Acid Sensing Ion Channels (ASICs) in NS20Y cells – potential role in neuronal differentiation

Abstract

Cultured neuronal cell lines can express properties of mature neurons if properly differentiated. Although the precise mechanisms underlying neuronal differentiation are not fully understood, the expression and activation of ion channels, particularly those of Ca2+-permeable channels, have been suggested to play a role. In this study, we explored the presence and characterized the properties of acid-sensing ion channels (ASICs) in NS20Y cells, a neuronal cell line previously used for the study of neuronal differentiation. In addition, the potential role of ASICs in cell differentiation was explored. Reverse Transcription Polymerase Chain Reaction and Western blot revealed the presence of ASIC1 subunits in these cells. Fast drops of extracellular pH activated transient inward currents which were blocked, in a dose dependent manner, by amiloride, a non-selective ASIC blocker, and by Psalmotoxin-1 (PcTX1), a specific inhibitor for homomeric ASIC1a and heteromeric ASIC1a/2b channels. Incubation of cells with PcTX1 significantly reduced the differentiation of NS20Y cells induced by cpt-cAMP, as evidenced by decreased neurite length, dendritic complexity, decreased expression of functional voltage gated Na+ channels. Consistent with ASIC1a inhibition, ASIC1a knockdown with small interference RNA significantly attenuates cpt-cAMP-induced increase of neurite outgrowth. In summary, we described the presence of functional ASICs in NS20Y cells and demonstrate that ASIC1a plays a role in the differentiation of these cells.

Large Acid-Evoked Currents, Mediated by ASIC1a, Accompany Differentiation in Human Dopaminergic Neurons

“…Transient activation of ASICs, thus, modulates synaptic transmission. In addition, ASIC1 was shown to modulate the density of dendritic spines (Zha et al, 2006 ) and neuronal differentiation in vitro (O’Bryant et al, 2016 ). Prolonged activation of ASICs occurs in the course of ischemic stroke or inflammation and contributes to cell death in these conditions (Friese et al, 2007 ; Xiong et al, 2012 ; Wang et al, 2015 ; Chassagnon et al, 2017 ).…”

ASIC1 promotes differentiation of neuroblastoma by negatively regulating Notch signaling pathway

“…NS20Y cell line, a mouse cholinergic neuroblastoma, was commonly used for determining neurite growth [ 25 – 27 ]. The NS20Y was adapted to undifferentiated growth in suspension culture while underwent differentiation by transferred to surface culture and treated with a variety of reagents including 8-(4-chlorophenylthio) adenosine 3’,5’-cyclic monophosphate (8-CPT-cAMP or CPT-cAMP) [ 28 – 29 ], retinoic acid or serum [ 30 – 32 ]. The NS20Y cell differentiation has crucial features which have been seen in normal neuronal development providing an appropriate model for investigating neuronal development.…”

Acute Ethanol Exposure Promotes Autophagy-Lysosome Pathway-Dependent ASIC1a Protein Degradation and Protects Against Acidosis-Induced Neurotoxicity

“…Protein isolation and Western blot analysis were performed as described previously [27,29]. Briefly, cells were treated with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor and phosphatase inhibitor cocktail).…”

“…1A and B, changing extracellular pH from 7.4 to 6.0 induced large transient inward currents. Amiloride (100 μM), a commonly used nonspecific inhibitor of ASICs [32,33,27], potently inhibited the acid induced currents. Perfusion of synthetic psalmotoxin-1 (PcTX1, 20 nM for 10 min), a spider toxin from the venom of tarantula Psalmopoeus cambridgei that specifically blocks the current mediated by homomeric ASIC1a and heteromeric ASIC1a/2b channels [34,32], also significantly reduced the amplitude of ASIC currents in NS20Y cells (Fig.…”

“…While alcohol abuse and addiction is a serious problem, a number of studies have shown that light to moderate alcohol intake is associated with a decreased risk of ischemic stroke [1][2][3][4]. cells, derived from the mouse neuroblastoma, were cultured in Dulbecco's Modified Eagle's Medium (Invitrogen), supplemented with 10% fetal bovine serum and antibiotics [27].…”