Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP.

Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 µM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.

Inhibiting the SARM1-NAD+ axis reduces oxidative stress-induced damage to retinal and nerve cells.

Retinal neurodegenerative diseases are a category of refractory blinding eye conditions closely associated with oxidative stress induced by mitochondrial dysfunction in retinal cells. SARM1, a core driver molecule leading to axonal degeneration, possesses NAD+ enzyme (NADase) activity. However, the role of the SARM1-NAD+ axis in oxidative stress-induced retinal cell death remains unclear. Here, we employed the SARM1 NADase inhibitor DSRM-3716 and established a glucose oxidase (GOx)-induced oxidative stress cell model. We found that compared to the GOx group, the DSRM-3716 pre-treated group reduced the hydrolysis of NAD+, inhibited the elevation of oxidative stress markers induced by GOx, decreased mitochondrial dysfunction, lowered the phosphorylation level of JNK, and attenuated the occurrence of pyroptosis in retinal and nerve cells, thereby providing protection for neurite growth. Further utilization of the JNK activator Anisomycin activated JNK, revealed that the JNK/c-Jun pathway down-regulated NMNAT2 expression. Consequently, it reduced cellular NAD+ synthesis, exacerbated mitochondrial dysfunction and cell pyroptosis, and reversed the protective effect of DSRM-3716 on cells. In summary, the inhibition of SARM1 NADase activity substantially mitigates oxidative damage to retinal cells and mitochondrial damage. Additionally, JNK simultaneously serves as both an upstream and downstream regulator in the SARM1-NAD+ axis, regulating retinal cell pyroptosis and neurite injury. Thus, this study provides new insights into the pathological processes of retinal cell oxidative stress and identifies potential therapeutic targets for retinal neurodegenerative diseases.

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products.

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

Armcx1 Reduces Neurological Damage Via a Mitochondrial Transport Pathway Involving Miro1 After Traumatic Brain Injury.

Armcx1 is a member of the ARMadillo repeat-Containing protein on the X chromosome (ARMCX) family, which is recognized to have evolutionary conserved roles in regulating mitochondrial transport and dynamics. Previous research has shown that Armcx1 is expressed at higher levels in mice after axotomy and in adult retinal ganglion cells after crush injury, and this protein increases neuronal survival and axonal regeneration. However, its role in traumatic brain injury (TBI) is unclear. Therefore, the aim of this study was to assess the expression of Armcx1 after TBI and to explore possible related mechanisms by which Armcx1 is involved in TBI. We used C57BL/6 male mice to model TBI and evaluated the role of Armcx1 in TBI by transfecting mice with Armcx1 small interfering RNA (siRNA) to inhibit Armcx1 expression 24 h before TBI modeling. Western blotting, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Nissl staining, transmission electron microscopy, adenosine triphosphate (ATP) level measurement, neuronal apoptosis analysis, neurological function scoring and the Morris water maze were performed. The results demonstrated that Armcx1 protein expression was elevated after TBI and that the Armcx1 protein was localized in neurons and astroglial cells in cortical tissue surrounding the injury site. In addition, inhibition of Armcx1 expression further led to impaired mitochondrial transport, abnormal morphology, reduced ATP levels, aggravation of neuronal apoptosis and neurological dysfunction, and decrease Miro1 expression. In conclusion, our findings indicate that Armcx1 may exert neuroprotective effects by ameliorating neurological injury after TBI through a mitochondrial transport pathway involving Miro1.

Adaptation of a Commercial NAD+ Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1.

Here, we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3-acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base-exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base-exchange activity.

Loss of Sarm1 reduces retinal ganglion cell loss in chronic glaucoma.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

SARM1 is responsible for calpain-dependent dendrite degeneration in mouse hippocampal neurons.

