Knockdown of Fidgetin Improves Regeneration of Injured Axons by a Microtubule-Based Mechanism

Abstract

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether fidgetin knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetin on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. Fidgetin knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration in vivo after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which fidgetin was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.

RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin

“…Studies have indicated that microtubule dynamics are largely regulated by microtubule-severing proteins [9,32,33]. Moreover, microtubule-severing proteins have been shown to be essential for neurite outgrowth and axon branching [34][35][36][37]. This led us to hypothesize that microtubule-severing proteins might be vital players in the neurite outgrowth directed by the RhoA signaling pathway.…”

The Struggle to Make CNS Axons Regenerate: Why Has It Been so Difficult?

“…A number of molecules affect microtubule dynamics by binding to them and having the ability to sever the microtubules, spastin atlastin fidgetin and katanin are examples. The level of these molecules is critical: in Drosophila if levels of spastin and atlastin are too high or too low regeneration fails [107], and fidgetin is a regeneration inhibitor [89]. Kinesins are generally thought of as transport molecules, but some can also affect microtubule dynamics.…”

Fidgetin-like 2 is a novel negative regulator of axonal growth and can be targeted to promote functional nerve regeneration after injury

“…It serves as the major track for vesicular transport through the axon shaft, 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]. Given its role in regulating axonal growth, targeting the MT cytoskeleton to enhance axon regeneration after nerve injury has been proposed as a potential therapeutic strategy [3][4][5][6][7][8][9][10][11]. This proposition is supported by studies demonstrating that modulation of MT dynamics in the axon can promote regeneration and attenuate degeneration: for example, multiple studies have found that low doses of MT stabilizing drugs (taxol and epothilones) promote axon regeneration and improve recovery of locomotor function after spinal cord injury (SCI) in rats [5][6][7]11].…”

“…This proposition is supported by studies demonstrating that modulation of MT dynamics in the axon can promote regeneration and attenuate degeneration: for example, multiple studies have found that low doses of MT stabilizing drugs (taxol and epothilones) promote axon regeneration and improve recovery of locomotor function after spinal cord injury (SCI) in rats [5][6][7]11]. Increasing the density of dynamic MTs in the axon by knocking down the MT severing enzyme fidgetin was also shown to promote axon regeneration after dorsal root crush in rats [9].…”

“…MT severing enzymes include katanin, spastin, and the fidgetin family (fidgetin, fidgetin-like 1 (FL1), and fidgetin-like 2 (FL2)). 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,[15][16][17][18][19].…”