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 3

FL2 depletion attenuates the effects of inhibitory substrates on adult DRG GC advancement during regeneration.

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FL2 depletion attenuates the effects of inhibitory substrates on adult D...
(A) Micrographs of GFP AV– and Cre AV–treated neurons immunostained for TUBB3 (green) 72 hours after plating on aggrecan stripes (red). Scale bar: 200 μm. (B) High-magnification image of a GFP AV–treated neurite turning in response to an aggrecan border and Cre AV–treated neurites crossing through the stripe border. Scale bar: 50 μm. (C) Fraction of neurites of GFP AV– and Cre AV–treated neurons that crossed aggrecan stripe borders (GFP AV mean = 0.26 ± 0.026; Cre AV mean = 0.41 ± 0.06. P = 0.0169, unpaired 2-tailed Welch’s t test, GFP n = 65 neurons, 257 neurite/border encounters; Cre AV n = 79 neurons, 200 neurite/border encounters. Experiment performed 4 times). (D) Immuno-micrographs showing an active, viable GC (left) and a collapsed GC (right), stained for MTs (TUBB3) in red and actin in green. Scale bar: 10 μm. (E) Fraction of collapsed GCs in untreated, GFP AV–treated, and Cre AV–treated neurons, in the presence and absence of Sema3A. GFP and Cre AV experiments performed in quadruplicate, untreated +/– Sema3A performed in triplicate (untreated, no Sema3A: 0.38 ± 0.04, n = 425 GCs; untreated, +Sema3A: 0.56 ± 0.03, n = 376; GFP AV, no Sema3A: 0.35 ± 0.01, n = 379; GFP AV, +Sema3A: 0.55 ± 0.04, n = 410; Cre AV, no Sema3A: 0.35 ± 0.05, n = 440; Cre AV, +Sema3A: 0.40 ± 0.02, n = 422. Mean ± SEM; *P < 0.05, **P < 0.01; unpaired 2-tailed Welch’s t test). Imaging and analyses performed in a blinded manner.