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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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WldS requires N70 and Nmnat1 biosynthetic activity for maximal protection of severed axons in vivo. UAS-regulated versions of WldS variants were expressed in OR22a+ ORNs with OR22a-Gal4, axons were severed, and the number of remaining intact GFP+ axons was scored at the time points indicated. Two to four independent insertion lines were tested for each UAS-regulated WldS variant molecule (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1); n ≥ 10 for individual lines; data were subsequently pooled and are presented here. For Nmnat1, **, P < 0.01 and ***, P < 0.001 (Nmnat1 vs. WldS at corresponding time points). For WldS, **, P < 0.01 (day 0 vs. day 30). Error bars represent SEM.
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fig3: WldS requires N70 and Nmnat1 biosynthetic activity for maximal protection of severed axons in vivo. UAS-regulated versions of WldS variants were expressed in OR22a+ ORNs with OR22a-Gal4, axons were severed, and the number of remaining intact GFP+ axons was scored at the time points indicated. Two to four independent insertion lines were tested for each UAS-regulated WldS variant molecule (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1); n ≥ 10 for individual lines; data were subsequently pooled and are presented here. For Nmnat1, **, P < 0.01 and ***, P < 0.001 (Nmnat1 vs. WldS at corresponding time points). For WldS, **, P < 0.01 (day 0 vs. day 30). Error bars represent SEM.

Mentions: To compare the protective effects of the aforementioned transgenes, we crossed each to the ORN-specific driver OR22a-Gal4, severed ORN axons, and determined the number of remaining intact axonal fibers present at 5, 10, 15, 20, and 30 d after injury (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1). Each neuroprotective molecule showed a unique pattern of protection, but this was indistinguishable among the other transgenic lines carrying the same transgene (Fig. S2), and therefore, axon protection data were pooled for each transgene (Fig. 3).


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 requires N70 and Nmnat1 biosynthetic activity for maximal protection of severed axons in vivo. UAS-regulated versions of WldS variants were expressed in OR22a+ ORNs with OR22a-Gal4, axons were severed, and the number of remaining intact GFP+ axons was scored at the time points indicated. Two to four independent insertion lines were tested for each UAS-regulated WldS variant molecule (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1); n ≥ 10 for individual lines; data were subsequently pooled and are presented here. For Nmnat1, **, P < 0.01 and ***, P < 0.001 (Nmnat1 vs. WldS at corresponding time points). For WldS, **, P < 0.01 (day 0 vs. day 30). Error bars represent SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: WldS requires N70 and Nmnat1 biosynthetic activity for maximal protection of severed axons in vivo. UAS-regulated versions of WldS variants were expressed in OR22a+ ORNs with OR22a-Gal4, axons were severed, and the number of remaining intact GFP+ axons was scored at the time points indicated. Two to four independent insertion lines were tested for each UAS-regulated WldS variant molecule (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1); n ≥ 10 for individual lines; data were subsequently pooled and are presented here. For Nmnat1, **, P < 0.01 and ***, P < 0.001 (Nmnat1 vs. WldS at corresponding time points). For WldS, **, P < 0.01 (day 0 vs. day 30). Error bars represent SEM.
Mentions: To compare the protective effects of the aforementioned transgenes, we crossed each to the ORN-specific driver OR22a-Gal4, severed ORN axons, and determined the number of remaining intact axonal fibers present at 5, 10, 15, 20, and 30 d after injury (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200808042/DC1). Each neuroprotective molecule showed a unique pattern of protection, but this was indistinguishable among the other transgenic lines carrying the same transgene (Fig. S2), and therefore, axon protection data were pooled for each transgene (Fig. 3).

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