1. Evaluation Summary:

    This paper explores the mechanism of vacor toxicity in neurons. The authors provide exciting and definitive data that vacor drives neurodegeneration by direct binding and activation of SARM1, a potent regulator of axon death. The work elucidates the vacor mechanism of action, provides strong in vitro and in vivo data that toxicity is entirely dependent on SARM1, and will advance the field in terms of how we understand vacor-induced toxicity, and provides a new model for testing anti-SARM1 therapeutics.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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  2. Public Review (Reviewer #1):

    Sarm is an important neurodegenerative protein. A series of papers over the past year described how Sarm is regulated, identifying an allosteric pocket that binds at least three NAD-related metabolites in order to inhibit or activate Sarm. The current study identifies and characterizes a fourth metabolite that binds in the allosteric pocket. Importantly, this is not a natural NAD-related metabolite, but instead is a metabolite of the neurotoxic pesticide vacor. Hence, this study shows that exogenous toxins can function by direct activation of Sarm. The study convincingly demonstrates that vacor toxicity is Sarm-dependent, identifies the relevant metabolite as vacor mononucleotide (VMN), shows that VMN directly binds and activates Sarm, and solves the structure of Sarm bound to VMN and compares this structure to the binding of the natural activator nicotinamide mononucleotide (NMN). The major strengths of this study are the high quality of the data and the demonstration that an exogenous toxin can activate Sarm. Surprisingly, the manuscript also shows that VMN can inhibit Sarm. The manuscript could be improved by working out how VMN can inhibit as well as activate Sarm.

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  3. Reviewer #2 (Public Review):

    This study provides definitive and convincing data that vacor, a neurotoxin, drives neurodegeneration by direct binding and activation of SARM1. The authors show that vacor is metabolized into VMN, which binds the ARM domain of SARM1 with high affinity, and drives activation of the SARM1 NAD hydrolase function to drive neurodegeneration. The study is rigorous, and uses a combination of biochemistry and structural biology with the ARM domain to make a convincing case that even in vivo, this activaiton is direct. The complete suppression of vacor-induced toxicity in DRGs and retina argues further that SARM1 is critical for neurodegeneration in these tissues and SARM1 blockade should be beneficial for patients. Finally, the model they have developed in the visual system will provide a nice assay for in vivo testing of small molecules for inhibition of SARM1.

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  4. Reviewer #3 (Public Review):

    In this manuscript, Loreto and colleagues show that vacor, a now banned pesticide, induces neurotoxicity via a Sarm1-dependent mechanism. Exposure to vacor induces both axon and soma degeneration that is blocked in Sarm1-deficient neurons. This is demonstrated in vitro in DRG and SCG neurons and in vivo in the eye with intravitreal injection of vacor. Importantly, the authors examine multiple potential mechanisms by which vacor may activate Sarm1 and elegantly demonstrate that the vacor metabolite VMN can directly bind to and activate Sarm1. The binding of VMN to Sarm1 is then examined at high resolution with crystallography studies with follow up mutational studies that confirm this model of interaction.

    Overall, the experiments conducted are thorough and examined with dose and time dependent studies in primary neurons. The in vivo studies with functional readouts are a very good complement to the in vitro experiments. The mechanisms experiments are also well designed and use complementary approaches to demonstrate that the vacor metabolite NMN is the direct activator of Sarm1. Lastly, the crystallography results provide a detailed perspective of the mechanism of binding of NMN to the ARM domain of Sarm1.

    These results have significant implications. First, they provide a mechanistic insight into the neurotoxicity of vacor and illustrate the critical function on Sarm1 in this context. Second, they identify vacor and NMN as reagents that are potent activators of Sarm1, a finding that other researchers will likely utilize for their studies on Sarm1. Third, the structure studies identify key residues that are involved in Sarm1 activation. This information will be very useful for the screening and identification of Sarm1 inhibitors. Sarm1 inhibition is being actively pursued by pharmaceutical companies as a therapeutic strategy for maintaining neuronal integrity in the context of neuronal injury or disease.

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