SARM1 activation triggers axon degeneration locally via NAD⁺ destruction

Abstract

Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif-containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD(+)) after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD(+), whereas SARM1-induced axon destruction could be counteracted by increased NAD(+) synthesis. SARM1-induced depletion of NAD(+) may explain the potent axon protection in Wallerian degeneration slow (Wld(s)) mutant mice.

Fig. 1

SARM1 functions following axon injury to promote destruction. A) Schematic showing how expression of SARM^ps-Frb-Ntev with Fkbp-Ctev allows rapamycin-induced complementation of split TEV and concomitant SARM^ps cleavage. B) Gel electrophoresis with anti-GFP immunoblot showing SARM^ps cleavage in DRG neurons induced by 100 nM rapamycin (rapa); FL=full length SARM^ps-Frb-Ntev-Cerulean; clv = cleaved form. α-Tubulin (αTub) was a loading control. C) Diagram of in vitro injury model: isolated DRG neurons were severed and axon degeneration was quantified after 24 hours from axon images. D) Requirement for SARM1 activity after axotomy to induce axon degeneration.. Axon degeneration is reported as the degeneration index (DI), a morphometric ratio of fragmented axon area to total axon area (13). Sarm1−/− DRG neurons treated with expression lentiviruses (control, SARMps-FrbNtev, and Fkbp-Ctev) were severed and treated with 100 nM rapamycin at various times (pre = 12 hours pre-injury). E) Micrographs show representative α-Tubulin stained axons corresponding to select treatment groups in (D). Scale bar = 50 micrometers. Error bars = SEM; * p < 0.01; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test.

Fig. 2

Axon degeneration and neuronal death induced by sTIR dimerization. A) Micrograph showing motor nerves of third instar Drosophila larvae. M12-Gal4 drives expression from mCD8-GFP (green) alone or with either UAS-SAM-TIR or UAS-SAM-TIR^mut in single motor axons in each nerve (red=HRP). UAS-SAM-TIR expression caused axon loss in 49/49 nerves as shown; whereas SAM-TIR with a disruptive TIR mutation led to degeneration in 0/70 nerves (χ2=119; p<0.001); scale bar=10 micrometers. B) Schematic showing sTIR dimerization by rapamycin or AP20187. C) Effect of sTIR, dimerized sTIR, and rapamycin on axon degeneration. α-Tubulin stained axons correspond to bars b and d. D) Effect of sTIR dimerization on neuronal viability quantified by ethidium homodimer exclusion after 24 hours. E) Effects of dimerization of sTIR or TIR domains of MYD88 or TLR4 on axon degeneration. F) Effects of sTIR dimerization on degeneration of Sarm1−/− axons physically disconnected from cell bodies. G) Left: diagram of axons growing through a diffusion barrier into an isolated fluid compartment. Right: micrographs of isolated distal axon segments after application of AP20187 globally or selectively to distal axons. Scale bar = 50 micrometers. Error bars = SEM; * p < 0.01; one-way ANOVA with Tukey’s post-hoc test.

Fig. 3

Loss of NAD+ underlies SARM1-induced destruction. A) Diagram of NAD+ synthesis and inhibition by FK866; Nam=nicotinamide, NR = nicotinamide riboside, Nrk=nicotinamide riboside kinase, NMN=nicotinamide mononucleotide. B) Axonal NAD+ concentration in cultured wild-type and Sarm1−/− DRG neurons following axotomy; normalized to wild-type control. C) NAD+ concentration in distal sciatic nerve segments from wild-type or Sarm1−/− animals following transection; wild-type n=5; Sarm1−/− n=9. D) Neuronal NAD+ and ATP concentrations following sTIR dimerization by AP20187; comparisons made to zero minutes control. E–F) Axon degeneration (E) and neuronal cell death (F) induced by sTIR homodimerization (AP20187) and inhibition by NAD+ synthetic enzymes with or without the Nampt inhibitor FK866 (10 nM); measured 24 hours after sTIR dimerization and FK866 application. G–H) Effect of NR (1 mM) on axon degeneration (G) and neuronal cell death (H) induced by sTIR homodimerization (AP20187) for 24 hours with or without NR. I) Micrographs showing sTIR-induced motor axon fragmentation in third instar Drosophila larvae blocked by cytosolic Nmnat1 (cytNmnat1) expression. M12-Gal4 drives expression from UAS-mCD8-GFP (green) and UAS-Fk^F36VTIR with or without UAS-cytNmnat1 in single motor axons in each nerve (red=HRP). Degeneration score = 76+/−4% (control) vs 11+/−2% (cytNmnat1); p<0.001 (t-test); scale bar=20 micrometers. Error bars = SEM; * p < 0.01; one-way ANOVA with Tukey’s post-hoc test.

Fig. 4

Effects of NAD+ breakdown on axon degeneration. A) Diagram of NAD+ manipulation using Tnkp dimerization. NAD+ loss induced by FkbpF36V-Tnkp dimerization is blocked by Tankyrase inhibitor XAV939 or NR and is exacerbated by FK866. B) Axon degeneration in response to NAD+ depletion by dimerized Tnkp and FK866 after 24 hours (bar d) and inhibition by Tankyrase inhibitor XAV939 (100 nM; bar e). Representative α-Tubulin-stained axons corresponding to bars b and d are shown; scale bar = 50 micrometers. C) Effect of NAD+ depletion by dimerized Tnkp + FK866 on axon degeneration in Sarm1−/− uninjured axons (bar b) or isolated (cut) Sarm1−/− axons (bar d). D) Effect of sTIR dimerization on endogenous (dotted lines) and exogenously introduced (solid lines) NAD+ or NaAD (control) in HTir cells. NaAD is undetectable in non-electroporated cells. E) Conversion of ¹⁴C-NAD+ in HTir cells to Nam 15 minutes after SARM1 TIR dimerization. NAD+ and Nam from cell extracts and extracellular media were resolved by thin layer chromatography. F) Top: effect of the PARP inhibitor olaparib (100 nM) on NAD+ loss induced by 1 mM H₂O₂ (10 min) or sTIR dimerization (10 min). Bottom: PAR formation after H₂O₂ treatment or sTIR dimerization in HTir cells expressing PARG shRNA and inhibition by olaparib. Error bars = SEM; * p < 0.01; one way ANOVA with Tukey’s post-hoc test.