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.
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We thank E. Janssen, C. Hoffmann (Department of Anatomy I, University of Cologne), F. Carnevali, F. Pierella (University of Ancona), S. Fearn and M. Botham (University of Southampton) for technical assistance, T. Vogt for supplying pHβAPr-1 plasmid and R. Martini for critically reading the manuscript. This work was supported by the Federal Ministry of Education and Research (FKZ: 01 KS 9502) and Center for Molecular Medicine, University of Cologne (ZMMK) (T.G.A.M., W.M., D.W., M.P.C.), a Wellcome Trust Biomedical Collaboration Grant (R.R.R., M.P.C.) and Consiglio Nazionale delle Ricerche Target Project "Biotechnology" (M.E., G.M.).
Higher magnification of preserved axon ultrastructure in distal sciatic nerve five days after transection. Electron micrograph (30,000x) of a transverse thin section 2-4 mm distal to the lesion in a 4836 homozygote. Typically, the myelinated axon contains fully preserved cytoskeleton and mitochondria. (JPG 222 kb)
The transgene construct and evidence for genomic integration. (a) The transgene construct. The chimeric cDNA was expressed with non-coding exon 1 of β-actin under the control of a human β-actin promoter and terminated with the SV40 polyadenylation signal. (b) Southern blot demonstrating transgene integration. Genomic DNA from homozygous transgenic mice and a non-transgenic 4830 littermate control was double-digested with BamHI and HindIII. Hybridization with 32P-labeled chimeric cDNA reveals the 1.1 kb transgene fragment specifically in transgenic mice. Larger fragments, also present in the control, are derived from endogenous sequences. Quantification of signal at lower loadings indicated transgene copy numbers of approximately 16 (line 4836), 12 (line 4830) and 4 (line 4839). Additional bands in line 4836 may indicate limited rearrangement. (JPG 56 kb)
Wld protein is not detectable in axons of sciatic nerve (a) Motor neurons in thoracic spinal cord of Wlds mice express the Wld protein in their nuclei. (b-d) Applying the same camera exposure parameters to transverse sciatic nerve sections labeled with anti-N70 (red) shows the absence of detectable Wld protein in axons and Schwann cells of Wlds (b, f), transgenic 4836 (c, g) and C57BL/6J (d, h) nerves. The faint red background signal indicates position of myelin sheaths surrounding axons and was equally strong in a control (e) from which the primary antibody was omitted. Schwann cell nuclei are detected with Hoechst dye (blue). (f-h) Same fields of view and labeling as in (b-d); only red channel shown. Scale bar, 50 μm (a), 25 μm (b-h). (JPG 336 kb)
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Mack, T., Reiner, M., Beirowski, B. et al. Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 4, 1199–1206 (2001). https://doi.org/10.1038/nn770
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