U6-shRNA-GFP AAV (Serotype 9)

Fidgetin is a microtubule-severing enzyme that regulates neurite growth, axon regeneration, and cell migration by trimming off the labile domain of microtubule polymers. Here, researchers constructed rat spinal cord injury and sciatic nerve injury models. Compared to spinal cord injury, the researchers found that expression levels of microtubule-destabilizing partially tyrosinated microtubules were consistently increased and expression levels of fidgetin were decreased after peripheral nerve injury. Depletion of fidgetin enhanced axonal regeneration after spinal cord injury, while the expression levels of terminal binding protein 3 (EB3) were significantly increased. Next, the researchers performed RNA interference to knockdown EB3 or fidgetin. Deletion of EB3 does not alter fidgetin expression. In contrast, deletion of fidgetin significantly increased the expression of tyrosinated microtubules and EB3. Deletion of fidgetin increases the amount of EB3 in neurite terminals, thereby increasing the levels of tyrosinated microtubules. Fidgetin trims tyrosinated tubulin by interacting with EB3. Upon deletion of fidgetin, the labile portion of microtubules was elongated, resulting in an increase in axonal length and the number of axonal branches. These findings suggest that fidgetin may serve as a novel therapeutic target to promote axonal regeneration after spinal cord injury.

Here, the researchers first established a spinal cord contusion model in adult rats. Fign knockdown and control mice were constructed using adeno-associated virus (AAV) serotype 9 of U6-shFign-green fluorescent protein (GFP) and control U6-shRNA-GFP AAV9, respectively (Figure 1C and D). After injury, behavioral recovery of rats was assessed using BBB scores. Results showed that Fign knockdown improved BBB scores, reaching significance at day 14 post-injury (Figure 1E). Fign knockdown efficiency at 14 days post-injury was confirmed by Western blotting (Figure 1F). Next, the effects of Fign knockdown were observed by immunohistochemistry 14 days after injury. In the Fign-shRNA treatment group (Figure 1G and H), the immunopositivity rate of NF200 in the injured area was significantly increased. These results indicate that Fign knockdown improves functional recovery and enhances axonal regeneration after SCI. Interestingly, knockdown of Fign resulted in a significant increase in the MT plus-end binding protein EB3, which was concentrated in tyrosine-tubulin-rich regions (Figure 1I and J).

Figure 1. Depletion of Fign improves functional recovery by increasing Tyr-tubulin. (Ma C, et al., 2023)