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.

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Figure 1: Preserved axon ultrastructure in distal sciatic nerve 5 days after transection.
Figure 2: Physiological and morphological evidence for preservation of axons and neuromuscular junctions.
Figure 3: Dose-dependence of axon protection by the Wld gene.
Figure 4: Axon protection 10–14 days after transection.
Figure 5: The intracellular location of the Wld protein.
Figure 6: NAD+ metabolism in WldS brain.

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References

  1. 1

    Waller, A. Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations of the alternatives produced thereby in the structure of their primitive fibres. Phil. Trans. R. Soc. Lond. 140, 423–429 (1850).

    Article  Google Scholar 

  2. 2

    Deckwerth, T. L. & Johnson, E. M. Jr. Neurites can remain viable after destruction of the neuronal soma by programmed cell death (apoptosis). Dev. Biol. 165, 63–72 (1994).

    CAS  Article  Google Scholar 

  3. 3

    Finn, J. T. et al. Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J. Neurosci. 20, 1333–1341 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Lunn, E. R., Perry, V. H., Brown, M. C., Rosen, H. & Gordon, S. Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve. Eur. J. Neurosci. 1, 27–33 (1989).

    CAS  Article  Google Scholar 

  5. 5

    Perry, V. H., Lunn, E. R., Brown, M. C., Cahusac, S. & Gordon, S. Evidence that the rate of Wallerian degeneration is controlled by a single autosomal dominant gene. Eur. J. Neurosci. 2, 408–413 (1990).

    CAS  Article  Google Scholar 

  6. 6

    Perry, V. H., Brown, M. C. & Lunn, E. R. Very slow retrograde and Wallerian degeneration in the CNS of C57BL/Ola mice. Eur. J. Neurosci. 3, 102–105 (1991).

    CAS  Article  Google Scholar 

  7. 7

    Glass, J. D., Brushart, T. M., George, E. B. & Griffin, J. W. Prolonged survival of transected nerve fibres in C57BL/Ola mice is an intrinsic characteristic of the axon. J. Neurocytol. 22, 311–321 (1993).

    CAS  Article  Google Scholar 

  8. 8

    Buckmaster, E. A., Perry, V. H. & Brown, M. C. The rate of Wallerian degeneration in cultured neurons from wild type and C57BL/WldS mice depends on time in culture and may be extended in the presence of elevated K+ levels. Eur. J. Neurosci. 7, 1596–1602 (1995).

    CAS  Article  Google Scholar 

  9. 9

    Zhang, Z., Fujiki, M., Guth, L. & Steward, O. Genetic influences on cellular reactions to spinal cord injury: a wound-healing response present in normal mice is impaired in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration. J. Comp. Neurol. 371, 485–495 (1996).

    CAS  Article  Google Scholar 

  10. 10

    Dal Canto, M. C. & Gurney, M. E. Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu, Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res. 676, 25–40 (1995).

    CAS  Article  Google Scholar 

  11. 11

    Perry, V.H. & Anthony, D.C. Axon damage and repair in multiple sclerosis. Phil. Trans. R. Soc. Lond. B Biol. Sci. 354, 1641–1647 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Glynn, P. Neural development and neurodegeneration: two faces of neuropathy target esterase. Prog. Neurobiol. 61, 61–74 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Wang, M. S., Wu, Y., Culver, D. G. & Glass, J. D. The gene for slow Wallerian degeneration (WldS) is also protective against vincristine neuropathy. Neurobiol. Dis. 8, 155–161 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Sagot, Y. et al. Bcl-2 overexpression prevents motoneuron cell body loss but not axonal degeneration in a mouse model of a neurodegenerative disease. J. Neurosci. 15, 7727–7733 (1995).

    CAS  Article  Google Scholar 

  15. 15

    Houseweart, M. K. & Cleveland, D. W. Bcl-2 overexpression does not protect neurons from mutant neurofilament-mediated motor neuron degeneration. J. Neurosci. 19, 6446–6456 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Coleman, M. P. et al. An 85-kb tandem triplication in the slow Wallerian degeneration (WldS) mouse. Proc. Natl. Acad. Sci. USA 95, 9985–9990 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Lyon, M. F., Ogunkolade, B. W., Brown, M. C., Atherton, D. J. & Perry, V. H. A gene affecting Wallerian nerve degeneration maps distally on mouse chromosome 4. Proc. Natl. Acad. Sci. USA 90, 9717–9720 (1993).

    CAS  Article  Google Scholar 

  18. 18

    Conforti, L. et al. A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse. Proc. Natl. Acad. Sci. USA 97, 11377–11382 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Emanuelli, M. et al. Human NMN adenylyltransferase: molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties. J. Biol. Chem. 276, 406–412 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Magni, G., Amici, A., Emanuelli, M., Raffaelli, N. & Ruggieri, S. Enzymology of NAD+ synthesis. Adv. Enzymol. Relat. Areas Mol. Biol. 73, 135–182 (1999).

    CAS  PubMed  Google Scholar 

  21. 21

    Conforti, L. et al. The major brain isoform of Kif1b lacks the putative mitochondria-binding domain. Mamm. Genome 10, 617–622 (1999).

    CAS  Article  Google Scholar 

  22. 22

    Zhao, C. et al. Charcot-Marie-Tooth disease type 2a caused by mutation in a microtubule motor kif1bbeta. Cell 105, 587–597 (2001).

