targeting-shRNA or SLC16A2-shRNA assayed for p-AKT and p-ERK1/2. (e–f) Densitometric quantification of p-AKT/total AKT, (G-H) p-ERK1/total ERK1, and (i–j) p-ERK2/total ERK2. (k) 3 days post-transduction with SLC16A2-shRNA, OPCs from NKX2.1-GFP + sorted cells were seeded on rat RGCs and were maintained for 7 days with T₃ (50 ng/mL), DITPA (10 ng/mL), or T₃ (50 ng/mL) + DITPA (10 ng/mL) treatment, then immunostained with mCherry, MBP and NF-200 Scale bar = 100 μm. Magnified images of single axons are shown on the right hand side of each image Scale bar = 20 μm. (l) The percentage of overall myelination and mCherry + myelination and (m) the percentages of ensheathing mCherry +/MBP + OLs among the different treatment groups. (n) Representative electron microscopic images of rat RGCs and OPCs co-cultures transduced with non-targeting, or, SLC16A2-shRNA upon DITPA treatment (10 ng/mL), showing compact myelin. Asterisks indicate axon fibers. Scale bar = 0.2 μm. One-way ANOVA with Tukey's post-hoc analysis; **P < 0.01; ****P < 0.0001; N.D.: not detected; (n = 9–10, mean ± SEM).