Abstract Text: Wallerian degeneration (WD) is a conserved mechanism of axonal self-destruction implicated in several neurological diseases from peripheral neuropathies to glaucoma, traumatic brain injury, and neurodegenerative diseases of the brain. Research on the regulation and execution of WD has pointed to the central role of NAD+ metabolism including the degradation of the NAD+ synthesizing enzyme NMNAT2, the buildup of its deleterious substrate NMN, and the activation of the NAD+ hydrolase SARM1; the latter is both necessary and sufficient for axonal degeneration. The contribution of WD in neurological diseases is currently attracting a lot of attention based on the notion that partially injured axons can exist in a potentially viable state for different lengths of time before their eventual submission to WD. Inhibition of WD may thus prevent degeneration and offer great therapeutic promise. Inspired by such a therapeutic promise, here we explored convenient and safe pharmacological strategies that modulate NMN and NAD+ metabolism as a means to inhibit WD in axotomy models in vitro. These include the inhibition of the NMN-synthesizing enzyme NAMPT, activation of the nicotinic acid riboside (NaR) salvage pathway, and inhibition of the NMNAT2-degrading stress MAPK (DLK) pathway. Results show that NAMPT and DLK inhibition cause a significant but time-dependent suppression of WD. NAMPT inhibition is sufficient to reduce SARM1 activity, protect axonal NAD+ levels and delay WD in a time-dependent manner. Supplementation with NaR leads to further reduction of SARM1 activity even with delayed treatment and prevents of axon fragmentation up to 4 days in vitro. Additional DLK inhibition further augments NAD+ metabolism and extends the protection of axon integrity to 6 days. The previous pharmacological treatments are also protective in WD models using human stem cell-derived neurons in vitro and in mice in vivo. Metabolic analyses in axons reveal that the axonal NAD+/NMN ratio is highly predictive of cADPR levels, consistent with previous cell-free evidence on the allosteric regulation of SARM1. Our findings establish the effect of translationally relevant small molecules in preventing the WD of injured axons via complex modulation of NAD+ metabolism and inhibition of SARM1.
