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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

Functional motor innervation. (A) ATX3WldS mice show robust preservation of neuromuscular function 3 d after nerve lesion. (i and ii) Isometric single twitch (i) and 20-Hz tetanic tension (ii) responses of isolated FDB to tibial nerve stimulation. (iii and iv) Averaged (16 sweeps) extracellular evoked EMG responses to stimulation of 3-d axotomized FDB from ATX3WldS (iii) and WldS (iv). The x axis represents milliseconds, and the y axis represents millinewtons or microvolts. Bars: (i) 200 ms and 2 mN; (ii) 700 ms and 5 mN; (iii) 6 ms and 200 µV; (iv) 12 ms and 200 µV. (B) Confocal microscopy of axotomized FDB from an ATX3WldS line 5 mouse in which physiological recordings indicated robust preservation of neuromuscular function. Most motor endplates (TRITC–α-bungarotoxin staining; red) were innervated by neurofilament-positive axon collaterals and motor nerve terminals (green), but ∼20% of endplates were unoccupied. (i) Low power z-series projection. (ii and iii) Higher magnification images correspond to the boxed areas in panel i.
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fig4: Functional motor innervation. (A) ATX3WldS mice show robust preservation of neuromuscular function 3 d after nerve lesion. (i and ii) Isometric single twitch (i) and 20-Hz tetanic tension (ii) responses of isolated FDB to tibial nerve stimulation. (iii and iv) Averaged (16 sweeps) extracellular evoked EMG responses to stimulation of 3-d axotomized FDB from ATX3WldS (iii) and WldS (iv). The x axis represents milliseconds, and the y axis represents millinewtons or microvolts. Bars: (i) 200 ms and 2 mN; (ii) 700 ms and 5 mN; (iii) 6 ms and 200 µV; (iv) 12 ms and 200 µV. (B) Confocal microscopy of axotomized FDB from an ATX3WldS line 5 mouse in which physiological recordings indicated robust preservation of neuromuscular function. Most motor endplates (TRITC–α-bungarotoxin staining; red) were innervated by neurofilament-positive axon collaterals and motor nerve terminals (green), but ∼20% of endplates were unoccupied. (i) Low power z-series projection. (ii and iii) Higher magnification images correspond to the boxed areas in panel i.

Mentions: We then applied more stringent tests to verify that ATX3WldS mice have a full WldS phenotype. 14 d after sciatic lesion, distal axons on semithin sections were structurally preserved, as in WldS (P > 0.05). In whole-mount nerves, many YFP-H–labeled axons retained continuity (Fig. 3 B and Fig. S1). Little but debris remained in wild-type nerves. We then confirmed that lesioned axons and their neuromuscular synapses remained functional for at least 3 d. Evoked action potentials in tibial nerve/flexor digitorum brevis (FDB) preparations provoked robust contractile and electromyographic responses (Fig. 4 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200807175/DC1) that were absent in controls (Video 2). Immunostaining for preserved presynaptic structures of 3-d axotomized neuromuscular junction showed most endplates partially or fully occupied by motor nerve terminals connected to intramuscular motor axons (Fig. 4 B).


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)

Functional motor innervation. (A) ATX3WldS mice show robust preservation of neuromuscular function 3 d after nerve lesion. (i and ii) Isometric single twitch (i) and 20-Hz tetanic tension (ii) responses of isolated FDB to tibial nerve stimulation. (iii and iv) Averaged (16 sweeps) extracellular evoked EMG responses to stimulation of 3-d axotomized FDB from ATX3WldS (iii) and WldS (iv). The x axis represents milliseconds, and the y axis represents millinewtons or microvolts. Bars: (i) 200 ms and 2 mN; (ii) 700 ms and 5 mN; (iii) 6 ms and 200 µV; (iv) 12 ms and 200 µV. (B) Confocal microscopy of axotomized FDB from an ATX3WldS line 5 mouse in which physiological recordings indicated robust preservation of neuromuscular function. Most motor endplates (TRITC–α-bungarotoxin staining; red) were innervated by neurofilament-positive axon collaterals and motor nerve terminals (green), but ∼20% of endplates were unoccupied. (i) Low power z-series projection. (ii and iii) Higher magnification images correspond to the boxed areas in panel i.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Functional motor innervation. (A) ATX3WldS mice show robust preservation of neuromuscular function 3 d after nerve lesion. (i and ii) Isometric single twitch (i) and 20-Hz tetanic tension (ii) responses of isolated FDB to tibial nerve stimulation. (iii and iv) Averaged (16 sweeps) extracellular evoked EMG responses to stimulation of 3-d axotomized FDB from ATX3WldS (iii) and WldS (iv). The x axis represents milliseconds, and the y axis represents millinewtons or microvolts. Bars: (i) 200 ms and 2 mN; (ii) 700 ms and 5 mN; (iii) 6 ms and 200 µV; (iv) 12 ms and 200 µV. (B) Confocal microscopy of axotomized FDB from an ATX3WldS line 5 mouse in which physiological recordings indicated robust preservation of neuromuscular function. Most motor endplates (TRITC–α-bungarotoxin staining; red) were innervated by neurofilament-positive axon collaterals and motor nerve terminals (green), but ∼20% of endplates were unoccupied. (i) Low power z-series projection. (ii and iii) Higher magnification images correspond to the boxed areas in panel i.
Mentions: We then applied more stringent tests to verify that ATX3WldS mice have a full WldS phenotype. 14 d after sciatic lesion, distal axons on semithin sections were structurally preserved, as in WldS (P > 0.05). In whole-mount nerves, many YFP-H–labeled axons retained continuity (Fig. 3 B and Fig. S1). Little but debris remained in wild-type nerves. We then confirmed that lesioned axons and their neuromuscular synapses remained functional for at least 3 d. Evoked action potentials in tibial nerve/flexor digitorum brevis (FDB) preparations provoked robust contractile and electromyographic responses (Fig. 4 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200807175/DC1) that were absent in controls (Video 2). Immunostaining for preserved presynaptic structures of 3-d axotomized neuromuscular junction showed most endplates partially or fully occupied by motor nerve terminals connected to intramuscular motor axons (Fig. 4 B).

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