WldS can delay Wallerian degeneration in mice when interaction with valosin-containing protein is weakened.

Axon degeneration is an early event in many neurodegenerative disorders. In some, the mechanism is related to injury-induced Wallerian degeneration, a proactive death program that can be strongly delayed by the neuroprotective slow Wallerian degeneration protein (Wld(S)) protein. Thus, it is important to understand the Wallerian degeneration mechanism and how Wld(S) blocks it. Wld(S) location is influenced by binding to valosin-containing protein (VCP), an essential protein for many cellular processes including membrane fusion and endoplasmic reticulum-associated degradation. In mice, the N-terminal 16 amino acids (N16), which mediate VCP binding, are essential for Wld(S) to protect axons, a role which another VCP binding sequence can substitute. In Drosophila, the Wld(S) phenotype is weakened by a similar N-terminal truncation and by knocking down the VCP homologue ter94. Neither null nor floxed VCP mice are viable so it is difficult to confirm the requirement for VCP binding in mammals in vivo. However, the hypothesis can be tested further by introducing a Wld(S) missense mutation, altering its affinity for VCP but minimizing the risk of disturbing other aspects of its structure or function. We introduced the R10A mutation, which weakens VCP binding in vitro, and expressed it in transgenic mice. R10AWld(S) fails to co-immunoprecipitate VCP from mouse brain, and only occasionally and faintly accumulates in nuclear foci for which VCP binding is necessary but not sufficient. Surprisingly however, axon protection remains robust and indistinguishable from that in spontaneous Wld(S) mice. We suggest that either N16 has an additional, VCP-independent function in mammals, or that the phenotype requires only weak VCP binding which may be driven forwards in vivo by the high VCP concentration.

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration.

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.