Background
Neuronal differentiation is essential for the development of the nervous system. A hallmark characteristic of differentiation is the sprouting of neurites which later become axons and dendrites. Major changes in membrane proteins are observed during the differentiation, maturation, and development of neurons, for example increased expression of acid-sensing ion channels (ASICs) [
1]. Although the precise mechanisms underlying neuronal differentiation are not fully understood, expression and activation of ion channels, particularly those which are Ca
2+-permeable, have been suggested to play an important role in the process [
2‐
4].
ASICs are proton-gated cation channels belonging to the degenerin/epithelial Na
+ channel (DEG/ENaC) superfamily. There are at least four genes that encode six alternatively spliced transcripts: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. ASIC1a, a primary subunit highly expressed in the central and peripheral neurons, is highly sensitive to decrease in extracellular pH [
5]. Studies using knockout mice have suggested that activation of ASIC1a contributes to synaptic plasticity, learning and memory [
6]. It is unclear however whether ASICs play any role in neuronal differentiation. In this study, we first explored the presence and characterized the properties of ASICs in NS20Y cells, a neuronal cell line that has been previously used to study neuronal differentiation. Next, we determined whether ASIC inhibition affects the differentiation of these cells. Our data provides strong evidence that functional ASIC1a channels are expressed in NS20Y cells and that activation of these channels may play a role in neuronal differentiation.
Methods
Cell culture
NS20Y cells, derived from the mouse neuroblastoma, were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen), supplemented with 10 % FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were plated at 10 - 20 % confluence on 35 mm dishes coated with poly-L-ornithine and maintained at 37 °C in a humidified incubator with 5 % CO2 - 95 % atmosphere. For differentiation, cells were treated with 1 mM cpt-cAMP for 72 h or 1 mM cpt-cAMP and 10 nM PcTX1 by adding reagents directly to cell media. The culture medium was not changed during the 72 h treatment. The pH during experiments were 7.62 in control, 7.63 in cpt-cAMP, and 7.64 in cpt-cAMP + PcTX1 treated medium.
Evaluation of neuronal differentiation
Cells in 35 mm dishes were examined at 400X magnification and photographed using phase contrast microscopy (Nikon). Cells were washed three times with extracellular solution (ECF) before photographs were taken. Neurite length and cell complexity were measured using Nikon Image Software (NIS) (Nikon Instruments, Inc., Melville, NY, USA). For each experiment, at least 5 random fields were selected for evaluation. Number of primary dendrites and total neurite length were quantified [
7]. In these experiments neurites are defined as any process that extends from the soma. Neurite length (in μm) was quantified by using a free hand line tool measuring the distance from the neurite tip to where the neurite joins the soma [
7]. Exclusion criteria included: 1) cell clusters typically greater than or equal to two, 2) where the total neurite length cannot be ascertained because neurites extend out of the field of view, 3) neurites that appear to have formed neurite-neurite or neurite-somatic connections, and 4) cases of extensively branched or overlapped neurites [
7].
Sholl analysis is a widely used method in neurobiology to quantify the complexity of dendritic arbors [
8]. The Sholl analysis of NS20Y cells was conducted by plotting the number of neurite intersections against the radial distance from the soma [
7].
Electrophysiology
Whole cell patch clamp recordings were performed as described previously [
9]. Patch pipettes were pulled by a two-step puller (PP83; Narishige, Tokyo, Japan) from thin walled borosilicate glass (1.5 mm diameter, World Precision Instruments, Sarasota, FL). The pipettes had a resistance of 3–4 MΩ when filled with intracellular solution: 140 mM CsF, 10 mM HEPES, 11 mM EGTA, 2 mM tetraethylammonium chloride, 1 mM CaCl
2, 2 mM MgCl
2 and 4 mM MgATP, pH 7.3 (adjusted with CsOH), 290–300 mOsm adjusted with sucrose. Extracellular solution contained: 140 mM NaCl, 5.4 KCl, 2 mM CaCl
2, 1 mM MgCl
2, 10 mM HEPES, 10 mM glucose, pH 7.4, 320–330 mOsm. When needed, 300 nM tetrodotoxin was used in ECF to block the Na
+ currents. PcTX1 (Peptide International) was dissolved in ddH
2O at 20 μM before adding to extracellular solutions. Amiloride was dissolved in dimethyl sulfoxide (DMSO) at 100 mM before adding to extracellular solutions to obtain final working concentrations. Tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN) and Zinc Chloride were dissolved in ddH
2O before adding to extracellular solutions. Unless described otherwise, all chemicals were purchased from Sigma.
