Abstract
Axon regeneration is abortive in the central nervous system following injury. Orchestrating microtubule dynamics has emerged as a promising approach to improve axonal regeneration. The microtubule severing enzyme spastin is essential for axonal development and regeneration through remodeling of microtubule arrangement. To date, however, little is known regarding the mechanisms underlying spastin action in neural regeneration after spinal cord injury. Here, we use glutathione transferase pulldown and immunoprecipitation assays to demonstrate that 14-3-3 interacts with spastin, both in vivo and in vitro, via spastin Ser233 phosphorylation. Moreover, we show that 14-3-3 protects spastin from degradation by inhibiting the ubiquitination pathway and upregulates the spastin-dependent severing ability. Furthermore, the 14-3-3 agonist Fusicoccin (FC-A) promotes neurite outgrowth and regeneration in vitro which needs spastin activation. Western blot and immunofluorescence results revealed that 14-3-3 protein is upregulated in the neuronal compartment after spinal cord injury in vivo. In addition, administration of FC-A not only promotes locomotor recovery, but also nerve regeneration following spinal cord injury in both contusion and lateral hemisection models; however, application of spastin inhibitor spastazoline successfully reverses these phenomena. Taken together, these results indicate that 14-3-3 is a molecular switch that regulates spastin protein levels, and the small molecule 14-3-3 agonist FC-A effectively mediates the recovery of spinal cord injury in mice which requires spastin participation.
The formation of the 14-3-3 and spastin protein complex requires phosphorylation of spastin at the S233 site.
14-3-3 is involved in the phosphorylation-ubiquitination crosstalk of spastin, thus impacting the protein stability.
14-3-3 agonists Fusicoccin-A can facilitate the repair of spinal cord injury in mice.
Activation of spastin function is a prerequisite for nerve regeneration and recovery of spinal cord injury in mice.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In light of the feedback from the editors and reviewers, we recognize that certain aspects of the title and key conclusions require further refinement. We have shown that 14-3-3, through its interaction with phosphorylated spastin, inhibits the degradation of spastin. Also, we have demonstrated that 14-3-3 can enhance spastin's microtubule-severing ability in cell lines. Furthermore, our work has illustrated the significant roles of 14-3-3 and spastin in the repair process of spinal cord injury. However, there is currently insufficient direct evidence to confirm the cooperation between 14-3-3 and spastin during axon regeneration and the recovery of spinal cord injury. Moreover, we have not provided conclusive evidence of their simultaneous action in injured axons, mediating changes in microtubule dynamics. Consequently, we have re-evaluated the manuscript's title and primary conclusions, and have made relevant modifications. For more detailed information, please refer to the reviewer's comments.
Abbreviations
- 5-HT
- 5-hydroxytryptamine
- Cdt2
- cell division cycle protein
- CNS
- central neuronal system
- DPI
- days post injury
- ECL
- enhanced chemiluminescence
- FOXO
- forkhead box transcription factors
- GFAP
- glial fibrillary acidic protein
- GFP
- green fluorescent protein
- HIPK2
- homeodomain interacting protein kinase 2
- HSP
- hereditary spastic paraplegia
- IP
- Immunoprecipitation
- MBP
- Myelin basic protein
- MS
- mass spectrometry
- MT
- microtubule
- ROS
- reactive oxygen species
