Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of 'slow Wallerian degeneration' (Wld (S)), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld(S). Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld(S)-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld(S) expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld(S) or NMNAT2.

Studies with the WldS mutant mouse have shown that axon and synapse pathology in several models of neurodegenerative diseases are mechanistically related to injury-induced axon degeneration (Wallerian degeneration). Crucially, an absence of SARM1 delays Wallerian degeneration as robustly as WldS, but their relative capacities to confer long-term protection against related, non-injury axonopathy and/or synaptopathy have not been directly compared. While Sarm1 deletion or WldS can rescue perinatal lethality and widespread Wallerian-like axonopathy in young NMNAT2-deficient mice, we report that an absence of SARM1 enables these mice to survive into old age with no overt phenotype, whereas those rescued by WldS invariantly develop a progressive neuromuscular defect in their hindlimbs from around 3 months of age. We therefore propose Sarm1 deletion as a more reliable tool than WldS for investigating Wallerian-like mechanisms in disease models and suggest that SARM1 blockade may have greater therapeutic potential than WLDS-related strategies.

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld(S) neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld(S) does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld(S) protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.

Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, Wld(S). The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles.

We identified a homozygous missense mutation in the gene encoding NAD synthesizing enzyme NMNAT2 in two siblings with childhood onset polyneuropathy with erythromelalgia. No additional homozygotes for this rare allele, which leads to amino acid substitution T94M, were present among the unaffected relatives tested or in the 60,000 exomes of the ExAC database. For axons to survive, axonal NMNAT2 activity has to be maintained above a threshold level but the T94M mutation confers a partial loss of function both in the ability of NMNAT2 to support axon survival and in its enzymatic properties. Electrophysiological tests and histological analysis of sural nerve biopsies in the patients were consistent with loss of distal sensory and motor axons. Thus, it is likely that NMNAT2 mutation causes this pain and axon loss phenotype making this the first disorder associated with mutation of a key regulator of Wallerian-like axon degeneration in humans. This supports indications from numerous animal studies that the Wallerian degeneration pathway is important in human disease and raises important questions about which other human phenotypes could be linked to this gene.

The three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members synthesize the electron carrier nicotinamide adenine dinucleotide (NAD+) and are essential for cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in mice, or in its ortholog dNmnat in Drosophila, results in axon outgrowth and survival defects. Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered protein stability and/or defects in NAD+ synthesis and chaperone functions. Thus, both patient NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown role for NMNAT2 in human neurological development and provide the first direct molecular evidence to support the involvement of Wallerian degeneration in a human axonal disorder. SIGNIFICANCE: Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) both synthesizes the electron carrier Nicotinamide Adenine Dinucleotide (NAD+) and acts a protein chaperone. NMNAT2 has emerged as a major neuron survival factor. Overexpression of NMNAT2 protects neurons from Wallerian degeneration after injury and declining levels of NMNAT2 have been implicated in neurodegeneration. While the role of NMNAT2 in neurodegeneration has been extensively studied, the role of NMNAT2 in human development remains unclear. In this work, we present the first human variants in NMNAT2 identified in two fetuses with severe skeletal muscle hypoplasia and fetal akinesia. Functional studies in vitro showed that the mutations impair both NMNAT2 NAD+ synthase and chaperone functions. This work identifies the critical role of NMNAT2 in human development.

SARM1 is an NAD(P) glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). Like other NAD(P)ases, it catalyzes multiple reactions that need to be fully investigated. Here, we compare these multiple activities for recombinant human SARM1, human CD38, and Aplysia californica ADP ribosyl cyclase. SARM1 has the highest transglycosidation (base exchange) activity at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclization. All SARM1 activities, including base exchange at neutral pH, are activated by an increased NMN:NAD ratio, at physiological levels of both metabolites. SARM1 base exchange occurs also in DRG neurons and is thus a very likely physiological source of calcium-mobilizing agent NaADP. Finally, we identify regulation by free pyridines, NADP, and nicotinic acid riboside (NaR) on SARM1, all of therapeutic interest. Understanding which specific SARM1 function(s) is responsible for axon degeneration is essential for its targeting in disease.