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. 2013 Mar 11;591(Pt 10):2579–2591. doi: 10.1113/jphysiol.2012.249151

Kv3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation

Takahiro Yasuda 1,2, Hartmut Cuny 1, David J Adams 1
PMCID: PMC3678044  PMID: 23478135

Abstract

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K+ (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.

Key points

  • In the adult mammalian brain, neural precursor cells (NPCs) play an important role in neuronal plasticity.

  • Although adult NPCs exhibit voltage-gated, delayed rectifier K+ (KDR) channel currents, the KDR channel subtype dominantly expressed in adult NPCs and its functional role have not been defined.

  • Using gene knockdown targeting Kv3.1 K+ channels, we show Kv3.1 is a dominant KDR subtype expressed in adult NPCs.

  • Kv3.1 knockdown significantly decreased adult NPC proliferation and reduced differentiation into neuroblasts.

  • Our findings provide new insight into a mechanism of adult neurogenesis and suggest that selective activation of Kv3.1 in adult NPCs may be a new therapeutic approach to treating neurodegenerative diseases.

Introduction

Adult neurogenesis occurs continually throughout life in a wide range of mammals, including humans. In the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles has been shown to maintain the ability to produce immature neurons (neuroblasts) (Curtis et al. 2007; Wang et al. 2011). Although the migration of neuroblasts through the rostral migratory stream and differentiation into neurons in the olfactory bulb has been shown to decline during infancy in humans (Sanai et al. 2011), the SVZ is a potential region for neurogenesis in brain injury and disease (Curtis et al. 2012). A number of studies have established that adult neurogenesis can be modulated by various factors, such as physical and mental stimuli, extrinsic molecules (e.g. growth factors, neurotrophic factors or morphogens), intracellular regulators (e.g. transcription or epigenetic factors) and pathological stimuli (Ming & Song, 2005; Zhao et al. 2008; Ma et al. 2010). Although there are numerous reports about identification of ion transport proteins expressed in different types of stem cells (Li & Deng, 2011), less research has examined the physiological role of membrane ion transport proteins, such as ion channels and transporters, in adult neurogenesis or neural stem/precursor cell (NPC) function (Yasuda & Adams, 2010; Swayne & Wicki-Stordeur, 2012).

Voltage-gated K+ (Kv) channels are predominantly distributed in neurons in the brain. In mature neurons, Kv channels play a critical role in membrane hyperpolarization after each action potential, thereby controlling the duration and repetitiveness of neuronal firing. Kv channels have been also found in glial cells in the brain, but their functional relevance in these cells is unclear. Kv channel currents are generally classified electrophysiologically and pharmacologically into two classes: (1) tetraethylammonium (TEA)-sensitive, relatively slowly inactivating or non-inactivating, delayed rectifier K+ (KDR) channel currents; or (2) 4-aminopyridine-sensitive, rapidly inactivating, A-type K+ (KA) channel currents.

Electrophysiological studies have revealed that NPCs in the adult mouse SVZ exhibit Kv channel currents in situ and in vitro (Liu et al. 2006; Yasuda et al. 2008; Lai et al. 2010). The Kv channel currents of adult NPCs are primarily KDR channel currents, with either minor or no contribution from KA channel currents in situ and in vitro (Liu et al. 2006; Yasuda et al. 2008; Lai et al. 2010). The number of KDR and KA channel currents in total Kv channel currents is different in neonatal and embryonic NPCs. Most (80%) neonatal mouse NPCs have almost equal amplitude of KA and KDR channel current density, whereas the remainder (20%) have only KDR channel currents in situ (Cesetti et al. 2009). Furthermore, embryonic human and rat NPCs more dominantly express KA currents than KDR currents in vitro (Piper et al. 2000; Cai et al. 2004; Liebau et al. 2006; Smith et al. 2008; Schaarschmidt et al. 2009). Therefore, it is suggested that functional expression of KDR channels is more characteristic of adult NPCs than embryonic and neonatal cells.

