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Neuritin Up-regulates Kv4.2 α -Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway *

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ABSTRACT

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4−/− mice but not in Nfatc2−/− mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4−/− mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.

No MeSH data available.


Neuritin induces dephosphorylation and nuclear accumulation of NFATc4.A, the levels of NFATc4 Ser168 and Ser170 phosphorylation (p-NFATc4) after neuritin treatment for 20 min in mice CGNs were examined by Western blotting. B and C, representative recording sample and statistical analysis showing effect of neuritin on nuclear accumulation of NFATc4 was examined by confocal microscopy in HeLa cells, which expressed IR activated by neuritin. The effects of CaN inhibitor CsA and IR inhibitor HNMPA are also shown. The percentage of nuclear NFATc4 in 100 cells was counted and presented. D, effect of neuritin on nuclear NFATc4 protein in mice CGNs by Western blotting analysis. E, effect of neuritin on cytoplasmic NFATc4 protein in mice CGNs by Western blotting analysis. *, p < 0.05; and ***, p < 0.001 for two groups connected with a straight line by one-way ANOVA followed by Fisher's post hoc test.
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Figure 3: Neuritin induces dephosphorylation and nuclear accumulation of NFATc4.A, the levels of NFATc4 Ser168 and Ser170 phosphorylation (p-NFATc4) after neuritin treatment for 20 min in mice CGNs were examined by Western blotting. B and C, representative recording sample and statistical analysis showing effect of neuritin on nuclear accumulation of NFATc4 was examined by confocal microscopy in HeLa cells, which expressed IR activated by neuritin. The effects of CaN inhibitor CsA and IR inhibitor HNMPA are also shown. The percentage of nuclear NFATc4 in 100 cells was counted and presented. D, effect of neuritin on nuclear NFATc4 protein in mice CGNs by Western blotting analysis. E, effect of neuritin on cytoplasmic NFATc4 protein in mice CGNs by Western blotting analysis. *, p < 0.05; and ***, p < 0.001 for two groups connected with a straight line by one-way ANOVA followed by Fisher's post hoc test.

Mentions: CaN dephosphorylates key Ser residues in the NFAT homology domain of NFATc4, including Ser168 and Ser170, and facilitates nuclear accumulation of NFATc4 (21). Thus, we examined the phosphorylation status and nuclear localization of NFATc4 upon neuritin stimulation in mice CGNs. After pretreatment of CGNs with neuritin for 20 min, phosphorylation on Ser168 and Ser170 of NFATc4 was significantly reduced by 22.12 ± 8.42% (n = 3, p < 0.05) compared with control cells (Fig. 3A). Meanwhile, either CaN inhibition or blocking neuritin signaling via the IR inhibitor hydroxy-2-naphthalenylmethyl phosphonic acid (HNMPA) (24, 25) abolished dephosphorylation of NFATc4 induced by neuritin. In the presence of 5 μm CsA or 100 μm HNMPA, phosphorylation on Ser168 and Ser170 of NFATc4 compared with controls was not significantly reduced by only 1.88 ± 5.00% (n = 3) and −4.80 ± 4.46% (n = 3), respectively (Fig. 3A). These data indicate that activation of CaN by neuritin promotes dephosphorylation of NFATc4.


Neuritin Up-regulates Kv4.2 α -Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway *
Neuritin induces dephosphorylation and nuclear accumulation of NFATc4.A, the levels of NFATc4 Ser168 and Ser170 phosphorylation (p-NFATc4) after neuritin treatment for 20 min in mice CGNs were examined by Western blotting. B and C, representative recording sample and statistical analysis showing effect of neuritin on nuclear accumulation of NFATc4 was examined by confocal microscopy in HeLa cells, which expressed IR activated by neuritin. The effects of CaN inhibitor CsA and IR inhibitor HNMPA are also shown. The percentage of nuclear NFATc4 in 100 cells was counted and presented. D, effect of neuritin on nuclear NFATc4 protein in mice CGNs by Western blotting analysis. E, effect of neuritin on cytoplasmic NFATc4 protein in mice CGNs by Western blotting analysis. *, p < 0.05; and ***, p < 0.001 for two groups connected with a straight line by one-way ANOVA followed by Fisher's post hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Neuritin induces dephosphorylation and nuclear accumulation of NFATc4.A, the levels of NFATc4 Ser168 and Ser170 phosphorylation (p-NFATc4) after neuritin treatment for 20 min in mice CGNs were examined by Western blotting. B and C, representative recording sample and statistical analysis showing effect of neuritin on nuclear accumulation of NFATc4 was examined by confocal microscopy in HeLa cells, which expressed IR activated by neuritin. The effects of CaN inhibitor CsA and IR inhibitor HNMPA are also shown. The percentage of nuclear NFATc4 in 100 cells was counted and presented. D, effect of neuritin on nuclear NFATc4 protein in mice CGNs by Western blotting analysis. E, effect of neuritin on cytoplasmic NFATc4 protein in mice CGNs by Western blotting analysis. *, p < 0.05; and ***, p < 0.001 for two groups connected with a straight line by one-way ANOVA followed by Fisher's post hoc test.
Mentions: CaN dephosphorylates key Ser residues in the NFAT homology domain of NFATc4, including Ser168 and Ser170, and facilitates nuclear accumulation of NFATc4 (21). Thus, we examined the phosphorylation status and nuclear localization of NFATc4 upon neuritin stimulation in mice CGNs. After pretreatment of CGNs with neuritin for 20 min, phosphorylation on Ser168 and Ser170 of NFATc4 was significantly reduced by 22.12 ± 8.42% (n = 3, p < 0.05) compared with control cells (Fig. 3A). Meanwhile, either CaN inhibition or blocking neuritin signaling via the IR inhibitor hydroxy-2-naphthalenylmethyl phosphonic acid (HNMPA) (24, 25) abolished dephosphorylation of NFATc4 induced by neuritin. In the presence of 5 μm CsA or 100 μm HNMPA, phosphorylation on Ser168 and Ser170 of NFATc4 compared with controls was not significantly reduced by only 1.88 ± 5.00% (n = 3) and −4.80 ± 4.46% (n = 3), respectively (Fig. 3A). These data indicate that activation of CaN by neuritin promotes dephosphorylation of NFATc4.

View Article: PubMed Central - PubMed

ABSTRACT

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4&minus;/&minus; mice but not in Nfatc2&minus;/&minus; mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4&minus;/&minus; mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.

No MeSH data available.