Figure 4. Regenerative axon growth from adult sensory neurons requires mTOR-independent protein synthesis.

(a, b) Adult DRG neurons were dissociated, cultured for 3 days and then replated to initiate axon growth anew, as depicted in Fig. 1a. Neurons were treated with rapamycin (20 nM), cycloheximide (5 μg/ml), or DMSO (control) either before (red bars in b) or after replating (blue bars in b), as indicated, and axon length was measured at 20 hr after replating. The schematics and symbols (upper left insets in a) are identical to Fig. 1d. Representative images of replated neurons are shown in a, and quantification of axon length from three independent experiments is shown in b. Scale bar, 200 μm. Error bars represent s.e.m. n.s., statistically insignificant difference, unpaired two-tailed student t test.

(c) Representative images of control (upper panels) and rapamycin-treated (lower panels) adult DRG neurons immunostained with TuJ1 and phospho-S6 antibodies. Note that rapamycin treatment completely prevented S6 phosphorylation, but had no effect on axon growth. Scale bar, 200 μm.

(d) Representative immunoblots from adult DRG neurons cultured for 3 days in the presence or absence of rapamycin. Actin antibodies were used as a loading control. Note that rapamycin treatment completely prevented S6 phosphorylation. Original immunoblot image is shown in Supplementary Fig. S11.