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 7

Regenerated myelinated and unmyelinated axons are present in the CN distal to the injury site following transection and FL2-siRNA wafer treatment.

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Regenerated myelinated and unmyelinated axons are present in the CN dist...
(A and D) Transmission electron microscopy (TEM) images, original magnification, ×5000, of a distal segment of a CN from an uninjured animal (A) and 1 from a transected and FL2-siRNA wafer–treated animal (D), 4 weeks after transection and treatment (harvest of control-siRNA wafer–treated distal nerve segments was not possible due to retraction of the distal nerve segment). Arrows point out myelinated large-diameter axons. Arrowheads point to some of the Remak bundles of unmyelinated small-diameter axons. Scale bar: 2 μm. SCN, Schwann cell nucleus. (B and E) TEM images, original magnification, ×20,000, of an uninjured (B) and transected and FL2-siRNA wafer–treated nerve (E) showing Remak bundles at higher magnification. Scale bar: 0.5 μm. (C and F) ×20,000 TEM images of uninjured CN (C) and a transected and FL2 siRNA wafer–treated nerve distal to injury site (F), showing myelinated axons. Scale bar: 0.5 μm. (G) Representative lanes from Western blot of corporal tissue lysates from control and FL2-siRNA wafer–treated animals, 1 month following bilateral transection of the CN, probed for nNOS and GAPDH (lanes rune on same gel but noncontiguous). (H) Relative levels of nNOS in the penile shaft analyzed by Western blot and normalized to GAPDH, 4 weeks after bilateral CN transection and treatment with FL2- or control-siRNA wafers. Only FL2-siRNA wafer–treated animals that exhibited regeneration in both CNs included in analysis. Control mean ± SEM = 1.00 ± 0.24, FL2 si = 1.5 ± 0.26, P = 0.18, unpaired 2-tailed Welch’s t test, control n = 5, FL2 si n = 4.