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FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption.

Nechipurenko IV, Broihier HT - J. Cell Biol. (2012)

Bottom Line: Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton.This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase.We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.

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Affiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA.

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foxO NMJs have expanded distribution of Ac-Tub staining. (A and C) Representative confocal images of NMJs 6/7 of the indicated genotypes colabeled with anti–Ac-Tub and anti-HRP. Bar, 20 µm. (B and D) Same NMJs as in A and C, respectively, stained for Ac-Tub only. (A′ and B′) Magnified views of boxes in A and B, respectively. At wild-type NMJs, anti–Ac-Tub intensity gradually declines toward terminal boutons (arrowheads). Bar, 40 µm. (C′ and D′) Magnified views of boxes in C and D, respectively. At foxO NMJs, the Ac-Tub signal is prominent in terminal boutons (arrowheads). (E) Quantification of anti–Ac-Tub staining at NMJs 6/7 in listed control and foxO backgrounds. At foxO21/foxO25, foxO21, and foxOΔ94 NMJs, the mean fraction of terminal boutons/NMJ with undetectable Ac-Tub signal is significantly decreased (Kruskal–Wallis, P < 0.0001) and that with strong Ac-Tub signal is significantly increased (Kruskal–Wallis, P < 0.0001) relative to ElavGal4/CS. The mean fractions of terminal boutons/NMJ with undetectable and strong Ac-Tub signal in the above alleles are also statistically different from those in futschK68/+;; foxO21 animals (Kruskal–Wallis, P < 0.05 for undetectable; P < 0.01 for strong). foxO represents foxO21/foxO25. n is the number of NMJs. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig4: foxO NMJs have expanded distribution of Ac-Tub staining. (A and C) Representative confocal images of NMJs 6/7 of the indicated genotypes colabeled with anti–Ac-Tub and anti-HRP. Bar, 20 µm. (B and D) Same NMJs as in A and C, respectively, stained for Ac-Tub only. (A′ and B′) Magnified views of boxes in A and B, respectively. At wild-type NMJs, anti–Ac-Tub intensity gradually declines toward terminal boutons (arrowheads). Bar, 40 µm. (C′ and D′) Magnified views of boxes in C and D, respectively. At foxO NMJs, the Ac-Tub signal is prominent in terminal boutons (arrowheads). (E) Quantification of anti–Ac-Tub staining at NMJs 6/7 in listed control and foxO backgrounds. At foxO21/foxO25, foxO21, and foxOΔ94 NMJs, the mean fraction of terminal boutons/NMJ with undetectable Ac-Tub signal is significantly decreased (Kruskal–Wallis, P < 0.0001) and that with strong Ac-Tub signal is significantly increased (Kruskal–Wallis, P < 0.0001) relative to ElavGal4/CS. The mean fractions of terminal boutons/NMJ with undetectable and strong Ac-Tub signal in the above alleles are also statistically different from those in futschK68/+;; foxO21 animals (Kruskal–Wallis, P < 0.05 for undetectable; P < 0.01 for strong). foxO represents foxO21/foxO25. n is the number of NMJs. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: Acetylation of α-tubulin at lysine 40 is a hallmark of stable neuronal MTs (Fukushima et al., 2009). foxO mutants were stained for acetylated α-tubulin (Ac-Tub) as a direct measure of synaptic MT stability. At a wild-type NMJ, the Ac-Tub signal is intense within the synaptic core and is much fainter or absent within terminal boutons, which contain a more dynamic MT pool (Fig. 4, A–B′; Viquez et al., 2006). Terminal boutons in foxO mutants display prominent anti–Ac-Tub staining (Fig. 4, C–D′). To enable quantification, we scored the proportion of terminal boutons at each NMJ with strong, weak, or undetectable Ac-Tub signals (Fig. 4 E). In foxO21 mutants, the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is 0.53 ± 0.05 compared with 0.14 ± 0.03 in control (Fig. 4 E). foxOΔ94 and foxOΔ2 homozygotes display similarly expanded Ac-Tub distributions, demonstrating that the phenotype is not allele specific (Fig. 4 E and Table S2). Neuronal knockdown of FoxO likewise increased the proportion of terminal boutons/NMJ with strong Ac-Tub signal—the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is 0.35 ± 0.05 in controls compared with 0.56 ± 0.03 in Elav>foxORNAi#1 mutants (Fig. 4 E). These findings support a neuronal role of FoxO in regulating synaptic MT stability. We next tested whether the phenotype is dominantly suppressed by Futsch. Indeed, the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is reduced from 0.53 ± 0.05 in foxO21 homozygotes to 0.27 ± 0.05 in futschK68/+;; foxO21 animals (Fig. 4 E). In concert with the MT looping data, these findings demonstrate that foxO attenuates MT stability.


FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption.