KDR channels are involved in adult NPC physiological function in vitro. Given that TEA inhibits KDR channel currents and neurosphere formation, Kv (KDR) channels play a critical role in cell growth of adult NPCs (Yasuda et al. 2008). However, in embryonic NPCs, KDR channels inhibited or had no effect on cell proliferation (Liebau et al. 2006; Schaarschmidt et al. 2009). KA channels, which are dominantly expressed in embryonic NPCs, were needed to proliferate embryonic human NPCs from aborted fetal brain tissue (Schaarschmidt et al. 2009), whereas in embryonic rat midbrain NPCs, KA channels were suggested to inhibit cell proliferation (Liebau et al. 2006).

In adult NPCs, expression of one KDR channel subtype, Kv3.1, has been identified by RT-PCR and immunostaining in neurospheres and the SVZ of the brain (Yasuda et al. 2008; Prüss et al. 2011). Pharmacological results implied that Kv3.1 plays a functional role in NPCs (Yasuda et al. 2008). Kv3.1 expression was also shown in embryonic rat and human NPCs (Cai et al. 2004; Liebau et al. 2006; Schaarschmidt et al. 2009), although another report showed no expression in embryonic rat NPCs (Smith et al. 2008) Kv3.1 channel activation in embryonic rat NPCs has also been suggested to inhibit neurosphere formation (Liebau et al. 2006). In previous studies, given that no selective Kv3.1 channel inhibitor was available, the non-specific KDR channel inhibitor TEA was used as a pharmacological tool in functional assays. In this context, although the functional relevance of Kv3.1 has been strongly suggested in adult and embryonic NPCs, the evidence for it is still not conclusive (Swayne & Wicki-Stordeur, 2012). Therefore, an alternative approach is necessary to confirm a functional role for Kv3.1 in NPCs, e.g. inhibition by anti-Kv3.1 antibody or gene knockout/knockdown.

In the present study, using siRNA-mediated gene knockdown to target Kv3.1, we show that Kv3.1 is a dominant KDR channel in adult NPCs and plays a pivotal role in NPC proliferation and differentiation. A preliminary report of some of these results has been published in abstract form (Yasuda et al. 2010).

Methods

Cell culture media

Serum-free basal media contains Dulbecco's modified Eagle's medium (DMEM)/F-12 (Gibco/Invitrogen, San Diego, CA, USA) supplemented with Hepes (5.6 mm), glucose (0.42%) and NaHCO3 (15 mm). Neurosphere assay (NSA) media was prepared by adding 10% mouse NeuroCult neural stem cell proliferation supplements (STEMCELL Technologies, Vancouver, Canada), 2% bovine serum albumin (BSA; Sigma-Aldrich, Sydney, Australia) and penicillin/streptomycin (200 U ml−1; Gibco/Invitrogen) to the NS basal media. For neurosphere formation, 20 ng ml−1 epidermal growth factor (EGF; BD Biosciences, Sydney, Australia), 10 ng ml−1 basic fibroblast growth factor (FGF-2; Roche Applied Science, Basel, Switzerland) and 2 μg ml−1 heparin (Sigma-Aldrich) were added to the NSA media. For neuronal differentiation, fetal bovine serum (FBS; JHR Biosciences, Melbourne, Australia) was added to the NSA media instead of growth factors.

Mouse brain dissection and adult NPC culture

CBA mice or doublecortin (DCX)-green fluorescent protein (GFP) transgenic mice (6–12 weeks old) (Walker et al. 2007) were killed as approved by the RMIT University Animal Ethics Committee, and their forebrain SVZ was isolated. After trypsin digestion and mechanical dissociation, cells were cultured in NSA media supplemented with growth factors. After 7–9 days, the primary cells were passaged and plated at a cell density of 104 cells cm−2. Serial passaging was then done every 4 days at the same cell density, unless otherwise stated.

