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Wld S requires Nmnat1 enzymatic activity and N16-VCP interactions to suppress Wallerian degeneration.

Avery MA, Sheehan AE, Kerr KS, Wang J, Freeman MR - J. Cell Biol. (2009)

Bottom Line: We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than Wld(S), and enzyme-dead versions of Nmnat1 and Wld(S) exhibit severely reduced axon-protective function.Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from Wld(S).Thus, nuclear Nmnat activity does not appear to be essential for Wld(S)-like axon protection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Slow Wallerian degeneration (Wld(S)) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of Wld(S) remains controversial. In this study, we characterize Wld(S)-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than Wld(S), and enzyme-dead versions of Nmnat1 and Wld(S) exhibit severely reduced axon-protective function. Interestingly, a 16-amino acid N-terminal domain of Wld(S) (termed N16) accounts for the differences in axon-sparing activity between Wld(S) and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in Wld(S) requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, Wld(S)-mediated suppression of Wallerian degeneration results from VCP-N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from Wld(S). Thus, nuclear Nmnat activity does not appear to be essential for Wld(S)-like axon protection.

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Related in: MedlinePlus

WldS variant proteins are stably expressed in Drosophila neurons. Three independent UAS-driven transgene insertion lines for each WldS variant molecule were crossed to elav-Gal4, and protein extracts from heads were assayed for expression using α-Nmnat1 antibodies. Multiple lines for each construct were found to express detectable levels of protein. The expression of each of these molecules in ORNs with OR22a-Gal4 did not lead to expression of proteins at levels detectable by Western blot analysis (not depicted). Molecular masses are shown in kilodaltons. MW, molecular weight.
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fig2: WldS variant proteins are stably expressed in Drosophila neurons. Three independent UAS-driven transgene insertion lines for each WldS variant molecule were crossed to elav-Gal4, and protein extracts from heads were assayed for expression using α-Nmnat1 antibodies. Multiple lines for each construct were found to express detectable levels of protein. The expression of each of these molecules in ORNs with OR22a-Gal4 did not lead to expression of proteins at levels detectable by Western blot analysis (not depicted). Molecular masses are shown in kilodaltons. MW, molecular weight.

Mentions: Multiple transgenic lines were established for each construct, and at least three lines bearing independently isolated transgene insertions were assayed for protein expression levels (Fig. 2) and functional protection (see following section). The efficiency of protein expression from each transgene was assayed by crossing individual UAS lines to the panneuronal driver elav-Gal4, preparing protein extracts from head, and assaying protein levels with α-Nmnat1 antibodies on Western blots. All WldS variants carry full-length Nmnat1, and thus, this antibody should recognize each with equal affinity. In some cases, each of the three transgenic lines for a given construct expressed very similar levels of protein (e.g., N16-Nmnat1 and WldS-ΔN16); for others, the total expression level of each protein varied among transgenic lines (e.g., WldS, Nmnat1, Nmnat1dead, and WldS-dead). Despite this fact, the in vivo axon-protective function of each molecule was in most cases strikingly similar among transgenic lines carrying different insertions of the same transgene (see following section).


Wld S requires Nmnat1 enzymatic activity and N16-VCP interactions to suppress Wallerian degeneration.

Avery MA, Sheehan AE, Kerr KS, Wang J, Freeman MR - J. Cell Biol. (2009)

WldS variant proteins are stably expressed in Drosophila neurons. Three independent UAS-driven transgene insertion lines for each WldS variant molecule were crossed to elav-Gal4, and protein extracts from heads were assayed for expression using α-Nmnat1 antibodies. Multiple lines for each construct were found to express detectable levels of protein. The expression of each of these molecules in ORNs with OR22a-Gal4 did not lead to expression of proteins at levels detectable by Western blot analysis (not depicted). Molecular masses are shown in kilodaltons. MW, molecular weight.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654119&req=5

fig2: WldS variant proteins are stably expressed in Drosophila neurons. Three independent UAS-driven transgene insertion lines for each WldS variant molecule were crossed to elav-Gal4, and protein extracts from heads were assayed for expression using α-Nmnat1 antibodies. Multiple lines for each construct were found to express detectable levels of protein. The expression of each of these molecules in ORNs with OR22a-Gal4 did not lead to expression of proteins at levels detectable by Western blot analysis (not depicted). Molecular masses are shown in kilodaltons. MW, molecular weight.
Mentions: Multiple transgenic lines were established for each construct, and at least three lines bearing independently isolated transgene insertions were assayed for protein expression levels (Fig. 2) and functional protection (see following section). The efficiency of protein expression from each transgene was assayed by crossing individual UAS lines to the panneuronal driver elav-Gal4, preparing protein extracts from head, and assaying protein levels with α-Nmnat1 antibodies on Western blots. All WldS variants carry full-length Nmnat1, and thus, this antibody should recognize each with equal affinity. In some cases, each of the three transgenic lines for a given construct expressed very similar levels of protein (e.g., N16-Nmnat1 and WldS-ΔN16); for others, the total expression level of each protein varied among transgenic lines (e.g., WldS, Nmnat1, Nmnat1dead, and WldS-dead). Despite this fact, the in vivo axon-protective function of each molecule was in most cases strikingly similar among transgenic lines carrying different insertions of the same transgene (see following section).

Bottom Line: We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than Wld(S), and enzyme-dead versions of Nmnat1 and Wld(S) exhibit severely reduced axon-protective function.Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from Wld(S).Thus, nuclear Nmnat activity does not appear to be essential for Wld(S)-like axon protection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Slow Wallerian degeneration (Wld(S)) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of Wld(S) remains controversial. In this study, we characterize Wld(S)-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than Wld(S), and enzyme-dead versions of Nmnat1 and Wld(S) exhibit severely reduced axon-protective function. Interestingly, a 16-amino acid N-terminal domain of Wld(S) (termed N16) accounts for the differences in axon-sparing activity between Wld(S) and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in Wld(S) requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, Wld(S)-mediated suppression of Wallerian degeneration results from VCP-N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from Wld(S). Thus, nuclear Nmnat activity does not appear to be essential for Wld(S)-like axon protection.

Show MeSH
Related in: MedlinePlus