NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein WldS prevent this rise. These data indicate that the mechanism by which NMNAT and the related WldS protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.

Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies.

The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration.

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld S ) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld S acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld S significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld S mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld S for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld S redistribution. Cytoplasmic Wld S fractionated predominantly with mitochondria and microsomes. We conclude that Wld S can act in one or more nonnuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

Axon and synapse degeneration are common components of many neurodegenerative diseases, and their rescue is essential for effective neuroprotection. The chimeric Wallerian degeneration slow protein (Wld S ) protects axons dose dependently, but its mechanism is still elusive. We recently showed that Wld S acts at a non-nuclear location and is present in axons. This and other recent reports support a model in which Wld S protects by extranuclear redistribution of its nuclear NMNAT1 portion. However, it remains unclear whether cytoplasmic NMNAT1 acts locally in axons and synapses or at a non-nuclear site within cell bodies. The potency of axon protection by non-nuclear NMNAT1 relative to Wld S also needs to be established in vivo. Because the N-terminal portion of Wld S (N70) localized to axons, we hypothesized that it mediates the trafficking of the NMNAT1 portion. To test this, we substituted N70 with an axonal targeting peptide derived from amyloid precursor protein, and fused this to NMNAT1 with disrupted nuclear targeting. In transgenic mice, this transformed NMNAT1 from a molecule unable to inhibit Wallerian degeneration, even at high expression levels, into a protein more potent than Wld S , able to preserve injured axons for several weeks at undetectable expression levels. Preventing NMNAT1 axonal delivery abolished its protective effect. Axonally targeted NMNAT1 localized to vesicular structures, colocalizing with extranuclear Wld S , and was cotransported at least partially with mitochondria. We conclude that axonal targeting of NMNAT activity is both necessary and sufficient to delay Wallerian degeneration, and that promoting axonal and synaptic delivery greatly enhances the effectiveness.