    CAS  Article  Google Scholar 

  23. 23

    Ribchester, R. R. et al. Persistence of neuromuscular junctions after axotomy in mice with slow Wallerian degeneration (C57BL/WldS). Eur. J. Neurosci. 7, 1641–1650 (1995).

    CAS  Article  Google Scholar 

  24. 24

    Gillingwater, T. H. & Ribchester, R. R. Age-dependent synapse withdrawal at axotomised neuromuscular junctions in WldS mutant mice. J. Physiol. (Lond.) 523P, 53P (2000).

    Google Scholar 

  25. 25

    Gillingwater, T. H. & Ribchester, R. R. Compartmental neurodegeneration and synaptic plasticity in the WldS mutant mouse. J. Physiol. (Lond.) 534, 627–639 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Brown, M. C., Booth, C. M., Lunn, E. R. & Perry, V. H. Delayed response to denervation in muscles of C57BL/Ola mice. Neuroscience 43, 279–283 (1991).

    CAS  Article  Google Scholar 

  28. 28

    Sagot, Y., Tan, S. A., Hammang, J. P., Aebischer, P. & Kato, A. C. GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice. J. Neurosci. 16, 2335–2341 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Burne, J. F., Staple, J. K. & Raff, M. C. Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J. Neurosci. 16, 2064–2073 (1996).

    CAS  Article  Google Scholar 

  30. 30

    Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).

    CAS  Article  Google Scholar 

  31. 31

    Saigoh, K. et al. Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice. Nat. Genet. 23, 47–51 (1999).

    CAS  Article  Google Scholar 

  32. 32

    Hamilton, M. H., Tcherepanova, I., Huibregtse, J. M. & McDonnell, D. P. Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates. J. Biol. Chem. 276, 26324–26331 (2001).

    CAS  Article  Google Scholar 

  33. 33

    Smith, S. The world according to PARP. Trends Biochem. Sci. 26, 174–179 (2001).

    CAS  Article  Google Scholar 

  34. 34

    Ha, H.C. & Snyder, S.H. Poly(ADP-ribose) polymerase is a mediator of necrotic death by ATP depletion. Proc. Natl. Acad. Sci. USA 96, 13978–13982 (1999).

    CAS  Article  Google Scholar 

  35. 35

    Nagayama, T. et al. Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia. J. Neurochem. 74, 1636–1645 (2000).

    CAS  Article  Google Scholar 

  36. 36

    Smith, K. J., Kapoor, R., Hall, S. M. & Davies, M. Electrically active axons degenerate when exposed to nitric oxide. Ann. Neurol. 49, 470–476 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Di Lisa, F. & Ziegler, M. Pathophysiological relevance of mitochondria in NAD(+) metabolism. FEBS Lett. 492, 4–8 (2001).

    CAS  Article  Google Scholar 

  38. 38

    Frei, R. et al. Loss of distal axons and sensory Merkel cells and features indicative of muscle denervation in hindlimbs of P0-deficient mice. J. Neurosci. 19, 6058–6067 (1999).

    CAS  Article  Google Scholar 

  39. 39

    Mi, W., Conforti, L. & Coleman, M. P. Genotyping methods to detect a unique neuroprotective factor for axons (WldS). J. Neurosci. Methods (in press).

  40. 40

    Valente, E.M. et al. Localization of a novel locus for autosomal recessive early- onset parkinsonism, PARK6, on human chromosome 1p35–p36. Am. J. Hum. Genet. 68, 895–900 (2001).

    CAS  Article  Google Scholar 

  41. 41

    Fujiki, M., Zhang, Z., Guth, L. & Steward, O. Genetic influences on cellular reactions to spinal cord injury: activation of macrophages/microglia and astrocytes is delayed in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration. J. Comp. Neurol. 371, 469–484 (1996).

    CAS  Article  Google Scholar 

  42. 42

    Gunning, P., Leavitt, J., Muscat, G., Ng, S. Y. & Kedes, L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc. Natl. Acad. Sci. USA 84, 4831–4835 (1987).

    CAS  Article  Google Scholar 

  43. 43

    Karnovsky, M. J. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A (1965).

    Google Scholar 

  44. 44

    Reynolds, E. E. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 (1963).

    CAS  Article  Google Scholar 

  45. 45

    Costanzo, E. M., Barry, J. A. & Ribchester, R. R. Co-regulation of synaptic efficacy at stable polyneuronally innervated neuromuscular junctions in reinnervated rat muscle. J. Physiol. (Lond.) 521, 365–374 (1999).

    CAS  Article  Google Scholar 

  46. 46

    Costanzo, E. M., Barry, J. A. & Ribchester, R. R. Competition at silent synapses in reinnervated skeletal muscle. Nat. Neurosci. 3, 694–700 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Ribchester, R. R., Mao, F. & Betz, W. J. Optical measurements of activity- dependent membrane recycling in motor nerve terminals of mammalian skeletal muscle. Proc. R. Soc. Lond. B Biol. Sci. 255, 61–66 (1994).

    CAS  Article  Google Scholar 

  48. 48

    Balducci, E. et al. Assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability. Anal. Biochem. 228, 64–68 (1995).

    CAS  Article  Google Scholar 

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Acknowledgements

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.).

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Correspondence to Michael P. Coleman.

Supplementary information

Supplementary Figure 1

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)

Supplementary Figure 2

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)

Supplementary Figure 3

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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