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

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.SIGNIFICANCE STATEMENT Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down fidgetin from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, fidgetin knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.