Whole cell patch clamp recordings were done with an Axopatch 200B amplifier and Digidata 1320 DAC unit. Unless indicated otherwise, cells were clamped at a holding potential of −60 mV. ASIC currents were elicited by pH drops from 7.4 to acidic pH values as indicated. Currents were activated at least 1 min apart to achieve a complete recovery from desensitization. A multi-barrel perfusion system (SF-77 Warner Instruments, Hamden, CT) was used to achieve a rapid exchange of extracellular solutions. To generate a current–voltage (I–V) relationship, voltage steps were initiated from −100 and +100 mV from a holding potential of −60 mV were applied at 1 s interval. The data were analyzed with pClamp and Sigma Plot software.
Western blot
Cells were treated with lysis buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1 % Triton X-100, protease and phosphatase inhibitor cocktail) and collected by scraping into individual aliquots. The samples were put on ice for 30 min, centrifuged at 12,000 g at 4 °C for 30 min, and then supernatants were collected. Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Thereafter, the proteins were mixed with Laemmli sample buffer and boiled at 95 °C for 5 min. Proteins were separated by 10 % SDS-PAGE, followed by electrotransfer to polyvinylidene difluoride membranes. Blots were probed with antibodies against ASIC1 (rabbit anti-mouse/human, 1:1,000; Gift from Dr. Xiangming Zha, University of South Alabama, Mobile, AL, USA) or beta-actin (1:2,000; Abcam, Cambridge, MA, USA), detected using horseradish peroxidase-conjugated secondary antibody (1:1,000; Cell Signaling, Danvers, MA, USA), and visualized by ECL (Amersham Biosciences Piscataway, NJ) and Blue Autoradiography film (MedSupply Partners, Atlanta, GA). The intensity of the protein bands were quantified using NIH Image J software.
Reverse Transcription – Polymerase Chain Reaction (RT-PCR)
RT-PCR was used to examine the expression of individual ASIC subunits, as described in our previous studies [
10]. Total RNAs were isolated from NS20Y cells with Trizol reagent (Invitrogen), according to the manufacturer’s protocol. Equal amount of total RNA was reverse transcribed and PCR amplified with Superscript II (Invitrogen) using specific primers for individual ASIC subunit. ASIC1a forward5′-TCCTATGAGCGGCTGTCTCT-3′, ASIC1a, reverse 5′-TGCTTTTCATCAGCCATCTG-3′, ASIC1b forward 5′-GGCCTTTGTCATAGCACTGGGTGC -3′, ASIC1b reverse 5′-TTCCCATACCGCGTGAAGACCAC -3′, ASIC2a forward 5′-CGCCAACACCTCTACTCTCC-3′, ASIC2a reverse 5′-TGCCATCCTCGCCTGAGTTA-3′, ASIC2b forward 5′-CCTTGGCTTGCTGTTGTCCT-3′ ASIC2b reverse 5′-TGCCATCCTCGCCTGAGTTA-3′, ASIC3 forward 5′-GTCTGGACCCTGCTGAACAT-3′, ASIC3 reverse 5′-GGCTCTGGATCAAAGTCGGG-3′, ASIC4 forward 5′-GGGCTAGCATCCTCACCTTG-3′, ASIC4 reverse (5′-GGCCCAGTTTCATGGGTACT-3′. RT positive (+) samples was run with the reverse transcriptase, while RT negative (−) samples were run without reverse transcriptase. The RT-PCR products were electrophoresed on 1.5 % agarose gel.
ASIC1a shRNA transfection
Short hairpin ASIC1a (shASIC1a) and control shRNA were purchased from SuperArray Bioscience Corporation (Frederick, MD), each vector contains the shRNA under control of U1 promoter and the GFP gene, for the enrichment of transiently transfected cells. NS20Y cells were transfected with 5 μg of each specific ASIC1a shRNA or control shRNA using Lipofectamine™ reagent in serum free OptiMEM-1 medium (Invitrogen, Carlsbad, CA) per 35 mm dish according to the manufacture’s instruction. After transfection, cells were grown for further 72 h in growth medium as indicated in each experiment before utilization.
Statistical analysis
Data were expressed as mean ± SEM. Where applicable, multiple groups were compared using analysis of variance (ANOVA). Two groups were compared using Student’s t-test (paired and unpaired where appropriate). Values of p < 0.05 were considered statistically significant.
Discussion
This is the first report, to our knowledge, of the presence of functional ASICs in NS20Y, a mouse neuroblastoma cell line. More importantly, we show that blocking the activity of ASICs inhibits neurite growth/neuronal differentiation. Cyclic-AMP is commonly used to differentiate NS20Y and other clonal cell lines [
19,
24]. The use of cyclic-AMPs induces increases in the activities of tyrosine hydroxylase, choline acetyltransferase, the content of poly(A)
+ cytoplasmic RNA, and causes changes in nuclear non-histone proteins [
7,
18,
25]. These molecular changes can be tracked by measuring changes in expression of differentially regulated molecules such as neuropeptides [
7]. As expected, treatment with cpt-cAMP resulted in an increased neurite extension, dendritic complexity and increase in Na
+ current.
