Fidgetin impacts axonal growth and branching in a local mTOR signal dependent manner.

Neurons require a constant increase in protein synthesis during axonal growth and regeneration. AKT-mTOR is a central pathway for mammalian cell survival and regeneration. Fidgetin (Fign) is an ATP-dependent microtubule (MT)-severing enzyme whose functions are associated with neurite outgrowth, axon regeneration and cell migration. Although most previous studies have indicated that depletion of Fign is involved in those biological activities by increasing labile MT mass, it remains unknown whether mTOR activation contributes to this process. Here, we showed that depletion of Fign enhanced p-mTOR/p-S6K activation, and the mTOR inhibitor Rapamycin inhibited axon outgrowth and p-rpS6 activation. We then investigated the effects of neuronal-specific Fign deletion in a rat spinal cord hemisection model by injecting syn-GFP Fign shRNA virus. BBB values revealed an improvement in functional recovery. The p-mTOR was activated along with neuronal Fign depletion. The syn-mCherry virus showed more sprouting neurites entering the injury region, which was confirmed by immunostaining GAP43 protein. Further, we showed that Fign siRNA treatment promoted axon outgrowth and branching, whose underlying mechanism was firstly attributed to local activation of the mTOR pathway, and increased MT dynamicity. Finally, considering L-leucine, promotes axonal growth and neuronal survival, we applied L-leucine with Fign depletion after spinal cord injury or in chondroitin sulfate proteoglycan inhibitory molecules. The phenomenon of synergistically augmented axon regeneration was observed. In summary, our results indicated a novel local mTOR pathway for fidgetin to impact axon growth and provided a combined strategy in SCI.

A comparative study of the expression patterns of Fign family members in zebrafish embryonic development.

During development, highly dynamic reconstruction of microtubules is involved in many cellular processes, including cell division, migration, morphological changes, and material transportation within cells. Microtubule severing proteins (MSPs), with the function of cutting microtubules into short parts, are important regulators in the reconstruction of microtubule arrays. Fidgetin (fign) and its family members fidgetin like 1 (fignl1) and fignl2 are MSPs, and knowledge on the expression patterns of fign family members will benefit our understanding of their primary roles in one specific stage during development. In this study, we compared the evolutionary relationships of fign family members and found that fignl2 is closer to fign than fignl1. We utilized the zebrafish model and in situ hybridization (ISH) to parallelly identify the expression features of fign family members. Our findings revealed that before 12 h post fertilization (hpf), the expression patterns of fign and fignl1 and fignl2 genes were similar, but differences arose thereafter. Fignl2 transcripts were present in more tissues and organs of zebrafish after 12 hpf and potentially exhibited more ubiquitous functions. This study is the first to assess systematic comparable data on the expression patterns of fign family members during development.

Microtubule Severing Protein Fignl2 Contributes to Endothelial and Neuronal Branching in Zebrafish Development.

Previously, fidgetin (fign) and its family members fidgetin-like 1 (fignl1) and fidgetin-like 2 (fignl2) were found to be highly expressed during zebrafish brain development, suggesting their functions in the nervous system. In this study, we report the effects of loss-of-function of these genes on development. We designed and identified single-guide RNAs targeted to generate fign, fignl1, and fignl2 mutants and then observed the overall morphological and behavioral changes. Our findings showed that while fign and fignl1 null mutants displayed no significant defects, fignl2 null zebrafish mutants displayed pericardial edema, reduced heart rate, and smaller eyes; fignl2 null mutants responded to the light-darkness shift with a lower swimming velocity. fignl2 mRNAs were identified in vascular endothelial cells by in situ hybridization and re-analysis of an online dataset of single-cell RNAseq results. Finally, we used morpholino oligonucleotides to confirm that fignl2 knockdown resulted in severe heart edema, which was caused by abnormal vascular branching. The zebrafish fignl2 morphants also showed longer axonal length and more branches of caudal primary neurons. Taken together, we summarize that Fignl2 functions on cellular branches in endothelial cells and neurons. This study reported for the first time that the microtubule-severing protein Fignl2 contributes to cell branching during development.