SARM1 is the central executioner of pathological axon degeneration, promoting axonal demise in response to axotomy, traumatic brain injury, and neurotoxic chemotherapeutics that induce peripheral neuropathy. SARM1 is an injury-activated NAD+ cleavage enzyme, and this NADase activity is required for the pro-degenerative function of SARM1. At present, SARM1 function is assayed by either analysis of axonal loss, which is far downstream of SARM1 enzymatic activity, or via NAD+ levels, which are regulated by many competing pathways. Here we explored the utility of measuring cADPR, a product of SARM1-dependent cleavage of NAD+, as an in cell and in vivo biomarker of SARM1 enzymatic activity. We find that SARM1 is a major producer of cADPR in cultured dorsal root ganglion (DRG) neurons, sciatic nerve, and brain, demonstrating that SARM1 has basal activity in the absence of injury. Following injury, there is a dramatic SARM1-dependent increase in the levels of axonal cADPR that precedes morphological axon degeneration. In vivo, there is also a rapid and large injury-stimulated increase in cADPR in sciatic and optic nerves. The increase in cADPR after injury is proportional to SARM1 gene dosage, suggesting that SARM1 activity is the prime regulator of cADPR levels. The role of cADPR as an important calcium mobilizing agent prompted exploration of its functional contribution to axon degeneration. We used multiple bacterial and mammalian engineered enzymes to manipulate cADPR levels in neurons but found no changes in the time course of axonal degeneration, suggesting that cADPR is unlikely to be an important contributor to the degenerative mechanism. Using cADPR as a SARM1 biomarker, we find that SARM1 can be partially activated by a diverse array of mitochondrial toxins administered at doses that do not induce axon degeneration. Hence, the subcritical activation of SARM1 induced by mitochondrial dysfunction may contribute to the axonal vulnerability common to many neurodegenerative diseases. Finally, we assay levels of both nerve cADPR and plasma neurofilament light chain (NfL) following nerve injury in vivo, and demonstrate that both biomarkers are excellent readouts of SARM1 activity, with cADPR reporting the early molecular changes in the nerve and NfL reporting subsequent axonal breakdown. The identification and characterization of cADPR as a SARM1 biomarker will help identify neurodegenerative diseases in which SARM1 contributes to axonal loss and expedite target validation studies of SARM1-directed therapeutics.

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

Peripheral axons can re-extend robustly after nerve injury. Soon after a nerve crush regenerating axons grow through the nerve segment distal to the lesion in close proximity to distal axons that are still morphologically and molecularly preserved. Hence, following the progress of regenerating axons necessitates markers that can distinguish between regenerating and degenerating axons. Here, we show that axonal levels of superior cervical ganglion 10 (SCG10) are dynamically regulated after axonal injury and provide an efficient method to label regenerating axons. In contrast to the rapid loss of SCG10 in distal axons (Shin et al., 2012b), we report that SCG10 accumulates in the proximal axons within an hour after injury, leading to a rapid identification of the lesion site. The increase in SCG10 levels is maintained during axon regeneration after nerve crush or nerve repair and allows for more selective labeling of regenerating axons than the commonly used markers growth-associated protein 43 (GAP43) and YFP. SCG10 is preferentially expressed in regenerating sensory axons rather than motor axons in the sciatic nerve. In a mouse model of slow Wallerian degeneration, SCG10 labeling remains selective for regenerating axons and allows for a quantitative analysis of delayed regeneration in this mutant. Taken together, these data demonstrate the utility of SCG10 as an efficient and selective marker of sensory axon regeneration.

Axonal degeneration contributes to the pathogenesis of many neurodegenerative disorders, motivating efforts to dissect the mechanism of pathological axon loss in order to develop therapies for axonal preservation. SARM1 is a particularly attractive therapeutic target, as it is an inducible NAD+ cleaving enzyme that is required for axon loss in multiple mouse models of traumatic and degenerative neurological disease. However, it is essential to establish whether SARM1 triggers axon degeneration in human neurons before proceeding with the development of SARM1-directed therapeutics. Here we combine genome engineering with the production of human stem cell-derived neurons to test the role of human SARM1 in traumatic and neurotoxic axon degeneration. We have generated two independent SARM1 knockout human iPSC lines that do not express SARM1 protein upon differentiation into neurons. We have developed a modified sensory neuron differentiation protocol that generates human sensory neurons with high yield and purity. We find that SARM1 is required for axon degeneration in response to both physical trauma and in a cellular model of chemotherapy-induced peripheral neuropathy. Finally, we identify cADPR as a biomarker of SARM1 enzyme activity in both healthy and injured human sensory neurons. These findings are consistent with prior molecular and cellular studies in mouse neurons, and highlight the therapeutic potential of SARM1 inhibition for the prevention and treatment of human neurological disease.