Beyond NAD Depletion: SARM1-Induced ATP Collapse Involves Direct ATP Degradation and Mitochondrial Dysfunction and Is Pharmacologically Reversible

Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is an inducible NAD-consuming enzyme and execution factor in axon degeneration. Rapid ATP collapse after SARM1 activation, however, is not fully explained by NAD depletion alone. We used SARM1-overexpressing HEK293 cells and the cell-permeant activator CZ-48 to examine SARM1-induced non-apoptotic cell death, termed sarmoptosis. CZ-48 induced cell death that was suppressed by HSP90/70-annotated ATP-competitive compounds, especially geldanamycin and VER-155008 (VER), without reducing SARM1 abundance. VER preserved NAD and ATP during SARM1 activation but failed to rescue FK866-mediated NAD starvation, thereby distinguishing CZ-48/SARM1-driven cytotoxicity from generic NAD depletion. In cell-free assays, purified SARM1 reduced ATP levels; this effect was enhanced by SARM1’s activator NMN and attenuated by its pharmacological inhibitors, although the in vitro activity was modest and the reaction products remain to be identified. ATPase-related perturbations, including thapsigargin and bafilomycin A1, also protected cells from CZ-48-induced death, further supporting a central role for ATP collapse in sarmoptosis. iTRAQ proteomics, MitoSOX Red staining, and DiOC6(3) staining revealed that CZ-48 treatment was associated with mitochondrial and metabolic remodeling, mitochondrial ROS accumulation, and mitochondrial depolarization, all of which were mitigated by VER. Collectively, these findings support a convergent ATP-collapse model in which SARM1 activation promotes NAD depletion, directly consumes ATP, and is associated with mitochondrial dysfunction that may amplify ATP-production failure.