Sterile alpha and toll/interleukin receptor motif-containing 1 (SARM1) is a critical regulator of axon degeneration that acts through hydrolysis of NAD+ following injury. Recent work has defined the mechanisms underlying SARM1's catalytic activity and advanced our understanding of SARM1 function in axons, yet the role of SARM1 signaling in other compartments of neurons is still not well understood. Here, we show in cultured hippocampal neurons that endogenous SARM1 is present in axons, dendrites, and cell bodies and that direct activation of SARM1 by the neurotoxin Vacor causes not just axon degeneration, but degeneration of all neuronal compartments. In contrast to the axon degeneration pathway defined in dorsal root ganglia, SARM1-dependent hippocampal axon degeneration in vitro is not sensitive to inhibition of calpain proteases. Dendrite degeneration downstream of SARM1 in hippocampal neurons is dependent on calpain 2, a calpain protease isotype enriched in dendrites in this cell type. In summary, these data indicate SARM1 plays a critical role in neurodegeneration outside of axons and elucidates divergent pathways leading to degeneration in hippocampal axons and dendrites.

Impacts of H2O2, SARM1 inhibition, and high NAm concentrations on Huntington's disease laser-induced degeneration.

Axonal degeneration is a key component of neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease, and amyotrophic lateral sclerosis. Nicotinamide, an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. However, studies on nicotinamide (NAm) supplementation in humans indicate that NAm has no protective effect. Sterile alpha and toll/interleukin receptor motif-containing protein 1 (SARM1) regulates several cell responses to axonal damage and has been implicated in promoting neuronal degeneration. SARM1 inhibition seems to result in protection from neuronal degeneration while hydrogen peroxide has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type and HD dorsal root ganglion cells treated with NAm, hydrogen peroxide (H2O2), and SARM1 inhibitor DSRM-3716 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured.

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II). RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination. CONCLUSIONS: Mutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

Deregulated mitochondrial quality control, the heel of Achilles in elucidating the role of autophagy in SARM1-mediated axon degeneration.

Autophagic dysfunction in neurodegenerative diseases is being extensively studied, yet the exact mechanism of macroautophagy/autophagy in axon degeneration is still elusive. A recent study by Kim et al. links autophagic stress to the sterile α and toll/interleukin 1 receptor motif containing protein 1 (SARM1)-dependent core axonal degeneration program, providing a new insight into the role of autophagy in axon degeneration. In the classical Wallerian axon degeneration model of axotomy, disruption of axonal transport destroys the coordinated activity of pro-survival and pro-degenerative factors in the axoplasm and activates the NADase activity of SARM1, thus triggering the axonal self-destruction program. However, the mechanism for SARM1 activation in the chronic neurodegenerative disorders is more complex. Mitochondrial defects and oxidative stress contribute to the activation of SARM1, while mitophagy can inhibit mitochondrial dysfunction and promote the clearance of SARM1 on mitochondria, thus protecting against neuronal degeneration. Therefore, in-depth elucidation of the underlying mechanisms of mitophagy during axonal degeneration can help develop promising strategies for the prevention and treatment of various neurodegenerative disorders.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy.

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport.

BACKGROUND: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. METHODS: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. RESULTS: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. CONCLUSION: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Targeting SARM1 improves autophagic stress-induced axonal neuropathy.

ABBREVIATIONS: AAV: adeno-associated virus; ATF3: activating transcription factor 3; ATG7: autophagy related 7; AVIL: advillin; cADPR: cyclic ADP ribose; CALC: calcitonin/calcitonin-related polypeptide; CMT: Charcot-Marie-Tooth disease; cKO: conditional knockout; DEG: differentially expressed gene; DRG: dorsal root ganglion; FE-SEM: field emission scanning electron microscopy; IF: immunofluorescence; NCV: nerve conduction velocity; PVALB: parvalbumin; RAG: regeneration-associated gene; ROS: reactive oxygen species; SARM1: sterile alpha and HEAT/Armadillo motif containing 1; SYN1: synapsin I.

Loss of SARM1 ameliorates secondary thalamic neurodegeneration after cerebral infarction.

