NMNAT2 is a key protein required for axon integrity whose rapid depletion following axon injury triggers Wallerian degeneration. The molecular mechanism controlling NMNAT2 turnover in neurons has not been fully understood. It has been believed that NMNAT2 degradation is accelerated in axons upon injury, and that NMNAT2 might have different regulatory mechanisms in neuronal soma and axons. 

In a study published online in Journal of Cell Biology, a research team led by Prof. FANG Yanshan from the Shanghai Institute of Organic Chemistry of the Chinese Academy of Sciences identified FBXO21 as the F-box protein and key factor mediating the ubiquitination and degradation of NMNAT2 in neurons, and found that the knockdown of Fbxo21 in mouse dorsal root ganglion neurons increases NMNAT2 levels and significantly delays Wallerian degeneration.

Researchers first demonstrated that the turnover rate of NMNAT2 protein in the soma and axons, as well as before and after injury, is comparable, suggesting a unified mechanism controlling NMNAT2 degradation. They then found that the rapid depletion of NMNAT2 in injured axons is mainly due to the lack of the supply of newly synthesized NMNAT2 protein from the soma. FBXO21 was found to regulate NMNAT2 stability and axon integrity in both soma and axon whether injured or not. 

Using denaturing immunoprecipitation and in vitro ubiquitination assays, researchers demonstrated that FBXO21 interacted with SKP1, CUL1 and RBX1 to form an SCF^FBXO21 E3 ligase complex that mediates NMNAT2 ubiquitination. They found that SCF^FBXO21 ubiquitinated NMNAT2 at K155 within the isoform-specific targeting and interaction domain (ISTID), and ubiquitination-deficient mutation K155R significantly prolonged the half-life of NMNAT2 and markedly enhanced its axonal protection. 

Interestingly, researchers found that swapping the ISTID of NMNAT1 or NMNAT3 with that of NMNAT2 resulted in rapid degradation of the originally very stable proteins, while introducing K155R mutation eliminated this effect. These results highlighted K155 ubiquitination within the ISTID as a key evolutionary determinant of the ultra-short half-life of NMNAT2, revealing the molecular and structural basis of the unique lability of NMNAT2.  

Finally, researchers generated Fbxo21 knockout mice, and revealed that NMNAT2 protein levels were specifically elevated in the nervous system. Using a mouse sciatic nerve injury model, they demonstrated that Fbxo21 knockout significantly delayed Wallerian degeneration in vivo.

The study reveals that FBXO21 plays a key role in regulating NMNAT2 stability, and demonstrates that the discovery of substrate-determining F-box protein FBXO21 for NMNAT2 is especially significant. It provides a potential target and intervention strategy for enhancing NMNAT2-dependent axon survival in neural injury and degenerative diseases.