Fidgetin-like 2 negatively regulates axonal growth and can be targeted to promote functional nerve regeneration.

Lisa Baker,Rabab A Charafeddine,Guillermo A Villegas,Joel Friedman,Adam H Kramer,Kelvin P Davies,Moses Tar,David J Sharp,Olga Vafaeva,Parimala Nacharaju

doi:10.1172/jci.insight.138484

Abstract

The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT-severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axon regeneration and a therapeutic target for promoting nerve regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether FL2 could be targeted to promote regeneration in a rodent model of cavernous nerve (CN) injury. The CNs are parasympathetic nerves that regulate blood flow to the penis, which are commonly damaged during radical prostatectomy (RP), resulting in erectile dysfunction (ED). Application of FL2-siRNA after CN injury significantly enhanced functional nerve recovery. Remarkably, following bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA, while no control-treated animals exhibited regeneration. These studies identify FL2 as a promising therapeutic target for enhancing regeneration after peripheral nerve injury and for mitigating neurogenic ED after RP — a condition for which, at present, only poor treatment options exist.

Highlights

The microtubule (MT) cytoskeleton is a major regulator of axon growth and guidance
To test whether fidgetin-like 2 (FL2) regulates axon growth, adult dorsal root ganglion (DRG) neurons harvested from FL2-flox homozygous mice were transduced ex vivo with adenovirus (AV) containing either a Cre recombinase plasmid (Cre AV) to excise the FL2 gene or GFP control plasmid (GFP AV)
We found that treatment with a recombinant semaphorin 3A (Sema3A)-Fc chimera caused an approximately 20% increase in the percentage of growth cone (GC) exhibiting a collapsed phenotype compared with neurons treated with Fc alone (Figure 3E), consistent with a previous study, which found that GCs of adult rat DRG neurons with a collapsed phenotype rose 18% with Sema3A treatment in vitro [46]

Summary

Introduction

The microtubule (MT) cytoskeleton is a major regulator of axon growth and guidance. It serves as the major track for vesicular transport through the axon shaft and provides structural support to the axon, and in the distal region of the axon, polymerizing MTs play a critical role in promoting axon elongation and in steering the growth cone in response to environmental cues [1, 2]. MT-severing enzymes, which are members of the ATPases associated with diverse cellular activities superfamily, cause breakages in MTs by forming hexameric rings around the C-terminal tails of tubulin and using energy from ATP hydrolysis to pull on the tails, thereby causing tubulin dimers to dissociate from the MT lattice [12, 13]. Through their severing activity, they regulate MT length, number, and branching and fine-tune the dynamics of the MT cytoskeleton [14, 15]. With the exception of FL2, all have been reported to play important roles in regulating axonal growth through their remodeling of the axonal MT array [9, 16,17,18,19,20,21]

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