(A) Schematic representation of the experimental design.
(B and C) Ribosomal decrease induced by FGF22 is dependent on ubiquitin proteasome system (UPS) but not dependent on autophagy. Spinal MNs axons were stimulated, at DIV 3/4, with either BSA (Ctr) or FGF22 (2 nM). Inhibitors of autophagy 3-MA (10 mM; B) and leupeptin (200 μM; C) or UPS inhibitors MG132 (1 μM; B) and epoxomicin (1 μM; C) were applied 6 h before the end of FGF22 stimulus. Neurons were immunostained against RibP P0 (green) and Tau (red). Scale bar is 2.5 μm.
(D–G) Quantitative data of the number of RibP P0 clusters per axonal length using different autophagy and UPS inhibitors. 3-MA (D), MG132 (E), leupeptin (F), and epoxomicin (G). Bars represent the mean ± SEM. Statistical significance by one-way ANOVA followed by Tukey’s post hoc test. (n.s., non-statistically significant.)
(H and I) Proposed model for ribosomal regulation in distal axons during axonal development (H) and upon axonal maturation (I). Upon axonal contact with a postsynaptic target or with synaptogenic molecules such as FGF22 (1), SVs accumulate within the nascent presynaptic button (2). After the immature axon establishes functional synapses, ribosome levels decrease significantly (3). A mechanism that is dependent on the ubiquitin proteasome system (UPS) is shown (4).