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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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Sir2 is not required for WldS-mediated protection of severed axons, and membrane-tethered WldS fails to suppress axon degeneration. (A) UAS-Sir2RNAi was driven in OR22a+ ORNs with OR22a-Gal4 in the presence or absence of WldS, and axons were assayed 15 d after injury. (B) The requirements for Sir2 in WldS-mediated axon protection were assayed 15 d after injury in WldS-expressing axons and WldS axons that lacked sir2 (sir24.5/sir25.26). WldS, n = 10; Sir2−/− + WldS, n = 6. Error bars represent SEM.
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fig6: Sir2 is not required for WldS-mediated protection of severed axons, and membrane-tethered WldS fails to suppress axon degeneration. (A) UAS-Sir2RNAi was driven in OR22a+ ORNs with OR22a-Gal4 in the presence or absence of WldS, and axons were assayed 15 d after injury. (B) The requirements for Sir2 in WldS-mediated axon protection were assayed 15 d after injury in WldS-expressing axons and WldS axons that lacked sir2 (sir24.5/sir25.26). WldS, n = 10; Sir2−/− + WldS, n = 6. Error bars represent SEM.

Mentions: In Drosophila, there is a single gene that bears strong sequence similarity to the mammalian sirtuins, Sir2; Sir2 is 55.9% identical to the Sirt1 at the amino acid level. The simplicity of the Drosophila sirtuin family offers the opportunity to directly reassess the requirements for sirtuins in WldS function in vivo. We first assayed the requirements for Drosophila Sir2 in WldS-mediated protection of severed axons using a UAS-regulated RNAi construct targeted to Sir2. We found that expression of Sir2RNAi in axons did not induce spontaneous degeneration and that ORN axons appeared to develop normally (unpublished data). Axotomized Sir2RNAi-expressing axons exhibited normal axon degeneration, arguing that Sir2 function is not required for Wallerian degeneration (Fig. 6 A). Moreover, expression of Sir2RNAi in WldS-expressing axons did not suppress the ability of WldS to protect severed axons (Fig. 6 A). Next, we confirmed our observation with Sir2RNAi genetically by crossing WldS into a Sir2- mutant background. Loss of function alleles of Sir2 are not available; however, removal of the Sir2 gene can be accomplished using a heteroallelic combination of the two partially overlapping deletions in sir25.26 and sir24.5 mutants (Newman et al., 2002). As with our Sir2RNAi experiments, we found that deletion of the Sir2 gene in a WldS background failed to suppress the ability of WldS to protect severed axons from degeneration (Fig. 6 B). These data argue strongly that WldS does not require the NAD+-binding histone deacetylase Sir2 to protect severed axons from autodestruction.


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)

Sir2 is not required for WldS-mediated protection of severed axons, and membrane-tethered WldS fails to suppress axon degeneration. (A) UAS-Sir2RNAi was driven in OR22a+ ORNs with OR22a-Gal4 in the presence or absence of WldS, and axons were assayed 15 d after injury. (B) The requirements for Sir2 in WldS-mediated axon protection were assayed 15 d after injury in WldS-expressing axons and WldS axons that lacked sir2 (sir24.5/sir25.26). WldS, n = 10; Sir2−/− + WldS, n = 6. Error bars represent SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Sir2 is not required for WldS-mediated protection of severed axons, and membrane-tethered WldS fails to suppress axon degeneration. (A) UAS-Sir2RNAi was driven in OR22a+ ORNs with OR22a-Gal4 in the presence or absence of WldS, and axons were assayed 15 d after injury. (B) The requirements for Sir2 in WldS-mediated axon protection were assayed 15 d after injury in WldS-expressing axons and WldS axons that lacked sir2 (sir24.5/sir25.26). WldS, n = 10; Sir2−/− + WldS, n = 6. Error bars represent SEM.
Mentions: In Drosophila, there is a single gene that bears strong sequence similarity to the mammalian sirtuins, Sir2; Sir2 is 55.9% identical to the Sirt1 at the amino acid level. The simplicity of the Drosophila sirtuin family offers the opportunity to directly reassess the requirements for sirtuins in WldS function in vivo. We first assayed the requirements for Drosophila Sir2 in WldS-mediated protection of severed axons using a UAS-regulated RNAi construct targeted to Sir2. We found that expression of Sir2RNAi in axons did not induce spontaneous degeneration and that ORN axons appeared to develop normally (unpublished data). Axotomized Sir2RNAi-expressing axons exhibited normal axon degeneration, arguing that Sir2 function is not required for Wallerian degeneration (Fig. 6 A). Moreover, expression of Sir2RNAi in WldS-expressing axons did not suppress the ability of WldS to protect severed axons (Fig. 6 A). Next, we confirmed our observation with Sir2RNAi genetically by crossing WldS into a Sir2- mutant background. Loss of function alleles of Sir2 are not available; however, removal of the Sir2 gene can be accomplished using a heteroallelic combination of the two partially overlapping deletions in sir25.26 and sir24.5 mutants (Newman et al., 2002). As with our Sir2RNAi experiments, we found that deletion of the Sir2 gene in a WldS background failed to suppress the ability of WldS to protect severed axons from degeneration (Fig. 6 B). These data argue strongly that WldS does not require the NAD+-binding histone deacetylase Sir2 to protect severed axons from autodestruction.

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