Combined pharmacological targeting of NAD⁺ metabolism preserves central and peripheral axons in an ex vivo model of Wallerian degeneration

Wallerian degeneration (WD) is a conserved axonal self-destruction program implicated in a spectrum of neurological conditions, including peripheral neuropathies, traumatic brain injury, and possibly neurodegenerative diseases. Wallerian degeneration is triggered by depletion of the NAD⁺-synthesizing enzyme NMNAT2, subsequent NMN accumulation, and activation of the NAD⁺-hydrolyzing SARM1, ultimately driving axonal fragmentation. While pharmacological inhibition of SARM1 is an emerging therapeutic strategy, current challenges due to paradoxical toxicity with orthosteric base-exchange SARM1 inhibitors highlight the need for alternative approaches. We have shown before that targeted modulation of NAD⁺ metabolism centered on NAMPT inhibition (FK866) and nicotinic acid riboside (NaR) supplementation, and enhanced by DLK-JNK inhibition (GNE-3511) can prevent or suppress SARM1 activation and robustly protect axotomized axons in vitro. In this paper, we investigated the efficacy of combined treatments that modulate SARM1 triggers and block DLK-JNK WD in an explant model of WD using sciatic and optic nerve segments. Our findings show that our combination treatment robustly preserved the integrity of axons in the nerve up to 7 days in culture, reduced ongoing degeneration of axoskeleton, and suppressed SARM1 NADase and DLK-JNK activation by metabolomic and immunoblot analyses. Effects were similar to those of the orthosteric inhibitor NB-3 and perhaps better for stabilizing neurofilaments. Delayed initiation of the combination regimen but not NB-3 were robustly protective, indicating the presence of a therapeutic window of at least 24 h. Our results demonstrate that targeted modulation of NAD⁺ metabolic pathways and inhibition of DLK-JNK signaling confers significant axon protection, further supporting the role of these approaches as viable therapeutic alternatives to direct orthosteric SARM1 inhibitors.