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

FL2 depletion results in a more dynamic MT array near the growth cone in regenerating neurites.

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FL2 depletion results in a more dynamic MT array near the growth cone in...
(A and B) Immuno-micrographs of DRG neurites of GFP AV– and Cre AV–treated neurons 2 days after replating, dual-stained for tubulin isoform β3 (TUBB3) (green) and tyrosinated tubulin (Tyr tub) (red) (A) or for TUBB3 (green) and acetylated tubulin (Act tub) (red). Brightness/contrast adjusted linearly to more clearly see acetylated tubulin stain (B). Scale bars: 50 μm. (C) Tyr/TUBB3 ratios in GFP AV– and Cre AV–treated neurons from 1 representative experiment, starting at the tip of the growth cone and moving toward the soma (dark line is the mean intensity ratio; shaded area represents the SEM). (D) Tyr/TUBB3 fluorescence intensity ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1.00 ± 0.03, Cre AV: 1.26 ± 0.05, n = 86 GFP, n = 101 Cre). (E) Distribution of Act/TUBB3 ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1 ± 0.06, Cre AV: 0.74 ± 0.04. n = 87 GFP; n = 116 Cre). (F) Act/TUBB3 ratios in the proximal to mid region of the axonal shaft (GFP AV: 1.00 ± 0.13; Cre AV: 0.94 ± 0.07, P = 0.68, n = 43 GFP, n = 51 Cre). Experiment performed in duplicate. (G) Inverted images of MTs in the growth cones (GCs) of GFP AV– and CRE AV–treated neurons, immunostained for TUBB3. Brightness/contrast adjusted to better visualize individual MTs. Scale bar: 10 μm. (H) Mean TUBB3 fluorescence intensities in the distalmost 50 μm of neurites (GFP: 1.00 ± 0.06, Cre: 1.18 ± 0.09, Welch’s t test, P = 0.12, n = 91 GFP, n = 101 Cre). Experiments performed in triplicate unless otherwise indicated. Data were analyzed using unpaired 2-tailed Welch’s t test. Bars represent mean ± SEM. ****P < 0.0001.