Local synthesis of Reticulon-1C lessens the outgrowth of injured axons and Spastin activity

The regenerative potential of developing cortical axons following injury depends on intrinsic mechanisms, such as axon-autonomous protein synthesis, that are still not fully understood. An emerging factor in this regenerative process is the bi-directional interplay between microtubule dynamics and the delicate morphology of the axonal endoplasmic reticulum (ER). Therefore, we hypothesize that locally synthesized ER structural proteins regulate microtubule dynamics and the regenerative response of cortical axons. This hypothesis is supported by RNA data-mining, which identified Reticulon-1 as the sole mRNA coding for an ER-shaping protein across eight axonal transcriptomes and found it to be downregulated following injury. Using compartmentalized microfluidic chambers, we demonstrate that local knockdown of Reticulon-1 mRNA increases the outgrowth while reducing the distal tubulin levels of injured axons. Furthermore, we show by live-cell imaging that axonal Reticulon-1 knockdown restores the loss of microtubule growth rate and length caused by injury. Interestingly, axonal inhibition of the microtubule-severing protein Spastin fully prevents the effects of axonal Reticulon-1 knockdown on outgrowth as well as distal tubulin levels, while not affecting most of the analyzed microtubule dynamic parameters. Further, we demonstrate that the Reticulon-1C isoform is locally synthesized within axons and provide evidence supporting its role in attenuating Spastin-mediated microtubule severing. These findings uncover a mechanism by which axonal protein synthesis provides fine control over the microtubule cytoskeleton in response to injury.