Ischemic stroke causes secondary neurodegeneration in the thalamus ipsilateral to the infarction site and impedes neurological recovery. Axonal degeneration of thalamocortical fibers and autophagy overactivation are involved in thalamic neurodegeneration after ischemic stroke. However, the molecular mechanisms underlying thalamic neurodegeneration remain unclear. Sterile /Armadillo/Toll-Interleukin receptor homology domain protein (SARM1) can induce Wallerian degeneration. Herein, we aimed to investigate the role of SARM1 in thalamic neurodegeneration and autophagy activation after photothrombotic infarction. Neurological deficits measured using modified neurological severity scores and adhesive-removal test were ameliorated in Sarm1-/- mice after photothrombotic infarction. Compared with wild-type mice, Sarm1-/- mice exhibited unaltered infarct volume; however, there were markedly reduced neuronal death and gliosis in the ipsilateral thalamus. In parallel, autophagy activation was attenuated in the thalamus of Sarm1-/- mice after cerebral infarction. Thalamic Sarm1 re-expression in Sarm1-/- mice increased thalamic neurodegeneration and promoted autophagy activation. Auotophagic inhibitor 3-methyladenine partially alleviated thalamic damage induced by SARM1. Moreover, autophagic initiation through rapamycin treatment aggravated post-stroke neuronal death and gliosis in Sarm1-/- mice. Taken together, SARM1 contributes to secondary thalamic neurodegeneration after cerebral infarction, at least partly through autophagy inhibition. SARM1 deficiency is a potential therapeutic strategy for secondary thalamic neurodegeneration and functional deficits after stroke.

SARM1 Promotes Neurodegeneration and Memory Impairment in Mouse Models of Alzheimer's Disease.

Neuroinflammation plays a crucial role in the pathogenesis and progression of Alzheimer's disease (AD). The Sterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) has been shown to promote axonal degeneration and is involved in neuroinflammation. However, the role of SARM1 in AD remains unclear. In this study, we found that SARM1 was reduced in hippocampal neurons of AD model mice. Interestingly, conditional knockout (CKO) of SARM1 in the central nervous system (CNS, SARM1Nestin-CKO mice) delayed the cognitive decline in APP/PS1 AD model mice. Furthermore, SARM1 deletion reduced the Aß deposition and inflammatory infiltration in the hippocampus and inhibited neurodegeneration in APP/PS1 AD model mice. Further investigation into the underlying mechanisms revealed that the signaling of tumor necrosis factor-α (TNF-α) was downregulated in the hippocampus tissues of APP/PS1;SARM1Nestin-CKO mice, thereby alleviating the cognitive decline, Aß deposition and inflammatory infiltration. These findings identify unrecognized functions of SARM1 in promoting AD and reveal the SARM1-TNF-α pathway in AD model mice.

Unexpected dynamics in femtomolar complexes of binding proteins with peptides.

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration.

Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson's disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration.

A phase transition reduces the threshold for nicotinamide mononucleotide-based activation of SARM1, an NAD(P) hydrolase, to physiologically relevant levels.

Axonal degeneration is a hallmark feature of neurodegenerative diseases. Activation of the NAD(P)ase sterile alpha and toll-interleukin receptor motif containing protein 1 (SARM1) is critical for this process. In resting neurons, SARM1 activity is inhibited, but upon damage, SARM1 is activated and catalyzes one of three NAD(P)+ dependent reactions: (1) NAD(P)+ hydrolysis to form ADP-ribose (ADPR[P]) and nicotinamide; (2) the formation of cyclic-ADPR (cADPR[P]); or (3) a base exchange reaction with nicotinic acid (NA) and NADP+ to form NA adenine dinucleotide phosphate. Production of these metabolites triggers axonal death. Two activation mechanisms have been proposed: (1) an increase in the nicotinamide mononucleotide (NMN) concentration, which leads to the allosteric activation of SARM1, and (2) a phase transition, which stabilizes the active conformation of the enzyme. However, neither of these mechanisms have been shown to occur at the same time. Using in vitro assay systems, we show that the liquid-to-solid phase transition lowers the NMN concentration required to activate the catalytic activity of SARM1 by up to 140-fold. These results unify the proposed activation mechanisms and show for the first time that a phase transition reduces the threshold for NMN-based SARM1 activation to physiologically relevant levels. These results further our understanding of SARM1 activation and will be important for the future development of therapeutics targeting SARM1.