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Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL.

Osório C, Chacón PJ, White M, Kisiswa L, Wyatt S, Rodríguez-Tébar A, Davies AM - Mol. Cell. Neurosci. (2014)

Bottom Line: In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation.Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation.The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AT Wales, United Kingdom.

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APRIL promotes axonal growth by activating GSK-3β signaling. (A) Representative Western blots probed for phospho-GSK-3α, phospho-GSK-3β and total GSK-3β in lysates of E18 hippocampal neurons treated with 100 ng/ml APRIL for the times indicated after first culturing the neurons for 3 days. (B) Densitometric quantification of levels of phospho-GSK-3β relative to total GSK-3β from 3 separate Western blot experiments (mean ± s.e.m., *** indicates P < 0.0001, statistical comparison with control, Mann–Whitney U test). (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro plasmids expressing either GFP alone or GFP together with the GSK3β S9A mutant. After transfection, the neurons were cultured for a further 18 h with and without APRIL (100 ng/ml) before GFP immunostaining. (D) Bar chart of the axonal growth under the experimental conditions illustrated in C. The mean ± s.e.m. of data obtained from > 150 neurons per condition compiled from at least three separate experiments are shown in D (*** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.
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f0040: APRIL promotes axonal growth by activating GSK-3β signaling. (A) Representative Western blots probed for phospho-GSK-3α, phospho-GSK-3β and total GSK-3β in lysates of E18 hippocampal neurons treated with 100 ng/ml APRIL for the times indicated after first culturing the neurons for 3 days. (B) Densitometric quantification of levels of phospho-GSK-3β relative to total GSK-3β from 3 separate Western blot experiments (mean ± s.e.m., *** indicates P < 0.0001, statistical comparison with control, Mann–Whitney U test). (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro plasmids expressing either GFP alone or GFP together with the GSK3β S9A mutant. After transfection, the neurons were cultured for a further 18 h with and without APRIL (100 ng/ml) before GFP immunostaining. (D) Bar chart of the axonal growth under the experimental conditions illustrated in C. The mean ± s.e.m. of data obtained from > 150 neurons per condition compiled from at least three separate experiments are shown in D (*** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.

Mentions: Glycogen synthase kinase-3 (GSK-3) proteins are among the many substrates of Akt kinases, and have been implicated in the regulation of multiple aspects of neural development (Hur and Zhou, 2010), including the control of axon growth from developing sensory and hippocampal neurons (Kim et al., 2006; Zhou et al., 2004). The two isoforms of GSK-3, GSK-3α and GSK-3β, can be phosphorylated on serine-21 and serine-9, respectively, by Akt kinases (Cross et al., 1995). This results in inactivation of GSK-3, and several studies suggest that inactivated GSK-3 promotes axon elongation from cultured sensory and hippocampal neurons by regulating the molecular machinery that controls actin rearrangement and microtubule assembly (Kim et al., 2006; Kumar et al., 2009; Namekata et al., 2012; Yoshimura et al., 2005; Zhou et al., 2004). To determine if treating hippocampal neurons with APRIL leads to GSK-3 phosphorylation, we cultured E18 pyramidal cells for 3 days before treating them with APRIL. Western blot analysis revealed that APRIL caused a rapid increase in the phosphorylation of phospho-S9 GSK-3β that was sustained for at least 60 min (Fig. 8A and B), but caused no change in the level of phospho-S21 GSK-3α.


Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL.

Osório C, Chacón PJ, White M, Kisiswa L, Wyatt S, Rodríguez-Tébar A, Davies AM - Mol. Cell. Neurosci. (2014)

APRIL promotes axonal growth by activating GSK-3β signaling. (A) Representative Western blots probed for phospho-GSK-3α, phospho-GSK-3β and total GSK-3β in lysates of E18 hippocampal neurons treated with 100 ng/ml APRIL for the times indicated after first culturing the neurons for 3 days. (B) Densitometric quantification of levels of phospho-GSK-3β relative to total GSK-3β from 3 separate Western blot experiments (mean ± s.e.m., *** indicates P < 0.0001, statistical comparison with control, Mann–Whitney U test). (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro plasmids expressing either GFP alone or GFP together with the GSK3β S9A mutant. After transfection, the neurons were cultured for a further 18 h with and without APRIL (100 ng/ml) before GFP immunostaining. (D) Bar chart of the axonal growth under the experimental conditions illustrated in C. The mean ± s.e.m. of data obtained from > 150 neurons per condition compiled from at least three separate experiments are shown in D (*** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.
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f0040: APRIL promotes axonal growth by activating GSK-3β signaling. (A) Representative Western blots probed for phospho-GSK-3α, phospho-GSK-3β and total GSK-3β in lysates of E18 hippocampal neurons treated with 100 ng/ml APRIL for the times indicated after first culturing the neurons for 3 days. (B) Densitometric quantification of levels of phospho-GSK-3β relative to total GSK-3β from 3 separate Western blot experiments (mean ± s.e.m., *** indicates P < 0.0001, statistical comparison with control, Mann–Whitney U test). (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro plasmids expressing either GFP alone or GFP together with the GSK3β S9A mutant. After transfection, the neurons were cultured for a further 18 h with and without APRIL (100 ng/ml) before GFP immunostaining. (D) Bar chart of the axonal growth under the experimental conditions illustrated in C. The mean ± s.e.m. of data obtained from > 150 neurons per condition compiled from at least three separate experiments are shown in D (*** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.
Mentions: Glycogen synthase kinase-3 (GSK-3) proteins are among the many substrates of Akt kinases, and have been implicated in the regulation of multiple aspects of neural development (Hur and Zhou, 2010), including the control of axon growth from developing sensory and hippocampal neurons (Kim et al., 2006; Zhou et al., 2004). The two isoforms of GSK-3, GSK-3α and GSK-3β, can be phosphorylated on serine-21 and serine-9, respectively, by Akt kinases (Cross et al., 1995). This results in inactivation of GSK-3, and several studies suggest that inactivated GSK-3 promotes axon elongation from cultured sensory and hippocampal neurons by regulating the molecular machinery that controls actin rearrangement and microtubule assembly (Kim et al., 2006; Kumar et al., 2009; Namekata et al., 2012; Yoshimura et al., 2005; Zhou et al., 2004). To determine if treating hippocampal neurons with APRIL leads to GSK-3 phosphorylation, we cultured E18 pyramidal cells for 3 days before treating them with APRIL. Western blot analysis revealed that APRIL caused a rapid increase in the phosphorylation of phospho-S9 GSK-3β that was sustained for at least 60 min (Fig. 8A and B), but caused no change in the level of phospho-S21 GSK-3α.

Bottom Line: In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation.Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation.The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AT Wales, United Kingdom.

Show MeSH
Related in: MedlinePlus