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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.

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

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APRIL-promoted axonal growth depends on PI-3 kinase activation. (A) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with a plasmid expressing GFP and were treated with 10 μM of LY294002 2 h prior to a further 18 h of incubation with and without APRIL (100 ng/ml) before GFP immunostaining. Separate cultures were treated with APRIL after transfection and control cultures received neither LY294002 nor APRIL. (B) Bar chart of the axonal growth under the experimental conditions illustrated in A. (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with plasmids expressing either GFP alone or GFP together with a dominant-negative form of p85α regulatory subunit of PI-3 kinase (p85α-DN). 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 B and D (** indicates P < 0.001, *** 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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f0030: APRIL-promoted axonal growth depends on PI-3 kinase activation. (A) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with a plasmid expressing GFP and were treated with 10 μM of LY294002 2 h prior to a further 18 h of incubation with and without APRIL (100 ng/ml) before GFP immunostaining. Separate cultures were treated with APRIL after transfection and control cultures received neither LY294002 nor APRIL. (B) Bar chart of the axonal growth under the experimental conditions illustrated in A. (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with plasmids expressing either GFP alone or GFP together with a dominant-negative form of p85α regulatory subunit of PI-3 kinase (p85α-DN). 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 B and D (** indicates P < 0.001, *** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.

Mentions: To test whether PI-3 kinase signaling contributes to APRIL-promoted axon growth, we first examined whether the selective PI-3 kinase inhibitor LY294002 (Vlahos et al., 1994) was able to prevent the APRIL-promoted axon elongation. In these experiments, the neurons were transfected with a GFP plasmid 2 days after plating and were pretreated for 2 h with LY294002 before adding APRIL to the culture medium and quantifying axon length 18 h later. Whereas axon length in cultures that received LY294002 alone was not significantly different from that in control cultures, LY294002 completely prevented the axon growth-enhancing effect of APRIL (Fig. 6A and B). To confirm the results of these pharmacological experiments, we transfected pyramidal cells with a construct that expresses both GFP and a dominant-negative form of the p85α regulatory subunit of PI-3 kinase that lacks the binding domain for the p110 catalytic subunit. The p85α mutant had a small, significant effect on axon extension from neurons not exposed to exogenous APRIL, and completely eliminated the axon growth-enhancing effect of exogenous APRIL (Fig. 6C and D). Taken together, these results suggest that PI3-kinase signaling is required for the effect of APRIL on axon growth.


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-promoted axonal growth depends on PI-3 kinase activation. (A) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with a plasmid expressing GFP and were treated with 10 μM of LY294002 2 h prior to a further 18 h of incubation with and without APRIL (100 ng/ml) before GFP immunostaining. Separate cultures were treated with APRIL after transfection and control cultures received neither LY294002 nor APRIL. (B) Bar chart of the axonal growth under the experimental conditions illustrated in A. (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with plasmids expressing either GFP alone or GFP together with a dominant-negative form of p85α regulatory subunit of PI-3 kinase (p85α-DN). 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 B and D (** indicates P < 0.001, *** 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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f0030: APRIL-promoted axonal growth depends on PI-3 kinase activation. (A) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with a plasmid expressing GFP and were treated with 10 μM of LY294002 2 h prior to a further 18 h of incubation with and without APRIL (100 ng/ml) before GFP immunostaining. Separate cultures were treated with APRIL after transfection and control cultures received neither LY294002 nor APRIL. (B) Bar chart of the axonal growth under the experimental conditions illustrated in A. (C) Images of representative E18 hippocampal pyramidal neurons that were transfected after 2 days in vitro with plasmids expressing either GFP alone or GFP together with a dominant-negative form of p85α regulatory subunit of PI-3 kinase (p85α-DN). 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 B and D (** indicates P < 0.001, *** indicates P < 0.0001 and n.s., non-significant, statistical comparison with control, Mann–Whitney U test). Scale bars = 100 μm.
Mentions: To test whether PI-3 kinase signaling contributes to APRIL-promoted axon growth, we first examined whether the selective PI-3 kinase inhibitor LY294002 (Vlahos et al., 1994) was able to prevent the APRIL-promoted axon elongation. In these experiments, the neurons were transfected with a GFP plasmid 2 days after plating and were pretreated for 2 h with LY294002 before adding APRIL to the culture medium and quantifying axon length 18 h later. Whereas axon length in cultures that received LY294002 alone was not significantly different from that in control cultures, LY294002 completely prevented the axon growth-enhancing effect of APRIL (Fig. 6A and B). To confirm the results of these pharmacological experiments, we transfected pyramidal cells with a construct that expresses both GFP and a dominant-negative form of the p85α regulatory subunit of PI-3 kinase that lacks the binding domain for the p110 catalytic subunit. The p85α mutant had a small, significant effect on axon extension from neurons not exposed to exogenous APRIL, and completely eliminated the axon growth-enhancing effect of exogenous APRIL (Fig. 6C and D). Taken together, these results suggest that PI3-kinase signaling is required for the effect of APRIL on axon growth.

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