Nuclear envelope (NE) is a Ca2+-storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+]n). However, how [Ca2+]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+/Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+]n-dependent PTEN/PI3K/Akt pathway.

Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation / Secondo, A; Esposito, A; Petrozziello, T; Boscia, F; Molinaro, P; Tedeschi, V; Pannaccione, Anna; Ciccone, R; Guida, N; Di Renzo, G; Annunziato, L. - In: CELL DEATH DISCOVERY. - ISSN 2058-7716. - 4:(2018), pp. 12-18. [doi: 10.1038/s41420-017-0018-1]

Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation

Secondo A
Conceptualization
;
Petrozziello T
Membro del Collaboration Group
;
Boscia F
Membro del Collaboration Group
;
Molinaro P
Membro del Collaboration Group
;
Tedeschi V
Membro del Collaboration Group
;
Pannaccione A
Membro del Collaboration Group
;
Ciccone R
Membro del Collaboration Group
;
Guida N
Conceptualization
;
Di Renzo G
Conceptualization
;
Annunziato L
2018

Abstract

Nuclear envelope (NE) is a Ca2+-storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+]n). However, how [Ca2+]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+/Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+]n-dependent PTEN/PI3K/Akt pathway.
2018
Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation / Secondo, A; Esposito, A; Petrozziello, T; Boscia, F; Molinaro, P; Tedeschi, V; Pannaccione, Anna; Ciccone, R; Guida, N; Di Renzo, G; Annunziato, L. - In: CELL DEATH DISCOVERY. - ISSN 2058-7716. - 4:(2018), pp. 12-18. [doi: 10.1038/s41420-017-0018-1]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/707953
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