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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 W258AWldS Tg mice. (A) Brain Western blots from W258AWldS Tg, WldS, and wild-type mice probed with Wld18. W258AWldS lines 2 and 4 express a 43-kD band, which is absent in wild type. (B) Wld18 immunofluorescence (red) of lumbar spinal cord. Motor neuron nuclear signal strength and distribution in W258AWldS lines match WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (C) Nmnat1 activity is unaltered in the W258AWldS brain. (D, i and ii) Semithin sections of W258AWldS distal sciatic nerve 72 h after lesion. Axons are degenerated, similar to wild-type or ΔN16WldS axons (Fig 2). (iii–vi) In mice crossed to YFP-H, tibial nerve axons lose continuity within 72 h of sciatic lesion, except in WldS (iii). (E) SCG explants untreated (i–iv) or treated (v–viii) with 100 nM FK866 for 72 h and then cut. Unlike wild-type axons (i, ii, v, and vi), untreated WldS axons are intact 72 h after being cut (iv) but when treated with FK866 are more degenerated (viii). (F) Quantification of continuous neurites (or neurite bundles). ***, P < 0.0001 (one-way analysis of variance followed by Bonferroni post-hoc test); n = 8 (wild type [WT]) and 9 (WldS). (G) NAD+ or NADP+ levels in wild-type and WldS explants ± FK866 at 1–100 nM. Mean of three different experiments. (C, F, and G) Mean ± SD. Bars: (B, D, i and ii, and E) 10 µm; (D, iii–vi) 100 µm.
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fig5: Rapid Wallerian degeneration in W258AWldS Tg mice. (A) Brain Western blots from W258AWldS Tg, WldS, and wild-type mice probed with Wld18. W258AWldS lines 2 and 4 express a 43-kD band, which is absent in wild type. (B) Wld18 immunofluorescence (red) of lumbar spinal cord. Motor neuron nuclear signal strength and distribution in W258AWldS lines match WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (C) Nmnat1 activity is unaltered in the W258AWldS brain. (D, i and ii) Semithin sections of W258AWldS distal sciatic nerve 72 h after lesion. Axons are degenerated, similar to wild-type or ΔN16WldS axons (Fig 2). (iii–vi) In mice crossed to YFP-H, tibial nerve axons lose continuity within 72 h of sciatic lesion, except in WldS (iii). (E) SCG explants untreated (i–iv) or treated (v–viii) with 100 nM FK866 for 72 h and then cut. Unlike wild-type axons (i, ii, v, and vi), untreated WldS axons are intact 72 h after being cut (iv) but when treated with FK866 are more degenerated (viii). (F) Quantification of continuous neurites (or neurite bundles). ***, P < 0.0001 (one-way analysis of variance followed by Bonferroni post-hoc test); n = 8 (wild type [WT]) and 9 (WldS). (G) NAD+ or NADP+ levels in wild-type and WldS explants ± FK866 at 1–100 nM. Mean of three different experiments. (C, F, and G) Mean ± SD. Bars: (B, D, i and ii, and E) 10 µm; (D, iii–vi) 100 µm.

Mentions: Tg-expressing lines 2 and 4 showed no increase in brain Nmnat activity over wild type, whereas activity in WldS heterozygotes increased two- to threefold (Fig. 5, A and C). As before, we confirmed protein expression in motor neuron nuclei (Fig. 5 B) and we lesioned sciatic nerves. At the 3-d (low stringency) time point, homogeneous axoplasm and unswollen mitochondria could no longer be identified in semithin sections (Fig. 5 D, i and ii), and neither line retained axon continuity (Fig. 5 D, v and vi). The third expressing line showed similar wild-type–like behavior (unpublished data). In contrast, WldS heterozygous mice expressing a similar level of WldS protein showed strong axon protection (Fig. 5 D, iii; and Fig. S1).


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 W258AWldS Tg mice. (A) Brain Western blots from W258AWldS Tg, WldS, and wild-type mice probed with Wld18. W258AWldS lines 2 and 4 express a 43-kD band, which is absent in wild type. (B) Wld18 immunofluorescence (red) of lumbar spinal cord. Motor neuron nuclear signal strength and distribution in W258AWldS lines match WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (C) Nmnat1 activity is unaltered in the W258AWldS brain. (D, i and ii) Semithin sections of W258AWldS distal sciatic nerve 72 h after lesion. Axons are degenerated, similar to wild-type or ΔN16WldS axons (Fig 2). (iii–vi) In mice crossed to YFP-H, tibial nerve axons lose continuity within 72 h of sciatic lesion, except in WldS (iii). (E) SCG explants untreated (i–iv) or treated (v–viii) with 100 nM FK866 for 72 h and then cut. Unlike wild-type axons (i, ii, v, and vi), untreated WldS axons are intact 72 h after being cut (iv) but when treated with FK866 are more degenerated (viii). (F) Quantification of continuous neurites (or neurite bundles). ***, P < 0.0001 (one-way analysis of variance followed by Bonferroni post-hoc test); n = 8 (wild type [WT]) and 9 (WldS). (G) NAD+ or NADP+ levels in wild-type and WldS explants ± FK866 at 1–100 nM. Mean of three different experiments. (C, F, and G) Mean ± SD. Bars: (B, D, i and ii, and E) 10 µm; (D, iii–vi) 100 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Rapid Wallerian degeneration in W258AWldS Tg mice. (A) Brain Western blots from W258AWldS Tg, WldS, and wild-type mice probed with Wld18. W258AWldS lines 2 and 4 express a 43-kD band, which is absent in wild type. (B) Wld18 immunofluorescence (red) of lumbar spinal cord. Motor neuron nuclear signal strength and distribution in W258AWldS lines match WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (C) Nmnat1 activity is unaltered in the W258AWldS brain. (D, i and ii) Semithin sections of W258AWldS distal sciatic nerve 72 h after lesion. Axons are degenerated, similar to wild-type or ΔN16WldS axons (Fig 2). (iii–vi) In mice crossed to YFP-H, tibial nerve axons lose continuity within 72 h of sciatic lesion, except in WldS (iii). (E) SCG explants untreated (i–iv) or treated (v–viii) with 100 nM FK866 for 72 h and then cut. Unlike wild-type axons (i, ii, v, and vi), untreated WldS axons are intact 72 h after being cut (iv) but when treated with FK866 are more degenerated (viii). (F) Quantification of continuous neurites (or neurite bundles). ***, P < 0.0001 (one-way analysis of variance followed by Bonferroni post-hoc test); n = 8 (wild type [WT]) and 9 (WldS). (G) NAD+ or NADP+ levels in wild-type and WldS explants ± FK866 at 1–100 nM. Mean of three different experiments. (C, F, and G) Mean ± SD. Bars: (B, D, i and ii, and E) 10 µm; (D, iii–vi) 100 µm.
Mentions: Tg-expressing lines 2 and 4 showed no increase in brain Nmnat activity over wild type, whereas activity in WldS heterozygotes increased two- to threefold (Fig. 5, A and C). As before, we confirmed protein expression in motor neuron nuclei (Fig. 5 B) and we lesioned sciatic nerves. At the 3-d (low stringency) time point, homogeneous axoplasm and unswollen mitochondria could no longer be identified in semithin sections (Fig. 5 D, i and ii), and neither line retained axon continuity (Fig. 5 D, v and vi). The third expressing line showed similar wild-type–like behavior (unpublished data). In contrast, WldS heterozygous mice expressing a similar level of WldS protein showed strong axon protection (Fig. 5 D, iii; and Fig. S1).

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