Nechipurenko IV, Broihier HT - J. Cell Biol. (2012)

foxO NMJs have expanded distribution of Ac-Tub staining. (A and C) Representative confocal images of NMJs 6/7 of the indicated genotypes colabeled with anti–Ac-Tub and anti-HRP. Bar, 20 µm. (B and D) Same NMJs as in A and C, respectively, stained for Ac-Tub only. (A′ and B′) Magnified views of boxes in A and B, respectively. At wild-type NMJs, anti–Ac-Tub intensity gradually declines toward terminal boutons (arrowheads). Bar, 40 µm. (C′ and D′) Magnified views of boxes in C and D, respectively. At foxO NMJs, the Ac-Tub signal is prominent in terminal boutons (arrowheads). (E) Quantification of anti–Ac-Tub staining at NMJs 6/7 in listed control and foxO backgrounds. At foxO21/foxO25, foxO21, and foxOΔ94 NMJs, the mean fraction of terminal boutons/NMJ with undetectable Ac-Tub signal is significantly decreased (Kruskal–Wallis, P < 0.0001) and that with strong Ac-Tub signal is significantly increased (Kruskal–Wallis, P < 0.0001) relative to ElavGal4/CS. The mean fractions of terminal boutons/NMJ with undetectable and strong Ac-Tub signal in the above alleles are also statistically different from those in futschK68/+;; foxO21 animals (Kruskal–Wallis, P < 0.05 for undetectable; P < 0.01 for strong). foxO represents foxO21/foxO25. n is the number of NMJs. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig4: foxO NMJs have expanded distribution of Ac-Tub staining. (A and C) Representative confocal images of NMJs 6/7 of the indicated genotypes colabeled with anti–Ac-Tub and anti-HRP. Bar, 20 µm. (B and D) Same NMJs as in A and C, respectively, stained for Ac-Tub only. (A′ and B′) Magnified views of boxes in A and B, respectively. At wild-type NMJs, anti–Ac-Tub intensity gradually declines toward terminal boutons (arrowheads). Bar, 40 µm. (C′ and D′) Magnified views of boxes in C and D, respectively. At foxO NMJs, the Ac-Tub signal is prominent in terminal boutons (arrowheads). (E) Quantification of anti–Ac-Tub staining at NMJs 6/7 in listed control and foxO backgrounds. At foxO21/foxO25, foxO21, and foxOΔ94 NMJs, the mean fraction of terminal boutons/NMJ with undetectable Ac-Tub signal is significantly decreased (Kruskal–Wallis, P < 0.0001) and that with strong Ac-Tub signal is significantly increased (Kruskal–Wallis, P < 0.0001) relative to ElavGal4/CS. The mean fractions of terminal boutons/NMJ with undetectable and strong Ac-Tub signal in the above alleles are also statistically different from those in futschK68/+;; foxO21 animals (Kruskal–Wallis, P < 0.05 for undetectable; P < 0.01 for strong). foxO represents foxO21/foxO25. n is the number of NMJs. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: Acetylation of α-tubulin at lysine 40 is a hallmark of stable neuronal MTs (Fukushima et al., 2009). foxO mutants were stained for acetylated α-tubulin (Ac-Tub) as a direct measure of synaptic MT stability. At a wild-type NMJ, the Ac-Tub signal is intense within the synaptic core and is much fainter or absent within terminal boutons, which contain a more dynamic MT pool (Fig. 4, A–B′; Viquez et al., 2006). Terminal boutons in foxO mutants display prominent anti–Ac-Tub staining (Fig. 4, C–D′). To enable quantification, we scored the proportion of terminal boutons at each NMJ with strong, weak, or undetectable Ac-Tub signals (Fig. 4 E). In foxO21 mutants, the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is 0.53 ± 0.05 compared with 0.14 ± 0.03 in control (Fig. 4 E). foxOΔ94 and foxOΔ2 homozygotes display similarly expanded Ac-Tub distributions, demonstrating that the phenotype is not allele specific (Fig. 4 E and Table S2). Neuronal knockdown of FoxO likewise increased the proportion of terminal boutons/NMJ with strong Ac-Tub signal—the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is 0.35 ± 0.05 in controls compared with 0.56 ± 0.03 in Elav>foxORNAi#1 mutants (Fig. 4 E). These findings support a neuronal role of FoxO in regulating synaptic MT stability. We next tested whether the phenotype is dominantly suppressed by Futsch. Indeed, the mean fraction of terminal boutons/NMJ with strong anti–Ac-Tub staining is reduced from 0.53 ± 0.05 in foxO21 homozygotes to 0.27 ± 0.05 in futschK68/+;; foxO21 animals (Fig. 4 E). In concert with the MT looping data, these findings demonstrate that foxO attenuates MT stability.

Bottom Line: Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton.This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase.We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA.

Show MeSH
Related in: MedlinePlus