FIGURE 6.

PI3K-mediated pathology was also not abrogated when mTOR was simultaneously ablated during adulthood from myelinating Schwann cells. (a) Semithin sections of sciatic nerves from control, PlpCre^ERT;mTOR-het;Pik^DD, and PlpCre^ERT;mTOR-KO;Pik^DD mice injected with Tm at 1 month of age and analyzed at 5 months postinjection (MPI) show numerous abnormal myelin figures (red arrowheads) in both the PlpCre^ERT;mTOR-het;Pik^DD and PlpCre^ERT;mTOR-KO;Pik^DD mice but not in the controls. (b) EM images at higher magnification show presence of abnormalities in both the PlpCre^ERT;mTOR-het;Pik^DD (i–iii) and PlpCre^ERT;mTOR-KO;Pik^DD (iv–vi), which includes (i,iv) myelin out-foldings with evaginating recurrent loops, (ii,v) redundant myelin, constricting and displacing the axon, and (iii,vi) multiple evaginating recurrent loops that appear as Schwann cell ensheathing multiple small-diameter axons. (c) Teased fiber preparation from sciatic nerve shows focal myelin thickening with abnormal structures in both the PlpCre^ERT;mTOR-het;Pik^DD and PlpCre^ERT;mTOR-KO;Pik^DD mice. Scale bar as indicated. Asterisks, axonal abnormalities. Representative images of sciatic nerves from 2–3 mice per genotype are shown. (d) Quantification of abnormal myelin figures in seminthin sections of sciatic nerves from mice at 5 MPI show a statistically significant increase in the percentage of affected axons in the PlpCre^ERT;mTOR-het;Pik^DD mice compared to control mice which failed to be rescue in the PlpCre^ERT;mTOR-KO;Pik^DD mice. Ten fields at 100x per genotype were analyzed from at least 2–3 mice per group. Error bars indicate SEM. **p < .01, one-way ANOVA. (e) Immunoblot analysis and quantification for p-mTOR^S2448 and p-p70S6K^T389 levels in total protein homogenates from sciatic nerve at 2MPI shows a significant increase in p-mTOR^S2448 and p-p70S6K^T389 levels in the PlpCre^ERT;Pik^DD mice compared to controls. While ablation of mTOR showed reduction trend of p-mTOR^S2448 levels in the PlpCre^ERT;mTOR-KO;Pik^DD compared to PlpCre^ERT;Pik^DD mice, the levels of p-p70S6K^T389 did not show such a trend. β-Actin was used as a loading control. N = 3 for control and PlpCre^ERT;Pik^DD and N = 2 for PlpCre^ERT;mTOR-KO;Pik^DD. Error bars indicate SEM. *p < .05, Student’s t-test [Color figure can be viewed at wileyonlinelibrary.com]