Local Synthesis of Reticulon-1C Lessens the Outgrowth of Injured Axons by Controlling Spastin Activity

Abstract

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 structural proteins of the axonal endoplasmic reticulum. Therefore, we hypothesize that locally synthesized structural proteins of the endoplasmic reticulum may regulate microtubule dynamics and the outgrowth of injured cortical axons. This hypothesis is supported by RNA data-mining, which identified Reticulon-1 as the sole ER-shaping protein consistently present in axonal transcriptomes and found it to be downregulated following cortical axon injury. Using compartmentalized microfluidic chambers, we demonstrate that local knockdown of Reticulon-1 mRNA enhances outgrowth while reducing the distal tubulin levels of injured cortical axons. Additionally, live cell imaging shows injury-induced reductions in microtubule growth rate and length, which are fully restored by axonal Reticulon-1 knockdown. Interestingly, axonal inhibition of the microtubule-severing protein Spastin fully prevents the effects of local Reticulon-1 knockdown on outgrowth and tubulin levels, while not affecting microtubule dynamics. Furthermore, we provide evidence supporting that the Reticulon-1C isoform is locally synthesized in injured axons and associates with Spastin to inhibit its severing activity. Our findings reveal a novel injury-dependent mechanism in which a locally synthesized ER-shaping protein lessens microtubule dynamics and the outgrowth of cortical axons.