Fidgetin-like 2 negatively regulates axonal growth and can be targeted to promote functional nerve regeneration
Lisa Baker, … , Kelvin P. Davies, David J. Sharp
Lisa Baker, … , Kelvin P. Davies, David J. Sharp
Published April 19, 2021
Citation Information: JCI Insight. 2021;6(9):e138484. https://doi.org/10.1172/jci.insight.138484.
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Research Article Neuroscience Reproductive biology

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

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.

Authors

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

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Figure 1

FL2 depletion accelerates the rate of axon regeneration in adult DRG neurons.

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FL2 depletion accelerates the rate of axon regeneration in adult DRG neu...
(A) Schematic of internal ribosome entry site–tdTomato (IRES-tdTomato) knockin and LoxP sites at Fignl2 endogenous locus. The Fignl2 gene is on chromosome 15 and is intronless, composed of 1 exon extending over 4.2 kB. An IRES and tdtomato (tdTOM) reporter gene were inserted after the stop codon between the coding sequence and 3′ UTR sequence. LoxP sites were inserted upstream of the start codon and after the reporter gene. (B) Fignl2 and tdTOM mRNA levels 1 week after transduction with GFP AV or Cre AV. RNA combined from 4 transduced cultures for each experiment. (C) Average length of longest neurites in control and FL2-knockout neurons 2 days after replating (GFP AV mean ± SEM: 1.00 ± 0.05; Cre AV: 1.51 ± 0.07. GFP AV n = 517, Cre AV n = 479). Experiment performed in quadruplicate. (D and E) Micrographs of GFP AV (D) and Cre AV (E) DRG neurons replated at low density 1 week after viral transduction, fixed 2 days later, and immunostained for tubulin (tubulin isoform β3) (scale bar: 200 μm). (F) Neurite outgrowth (sum length of all neurites per neuron) in GFP AV– and Cre AV–treated neurons 2 days after replating, normalized to the control mean (GFP AV mean ± SEM: 1.00 ± 0.10; Cre AV: 1.74 ± 0.18). GFP n = 162, Cre AV n = 174. (G) Numbers of primary, secondary, and tertiary neurites on GFP AV– and Cre AV–treated neurons (primary: GFP AV: 3.26 ± 016, Cre AV: 3.55 ± 0.18, P = 0.24, secondary: GFP AV: 2.01 ± 0.24, Cre AV: 2.47 ± 0.24, P = 0.17; tertiary: GFP AV: 0.72 ± 0.25; Cre AV: 1.3 ± 0.27, P = 0.12. GFP n = 159, Cre AV n = 173). Experiments performed in triplicate unless otherwise noted. Data were analyzed using unpaired 2-tailed Welch’s t test (morphometric data) or 2-tailed Student’s t test (qPCR data). Bars represent mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001.