Real-time quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from primary or passaged neurospheres using the Absolutely RNA Nanoprep kit (Agilent Technologies, Santa Clara, CA, USA) and then reversely transcribed using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). SensiMix SYBR No-ROX Kit (Bioline, London, UK) was used to prepare PCR mixtures with cDNAs and gene-specific primers for mouse Kv subtypes (Table 1). PCR was implemented using a real-time PCR machine (Rotor-Gene 3000; Corbett Research, Sydney, Australia). The cDNAs of succinate dehydrogenase complex, subunit A, ubiquitin C and ribosomal protein L13a housekeeping genes as internal references were amplified in parallel control reactions using gene-specific primers (Mouse Normalisation Gene Panel; Bioline). Comparative quantitative analysis of Kv channel mRNA expression levels was performed with Rotor-Gene 6 software (Corbett).

Table 1.

Primer sequences used for qRT-PCR

Channel Primer sequence
Kv2.1 (pair 1) F: 5′-AAGGAGCAGATGAACGAGGA
R: 5′-CGAGGAAGAGGATGAGCAAG
Kv2.1 (pair 2) F: 5′-CTGGAGAAGCCCAACTCAT
R: 5′-CTGTAGCTCAGGCAGTGTG
Kv2.2 F: 5′-AGGAAATGTGTGCGCTCTCT
R: 5′-AGCCTCTACGTGTGCCAACT
Kv3.1 (pair 1) F: 5′-GAGGACGAGCTGGAGATGAC
R: 5′-CAGGGCCAGGAAGATGATAA
Kv3.1 (pair 2) F: 5′-CCTACTCATCCCGCTACG
R: 5′-AGTCTCCAGACAGAAGGTTG
Kv3.2 F: 5′-ACCCTGGTGATGATGAGGAC
R: 5′-AGCACCCTCAGACCTACGAA
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Small interfering RNA (siRNA)-mediated Kv3.1 channel knockdown

Two duplex siRNAs, MSS236945 (#945) and MSS236946 (#946) of Stealth Select RNAi siRNAs (Invitrogen), against the mouse Kv3.1 channel gene (Kcnc1) were used for gene knockdown. A non-targeting siRNA, a duplex siRNA comprising a sequence not homologous with anything in vertebrate transcriptome (Stealth RNAi siRNA negative control med GC; Invitrogen) was transfected for an ‘siRNA control’ group as negative control. Mock-transfected cells were also prepared by carrying out the same transfection protocol without siRNA as mock control. The siRNAs (100 nm or 300 nm final concentration) were electroporated into (2–5) × 105 cells of dissociated NPCs using the Amaxa Nucleofector II system in combination with the Mouse Neural Stem Cell Nucleofector Kit (both Lonza, Cologne, Germany), following the manufacturer's instructions. Transfected cells were cultured in NSA medium supplemented with EGF and FGF-2 for 1–2 days before being used for each assay.

Electrophysiological patch-clamp recordings

Dissociated NPCs were plated on cover slips coated with poly-l-ornithine (Sigma-Aldrich) at a cell density of approximately 5 × 103 cells per well and cultured for 2–3 days before electrophysiological recordings started. In siRNA transfection experiments, cells were cultured overnight after transfection, seeded on coverslips with a mild trypsin digestion, then cultured for another 2–3 days before use. Membrane current and voltage were recorded using the whole-cell patch-clamp technique and an Axopatch 200A patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA). Patch electrodes were pulled from borosilicate glass capillaries and had resistances of 1.5–3.0 MΩ when filled with an intracellular solution containing (in mm): 105 potassium gluconate, 25 KCl, 1 CaCl2, 2 MgCl2, 5 EGTA, 2 Na2ATP and 10 Hepes, pH 7.3 with KOH. Cells were maintained in a physiological saline solution containing (in mm): 125 NaCl, 3 KCl, 1 CaCl2, 2 MgCl2, 10 glucose and 10 Hepes, pH 7.3 with NaOH. All experiments were performed at room temperature (21–24°C). Data were obtained at 5–10 kHz using a Digidata 1322A interface (Molecular Devices), linked to a computer equipped with pCLAMP 9.0 software (Molecular Devices) and filtered at 2–5 kHz. Leak currents were subtracted (–P/4) during voltage-clamp recordings for Kv channel currents. The liquid junction potential was calculated as 10.4 mV and corrected. Resting membrane potential (VR) and input resistance (Rin) were determined from voltage responses to a hyperpolarizing current pulse of 0–200 pA. Data were analysed using Clampfit 9.2 (Molecular Devices).

