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Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

Conforti L, Wilbrey A, Morreale G, Janeckova L, Beirowski B, Adalbert R, Mazzola F, Di Stefano M, Hartley R, Babetto E, Smith T, Gilley J, Billington RA, Genazzani AA, Ribchester RR, Magni G, Coleman M - J. Cell Biol. (2009)

Bottom Line: Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection.Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function.Thus, neither domain is effective without the function of the other.

View Article: PubMed Central - PubMed

Affiliation: Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, England, UK.

ABSTRACT
The slow Wallerian degeneration (Wld(S)) 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 Wld(S)-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 Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

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Related in: MedlinePlus

Rapid Wallerian degeneration in ΔN16WldS Tg mice. (a–d) Semithin sections of distal sciatic nerve 5 d after lesion. (a) Axons are well preserved in WldS heterozygotes, with intact myelin sheaths, uniform and regularly spaced cytoskeleton, and normal-shaped mitochondria. (b) Wild-type axons are degenerated, with collapsed myelin and disorganized or vacuolized cytoskeleton. (c and d) ΔN16WldS line 1 homozygous and line 2 hemizygous nerves are indistinguishable from wild type. (e–h) Tibial nerves from mice crossed to YFP-H show rapid loss of axon continuity in ΔN16WldS. (e) WldS heterozygotes 3 d after lesion show axon continuity. (f–h) In contrast, all lesioned wild-type and ΔN16WldS line 1 and line 2 axons lose continuity within 72 h. (i–l) Transmission electron microscopy of distal sciatic nerve 3 d after lesion. (i) Myelinated and unmyelinated axons are well preserved in WldS heterozygotes, which are indistinguishable from unlesioned nerves (l). (j) In wild type, myelin collapsed to form ovoids, the cytoskeleton is floccular or absent, and mitochondria are swollen or absent. (k) Nerves from ΔN16WldS line 1 homozygotes are indistinguishable from wild types. Bars: (a–d) 20 µm; (e–h) 50 µm; (i–l) 2 µm.
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fig2: Rapid Wallerian degeneration in ΔN16WldS Tg mice. (a–d) Semithin sections of distal sciatic nerve 5 d after lesion. (a) Axons are well preserved in WldS heterozygotes, with intact myelin sheaths, uniform and regularly spaced cytoskeleton, and normal-shaped mitochondria. (b) Wild-type axons are degenerated, with collapsed myelin and disorganized or vacuolized cytoskeleton. (c and d) ΔN16WldS line 1 homozygous and line 2 hemizygous nerves are indistinguishable from wild type. (e–h) Tibial nerves from mice crossed to YFP-H show rapid loss of axon continuity in ΔN16WldS. (e) WldS heterozygotes 3 d after lesion show axon continuity. (f–h) In contrast, all lesioned wild-type and ΔN16WldS line 1 and line 2 axons lose continuity within 72 h. (i–l) Transmission electron microscopy of distal sciatic nerve 3 d after lesion. (i) Myelinated and unmyelinated axons are well preserved in WldS heterozygotes, which are indistinguishable from unlesioned nerves (l). (j) In wild type, myelin collapsed to form ovoids, the cytoskeleton is floccular or absent, and mitochondria are swollen or absent. (k) Nerves from ΔN16WldS line 1 homozygotes are indistinguishable from wild types. Bars: (a–d) 20 µm; (e–h) 50 µm; (i–l) 2 µm.

Mentions: 5 d after sciatic nerve lesion, WldS heterozygotes retained 69.7 ± 1.8% of axons with normal cytoskeleton, unswollen mitochondria, and a regular myelin sheath of normal thickness compared with only 1.2 ± 0.4% in wild-type mice (Fig. 2 and Fig. S1). In contrast, ΔN16WldS was indistinguishable from wild type. Line 1 homozygotes and line 2 hemizygotes showed 2.3 ± 0.2% and 0.9 ± 0.2% surviving axons, respectively (Fig. 2, a–d; and Fig. S1). Axons lost continuity, as assessed by crossing to YFP-H Tg mice (Beirowski et al., 2004), by 3 d (Fig. 2, g and h), which is a low stringency test ruling out even a weak protective phenotype. Axons in WldS heterozygotes maintain continuity even at 5–14 d (Fig. 3). Finally, ultrastructural experiments confirmed that axons of ΔN16WldS mice retained little, if any, normal cytoskeleton and organelles after 3 d (Fig. 2, i–l). Thus, N16, which contains a VBM, is essential for WldS to delay Wallerian degeneration.


Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

Conforti L, Wilbrey A, Morreale G, Janeckova L, Beirowski B, Adalbert R, Mazzola F, Di Stefano M, Hartley R, Babetto E, Smith T, Gilley J, Billington RA, Genazzani AA, Ribchester RR, Magni G, Coleman M - J. Cell Biol. (2009)

Rapid Wallerian degeneration in ΔN16WldS Tg mice. (a–d) Semithin sections of distal sciatic nerve 5 d after lesion. (a) Axons are well preserved in WldS heterozygotes, with intact myelin sheaths, uniform and regularly spaced cytoskeleton, and normal-shaped mitochondria. (b) Wild-type axons are degenerated, with collapsed myelin and disorganized or vacuolized cytoskeleton. (c and d) ΔN16WldS line 1 homozygous and line 2 hemizygous nerves are indistinguishable from wild type. (e–h) Tibial nerves from mice crossed to YFP-H show rapid loss of axon continuity in ΔN16WldS. (e) WldS heterozygotes 3 d after lesion show axon continuity. (f–h) In contrast, all lesioned wild-type and ΔN16WldS line 1 and line 2 axons lose continuity within 72 h. (i–l) Transmission electron microscopy of distal sciatic nerve 3 d after lesion. (i) Myelinated and unmyelinated axons are well preserved in WldS heterozygotes, which are indistinguishable from unlesioned nerves (l). (j) In wild type, myelin collapsed to form ovoids, the cytoskeleton is floccular or absent, and mitochondria are swollen or absent. (k) Nerves from ΔN16WldS line 1 homozygotes are indistinguishable from wild types. Bars: (a–d) 20 µm; (e–h) 50 µm; (i–l) 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654131&req=5

fig2: Rapid Wallerian degeneration in ΔN16WldS Tg mice. (a–d) Semithin sections of distal sciatic nerve 5 d after lesion. (a) Axons are well preserved in WldS heterozygotes, with intact myelin sheaths, uniform and regularly spaced cytoskeleton, and normal-shaped mitochondria. (b) Wild-type axons are degenerated, with collapsed myelin and disorganized or vacuolized cytoskeleton. (c and d) ΔN16WldS line 1 homozygous and line 2 hemizygous nerves are indistinguishable from wild type. (e–h) Tibial nerves from mice crossed to YFP-H show rapid loss of axon continuity in ΔN16WldS. (e) WldS heterozygotes 3 d after lesion show axon continuity. (f–h) In contrast, all lesioned wild-type and ΔN16WldS line 1 and line 2 axons lose continuity within 72 h. (i–l) Transmission electron microscopy of distal sciatic nerve 3 d after lesion. (i) Myelinated and unmyelinated axons are well preserved in WldS heterozygotes, which are indistinguishable from unlesioned nerves (l). (j) In wild type, myelin collapsed to form ovoids, the cytoskeleton is floccular or absent, and mitochondria are swollen or absent. (k) Nerves from ΔN16WldS line 1 homozygotes are indistinguishable from wild types. Bars: (a–d) 20 µm; (e–h) 50 µm; (i–l) 2 µm.
Mentions: 5 d after sciatic nerve lesion, WldS heterozygotes retained 69.7 ± 1.8% of axons with normal cytoskeleton, unswollen mitochondria, and a regular myelin sheath of normal thickness compared with only 1.2 ± 0.4% in wild-type mice (Fig. 2 and Fig. S1). In contrast, ΔN16WldS was indistinguishable from wild type. Line 1 homozygotes and line 2 hemizygotes showed 2.3 ± 0.2% and 0.9 ± 0.2% surviving axons, respectively (Fig. 2, a–d; and Fig. S1). Axons lost continuity, as assessed by crossing to YFP-H Tg mice (Beirowski et al., 2004), by 3 d (Fig. 2, g and h), which is a low stringency test ruling out even a weak protective phenotype. Axons in WldS heterozygotes maintain continuity even at 5–14 d (Fig. 3). Finally, ultrastructural experiments confirmed that axons of ΔN16WldS mice retained little, if any, normal cytoskeleton and organelles after 3 d (Fig. 2, i–l). Thus, N16, which contains a VBM, is essential for WldS to delay Wallerian degeneration.

Bottom Line: Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection.Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function.Thus, neither domain is effective without the function of the other.

View Article: PubMed Central - PubMed

Affiliation: Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, England, UK.

ABSTRACT
The slow Wallerian degeneration (Wld(S)) 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 Wld(S)-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 Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

Show MeSH
Related in: MedlinePlus