To explore a potential role of ASICs in the differentiation of NS20Y cells, we first determined whether NS20Y cells express ASICs. RT-PCR detected the presence of both ASIC1a and ASIC1b transcripts and Western blot confirmed the presence of ASIC1 protein. The presence of ASIC1a subunit was expected as it is fairly ubiquitous in the central and peripheral neuronal tissues [
26‐
28]. While the presence of ASIC1b in NS20Y cells was surprising, it was not unaccounted for as this cell line has heterogeneous origins, composition, and is mainly found in the peripheral nervous system [
14,
24].
Acid-sensing ion channels were further characterized using the whole-cell patch clamp technique. In all cells examined, lowering the extracellular pH from 7.4 to pH 6.0 evoked a transient inward current at a holding potential of −60 mV. The properties of acid-activated currents in NS20Y cells resemble ASIC currents in cultured primary CNS neurons (human, mouse, and rat) [
9,
29,
30]. For example, ASICs in NS20Y were pharmacologically blocked by the non-specific inhibitor amiloride, and the specific inhibitor PcTX1. The concentration response data of amiloride in NS20Y cells is consistent with previously established IC
50 for amiloride blockade of ASICs in CNS neurons [
9,
30]. In addition, ASIC currents were inhibited by zinc but potentiated by zinc chelation. Zinc inhibition is consistent with the presence of ASIC1a containing channels [
16,
17]. It is plausible that homomeric ASIC1b and heteromeric ASIC1a/1b formations may occur, these configurations of ASICs are not sensitive to PcTX1, which is not the case for the current in NS20Y cells where application of PcTX1 inhibited ~70 % of the current. Although PcTX1 also inhibits the current mediated by heteromeric ASIC1a/ASIC2b channels [
11,
15], our RT-PCR result did not show clear expression of 2b transcript. Taking together, our data suggest that homomeric ASIC1a channels are predominantly responsible for acid induced currents in NS20Y cells.
Having established the presence of functional ASICs in NS20Y cells, we explored the potential role of these channels in neuronal neuritogenesis. Neuritogenesis was defined as the extension and branching of neurites (length and complexity), similar to other reports in the field [
7,
19]. These parameters were quantified by counting the number of neurites/cell and their length from soma to furthest tip. We noted that while control cells remained small and lacked of extensive branches, cells treated with 1 mM cpt-cAMP increased neurite number, length, overall soma size, and arborization. When PcTX1 was used to inhibit the ASIC1a channels cells treated with cpt-cAMP failed to exhibit the same increase of neurite growth. Similarly, knock-down the expression of ASIC1a resulted in a suppression of cpt-cAMP induced neurite extension. Together, these data suggest that ASIC1a may play an important role in neuronal differentiation of NS20Y cells.
ASIC1a has been suggested to play a role in synaptic plasticity, learning and memory [
6], and in acidosis-mediated cell death [
9,
31]. Since Ca
2+ permeability/signaling plays a pervasive role in neuronal maturation, dendritic arborization, and axon outgrowth [
18,
32,
33], it is plausible that ASIC1a activation may play a role in neuritogenesis and neuronal differentiation. ASICs are spatially distributed and co-localize with postsynaptic density protein-95 (PSD-95) at the soma, along dendritic shafts and spines and importantly at the synapses, suggesting the possible involvement of ASIC in normal synaptic transmission and plasticity [
6]. Indeed, when ASICs are removed from the synapse, long term potentiation (LTP), the molecular model for learning memory, is impaired [
6]. Previous studies have shown that expression of ASIC1a modulates the density of dendritic spines [
34], which may partially explain its role in synaptic plasticity. Our current study suggests that ASIC1a may also play a role in neuronal differentiation and maturation, which may, at least partially, account for its role in synaptic transmission.
NS20Y is a cholinergic cell line which resembles many properties of neurons when differentiated; however, it cannot represent all properties of native neurons and therefore has limitations in neuronal differentiation investigation. Future studies will explore the role of ASIC1a in differentiation/maturation of native neurons.
Abbreviations
ASICs, acid sensing ion channels; CHO, Chinese Hamster ovarian; CHO-ASIC1a, Chinese Hamster ovarian transfected with ASIC1a; cpt-cAMP, 8 - (4-Chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate, sodium salt; DEG/ENaC, degenerin/epithelial Na+ channel; ECF, extracellular fluid; LTP, long term potentiation; PcTX1, psalmotoxin 1; RT-PCR, reverse transcription polymerase chain reaction; TPEN, tetrakis-(2-Pyridylmethyl) ethylenediamine
Acknowledgements
We thank Dr. An Zhou for the cell line, NS20Y, Dr. Fengxia Yan for assistance in statistical analysis, Dr. Yan Huang and Zhao Zhen for technical assistance.