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

Abstract

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.

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

“…Mean values were increased 3.2-3.5 times, slightly more (although not significantly) than Wld S heterozygotes ( Figure 1b) and only a little less than the four-fold increase reported in Wld S homozygotes. 15 Wallerian degeneration was tested in sciatic nerve, so we first confirmed transgene expression in neurons projecting axons to this nerve. RT-PCR confirmed the presence of a transgene-specific Nmnat1 mRNA in lumbar DRG (Figure 1c).…”

“…C57BL/Wld S heterozygotes express increased Nmnat1 as part of the Wld S protein and show a strong axon protection phenotype. 13,15,22 Western blots of sagittally divided half-brain homogenates, probed with antiNmnat1 antibody 183, showed that lines 881, 891, 2460, 7103 and 7104 overexpressed Nmnat1 (Figure 1a). Nonexpressing line 884 (data not shown) was not studied further.…”

“…9,15,22 We therefore chose these lines for most of our subsequent investigations, but used also the other expressing lines, 891, 2460 and 7103, in some assays.…”

“…6 Our groups showed that the gene identified by positional cloning [12][13][14] delays Wallerian degeneration in transgenic mice and virally transduced dorsal root ganglion (DRG) explant cultures. 15,16 Wld S encodes a fusion protein containing 70 N-terminal amino acids of ubiquitination factor Ube4b, full-length nicotinamide adenine dinucleotide (NAD þ )-synthesising enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), and a unique 18-amino-acid joining sequence. 15 Surprisingly, Wld S is abundant in neuronal nuclei 8,11,15,18 and so far undetectable in axons in vivo.…”

“…15,16 Wld S encodes a fusion protein containing 70 N-terminal amino acids of ubiquitination factor Ube4b, full-length nicotinamide adenine dinucleotide (NAD þ )-synthesising enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), and a unique 18-amino-acid joining sequence. 15 Surprisingly, Wld S is abundant in neuronal nuclei 8,11,15,18 and so far undetectable in axons in vivo. However, it does enter neurites in virally transduced primary cultures.…”