FIGURE 5.

Sciatic nerve injury induces a decrease of PDCD4 levels locally in axons next to the injury site and an up-regulation (and activation) of p-p70S6K, a component of mTOR-PDCD4 pathway. PDCD4 could be locally synthetized in peripheral axons as a possible mechanism for protein level regulation. (A) A complete transection of the sciatic nerve was performed in adult rats using the contralateral nerve as a control condition. The analyzed regions are labeled as proximal and distal (or distal injury) in reference to cell bodies. (B) Eighteen hours post injury, the sciatic nerves were extracted from the animals and the levels of PDCD4 were analyzed by immunohistochemistry (scale bar, 5 µm). Signal quantification shows no differences on PDCD4 levels between regions in the control condition (distal vs. proximal), but in the injury to the sciatic nerves, a significant decrease in PDCD4 levels at the injury region was detected (distal injury vs. proximal). These changes are specific to PDCD4 because other proteins in the axon-like F-Actin (evidenced by phalloidin) and MAG protein did not have differences on expression levels comparing distal injury versus proximal (**P ≤ 0.01, two-tailed Mann–Whitney test, n = 3, error bars: SEM). White dotted ROIs correspond to examples of quantified axonal regions. (C) As in B, levels of PDCD4 and p70S6K were analyzed by immunohistochemistry comparing injury axons vs control (scale bar, 10 µm). By signal quantification we detect an increase in the phosphorylated form of p70S6K, a direct upstream regulator of PDCD4 expression on the mTOR pathway (***P ≤ 0.001, two-tailed Mann–Whitney test, n = 2, error bars: SEM). White dotted ROIs correspond to examples of axonal quantified regions. (D) Maximum intensity projection images showing Puro-PLA signal for PDCD4 inside the axoplasm at 30 min of puromycin incubation and the associated control condition without puromycin. The green spots inside the axoplasm reveals that PDCD4 is newly synthetized in the axon. The right panel shows the signal quantification at two different time points of puromycin incubation compared to the control (***P ≤ 0.001 and *P ≤ 0.02, ANOVA and Mann–Whitney test, n = 2, error bars: SEM). White dotted regions define the limits of the axoplasm region. Note that in all the cases, “n” are from independent biological replicates.