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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

Overexpression of ΔN16WldS and ATX3WldS in Tg mice. (A and B) Western blots of ΔN16WldS and ATX3WldS brains probed with Wld18. ΔN16WldS lines 1 and 2 alone express a protein slightly smaller than WldS. The 43-kD ATX3WldS band matches that in WldS. (C) Brain Western blots of total homogenate and nuclear and cytoplasmic fractions of WldS, ΔN16WldS (line 1), and ATX3WldS (line 6). The graph shows integrated band intensities of nuclear and cytoplasmic fractions normalized to H1 and β-actin, respectively. These normalized figures were then expressed as a percentage of the total homogenate signal and normalized to the same respective markers (mean ± SD; n = 3). Statistical analysis was performed on the nuclear versus supernatant ratio using a Mann-Whitney test followed by a Bonferroni post-hoc test. (D) Immunofluorescence of lumbar spinal cord sections with Wld18 (red) and DAPI. Motor and interneuron nuclear signals in Tg ΔN16WldS (i–iv) and ATX3WldS (v–viii) show similar strength and distribution as WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (E and F) Transgene products are enzymatically active. Nmnat activity is very significantly increased compared with wild-type (WT) brains in hemizygotes and homozygotes of all expressing Tg lines as well as WldS heterozygotes. Mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.
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fig1: Overexpression of ΔN16WldS and ATX3WldS in Tg mice. (A and B) Western blots of ΔN16WldS and ATX3WldS brains probed with Wld18. ΔN16WldS lines 1 and 2 alone express a protein slightly smaller than WldS. The 43-kD ATX3WldS band matches that in WldS. (C) Brain Western blots of total homogenate and nuclear and cytoplasmic fractions of WldS, ΔN16WldS (line 1), and ATX3WldS (line 6). The graph shows integrated band intensities of nuclear and cytoplasmic fractions normalized to H1 and β-actin, respectively. These normalized figures were then expressed as a percentage of the total homogenate signal and normalized to the same respective markers (mean ± SD; n = 3). Statistical analysis was performed on the nuclear versus supernatant ratio using a Mann-Whitney test followed by a Bonferroni post-hoc test. (D) Immunofluorescence of lumbar spinal cord sections with Wld18 (red) and DAPI. Motor and interneuron nuclear signals in Tg ΔN16WldS (i–iv) and ATX3WldS (v–viii) show similar strength and distribution as WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (E and F) Transgene products are enzymatically active. Nmnat activity is very significantly increased compared with wild-type (WT) brains in hemizygotes and homozygotes of all expressing Tg lines as well as WldS heterozygotes. Mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Mentions: First, we tested the need for the VCP-binding sequence, expressing WldS without amino acids 2–16 in ΔN16WldS Tg mice (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807175/DC1). Two of the four lines expressed protein on brain Western blots (Fig. 1 A; Samsam et al., 2003). Line 1 homozygotes expressed similar levels to WldS heterozygotes. Line 2 could not breed to homozygosity. Hemizygotes expressed slightly less, but still at a level where WldS delays axon degeneration significantly (Mack et al., 2001). Nmnat activity matched or exceeded that in WldS (Fig. 1 E), and lumbar spinal cord motor neurons, whose axons in the sciatic nerve we lesioned, express the protein (Fig. 1 D). The ΔN16WldS variant showed a clear cytoplasmic signal, with no significant difference in nuclear/cytoplasmic distribution relative to WldS at n = 3 (Fig. 1, C and D, i–iv).


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)

Overexpression of ΔN16WldS and ATX3WldS in Tg mice. (A and B) Western blots of ΔN16WldS and ATX3WldS brains probed with Wld18. ΔN16WldS lines 1 and 2 alone express a protein slightly smaller than WldS. The 43-kD ATX3WldS band matches that in WldS. (C) Brain Western blots of total homogenate and nuclear and cytoplasmic fractions of WldS, ΔN16WldS (line 1), and ATX3WldS (line 6). The graph shows integrated band intensities of nuclear and cytoplasmic fractions normalized to H1 and β-actin, respectively. These normalized figures were then expressed as a percentage of the total homogenate signal and normalized to the same respective markers (mean ± SD; n = 3). Statistical analysis was performed on the nuclear versus supernatant ratio using a Mann-Whitney test followed by a Bonferroni post-hoc test. (D) Immunofluorescence of lumbar spinal cord sections with Wld18 (red) and DAPI. Motor and interneuron nuclear signals in Tg ΔN16WldS (i–iv) and ATX3WldS (v–viii) show similar strength and distribution as WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (E and F) Transgene products are enzymatically active. Nmnat activity is very significantly increased compared with wild-type (WT) brains in hemizygotes and homozygotes of all expressing Tg lines as well as WldS heterozygotes. Mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2654131&req=5

fig1: Overexpression of ΔN16WldS and ATX3WldS in Tg mice. (A and B) Western blots of ΔN16WldS and ATX3WldS brains probed with Wld18. ΔN16WldS lines 1 and 2 alone express a protein slightly smaller than WldS. The 43-kD ATX3WldS band matches that in WldS. (C) Brain Western blots of total homogenate and nuclear and cytoplasmic fractions of WldS, ΔN16WldS (line 1), and ATX3WldS (line 6). The graph shows integrated band intensities of nuclear and cytoplasmic fractions normalized to H1 and β-actin, respectively. These normalized figures were then expressed as a percentage of the total homogenate signal and normalized to the same respective markers (mean ± SD; n = 3). Statistical analysis was performed on the nuclear versus supernatant ratio using a Mann-Whitney test followed by a Bonferroni post-hoc test. (D) Immunofluorescence of lumbar spinal cord sections with Wld18 (red) and DAPI. Motor and interneuron nuclear signals in Tg ΔN16WldS (i–iv) and ATX3WldS (v–viii) show similar strength and distribution as WldS heterozygotes. Identical laser intensities and camera settings were used for each image. (E and F) Transgene products are enzymatically active. Nmnat activity is very significantly increased compared with wild-type (WT) brains in hemizygotes and homozygotes of all expressing Tg lines as well as WldS heterozygotes. Mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.
Mentions: First, we tested the need for the VCP-binding sequence, expressing WldS without amino acids 2–16 in ΔN16WldS Tg mice (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807175/DC1). Two of the four lines expressed protein on brain Western blots (Fig. 1 A; Samsam et al., 2003). Line 1 homozygotes expressed similar levels to WldS heterozygotes. Line 2 could not breed to homozygosity. Hemizygotes expressed slightly less, but still at a level where WldS delays axon degeneration significantly (Mack et al., 2001). Nmnat activity matched or exceeded that in WldS (Fig. 1 E), and lumbar spinal cord motor neurons, whose axons in the sciatic nerve we lesioned, express the protein (Fig. 1 D). The ΔN16WldS variant showed a clear cytoplasmic signal, with no significant difference in nuclear/cytoplasmic distribution relative to WldS at n = 3 (Fig. 1, C and D, i–iv).

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