It has been suggested that axonal protective function of WldS is mediated by its effect on bioenergetics . Similarly, we found that WldS MEFs have increased ATP content at basal level compared with wild-type MEFs, and WldS significantly delays the decrease of ATP induced by paraquat. These results implicate that WldS might also exert cellular protective function through its bioenergetic role. Furthermore, we found that NAD synthesis activity of WldS is essential for its protective function against cytotoxicity induced by paraquat. Similarly, the activity of WldS to synthesize NAD has been reported to be responsible for its axon sparing ability . It has also been reported that WldS exerts its axon-protective function by compensating for the fast proteasome-dependent degradation of Nmnat2, an important enzyme in NAD synthesis . In this study, we found that the proteasome inhibitor MG- 132 had no effect on paraquat-induced cytotoxicity and WldS- mediated protection in MEFs , suggesting that proteasome-dependent degradation of Nmnat2 is not involved in WldS-mediated protection against paraquat. Previous studies have shown that NAD levels are not upregulated in the brain of WldS mice or axons of dorsal root ganglia overexpressing WldS, but WldS delays the decline of NAD levels in degenerating axons . Consistently, we found that WldS didn��t upregulate the NAD levels in MEFs, but significantly attenuated the decline of NAD levels induced by paraquat. NMN can be directly converted to NAD by WldS or Nmnats . We found, treatment with NAD or NMN also protected cells against cytotoxicity and decline of NAD levels induced by INCB28060 supplier paraquat . These findings suggest the protective role of intracellular NAD against paraquat. Consistently, upregulation or replenishment of intracellular NAD has been reported to promote cell survival under various conditions . Further studies focused on NAD synthesis pathway will provide more information about how NAD level is regulated to maintain cell survival. NAD is an essential substrate for various enzymes such as SIRT1 to exert their functions . The activity of NADdependent deacetylase SIRT1 has been shown to be regulated by NAD ABT-199 customer reviews biosynthesis via Nampt in various biological processes . In addition, NAD depletion induced by PARP activation reduced SIRT1 deacetylase activity, contributing to myocyte cell death during heart failure .