SUMMARY Axonal degeneration is an early and prominent feature of many neurological disorders. SARM1 is the central executioner of the axonal degeneration pathway that culminates in depletion of axonal NAD+; yet the identity of the underlying NAD+ depleting enzyme(s) is unknown. Here, in a series of experiments using purified proteins from mammalian cells, bacteria, and a cell-free protein translation system, we show that the SARM1-TIR domain itself has intrinsic NADase activity – cleaving NAD+ into ADP Ribose (ADPR), cyclic ADPR, and Nicotinamide, with Nicotinamide serving as a feedback inhibitor of the enzyme. Using traumatic and vincristine-induced injury models in neurons, we demonstrate that the NADase activity of full-length SARM1 is required in axons to promote axonal NAD+ depletion and axonal degeneration after injury. Hence, the SARM1 enzyme represents a novel therapeutic target for axonopathies. Moreover, the widely utilized TIR-domain is a protein motif that can possess enzymatic activity.

The Toll/interleukin-1 receptor (TIR) domain is the signature signaling domain of Toll-like receptors (TLRs) and their adaptors, serving as a scaffold for the assembly of protein complexes for innate immune signaling [1, 2]. TIR domain proteins are also expressed in plants, where they mediate disease resistance [3, 4], and in bacteria, where they have been associated with virulence [5-9]. In pursuing our work on axon degeneration [10], we made the surprising discovery that the TIR domain of SARM1 (sterile alpha and TIR motif containing 1), a TLR adaptor protein, has enzymatic activity [11]. Upon axon injury, the SARM1 TIR domain cleaves nicotinamide adenine dinucleotide (NAD), destroying this essential metabolic co-factor to trigger axon destruction [11, 12]. Whereas current studies of TIR domains focus on their scaffolding function, our findings with SARM1 inspired us to ask whether this enzymatic activity is the primordial function of the TIR domain. Here we show that ancestral prokaryotic TIR domains constitute a new family of NADase enzymes. Using purified proteins from a cell-free translation system, we find that TIR domain proteins from both bacteria and archaea cleave NAD into nicotinamide and ADP-ribose (ADPR), with catalytic cleavage executed by a conserved glutamic acid. A subset of bacterial and archaeal TIR domains generates a non-canonical variant cyclic ADPR (cADPR) molecule, and the full-length TIR domain protein from pathogenic Staphylococcus aureus induces NAD loss in mammalian cells. These findings suggest that the primordial function of the TIR domain is the enzymatic cleavage of NAD and establish TIR domain proteins as a new class of metabolic regulatory enzymes.

Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration.DOI: http://dx.doi.org/10.7554/eLife.19749.001

Highlights d Inhibitors of SARM1 NADase recapitulate SARM1 À/À phenotype and protect injured axons d Transient mitochondrial injury generates an intermediate state of axonal damage d Axons entering the intermediate state of damage are fated to subsequent degeneration d SARM1 inhibitors allow recovery of axons already in the intermediate injury state

The sensitivity of an organism and its cells to hypoxic injury varies widely; yet, few genetic determinants of metazoan hypoxic sensitivity have been identified. We report here the isolation of a profoundly hypoxia resistant mutant and its identification as a reduction-of-function allele of rrt-1, which encodes an arginyl-tRNA synthetase. rrt-1 knockdown before or after the hypoxic injury rescues animals from death. RNAi knockdown of most other aminoacyl-tRNA synthetases also confers hypoxia resistance, the level of which inversely correlates with translation rate. rrt-1(RNAi) blocked hypoxic induction of the unfolded protein response and tunicamycin toxicity. Disruption of the unfolded protein response partially suppressed the hypoxia resistance of rrt-1(lf). The data support a model where translational suppression induces hypoxia resistance, in part by reducing unfolded protein toxicity.

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus–mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for 10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss.

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Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.