Cell growth assay

Dissociated neurosphere cells (2.5 × 105 cells per group) were cultured for 4 days in the absence or presence of TEA, before cells were counted. In mRNA knockdown experiments, dissociated neurosphere cells (5 × 105 cells per group) were transfected with siRNAs at a concentration of 300 nm and cultured overnight. After a mild trypsin digestion, cells were passaged and cultured for 4 more days, then counted.

Apoptosis and proliferation assays

For the apoptosis assay, dissociated NPCs were plated onto poly-l-ornithine-coated 96-well plates at a cell density of 4 × 103 cells per well and cultured for 18 h in the absence or presence of drugs, in NSA media supplemented with growth factors. Cells were incubated with 1 μm of the apoptotic cell marker YO-PRO-1 (Invitrogen), 0.01 ng ml−1 propidium iodide (PI; Sigma-Aldrich) and 0.05 ng ml−1 Hoechst 33342 (Invitrogen) for 30 min at 37°C. Fluorescent images (25 imaging sites per well) were taken using an automated acquisition and analysis system, ImageXpress Micro (IXM; Molecular Devices). The size of each imaging site was approximately 333 μm × 450 μm. At least two wells were prepared for each treatment group. For the proliferation assay, growing neurospheres were incubated with 30 μm 5-bromo-2-deoxyuridine (BrdU, Sigma-Aldrich) for 4 h at 37°C. After incubation, neurospheres were enzymatically digested and seeded onto poly-l-ornithine-coated 24-well plates. Immunostaining of BrdU incorporated into nuclei followed a standard protocol. This included denaturing DNA with HCl, treating it with a biotinylated primary antibody against BrdU (1:250, Invitrogen) and incubating it with streptavidine-Cy3 (1:1000, Invitrogen). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:500; Invitrogen). Fluorescent images (154 imaging sites per well) were taken using an IXM, as for the apoptosis assay. Imaging data were analysed quantitatively using MetaXpress software (Molecular Devices).

Neuronal differentiation assay

NPCs for the neuronal differentiation assay were dissected from DCX-GFP transgenic mice. After 2–4 passages, cultured neurospheres were dissociated and seeded onto poly-l-ornithine- and laminin-coated 96-well plates at a cell density of (4–5) × 104 cells per well. For the next 5 days, cells were forced to differentiate under the following conditions. On the first day, they differentiated in NSA medium supplemented with 5% FBS. Over the next 4 days, they differentiated in NSA media with 0.5% fetal calf serum. For a knockdown study, cells ((3–4) × 105 cells per group) were transfected with 300 nm siRNAs and cultured for 2 days in NSA media supplemented with EGF and FGF-2 as a normal growth condition. Cells were then dissociated and cultured for another 5 days under the differentiation conditions specified above. After staining cell nuclei with Hoechst 33342, images of nuclei and GFP-positive newly generated neuroblasts (immature neurons) were captured with IXM. Each treatment group consisted of at least three wells and each well had 25 imaging sites. Captured images were analysed with MetaXpress software, evaluating the neuroblast population among total differentiated cells.

Data analysis

Numerical data were statistically analysed using Prism 4.0 software (GraphPad, Software Inc., La Jolla, CA, USA) and presented as mean ± standard error of the mean (SEM). A paired t test or one-way analysis of variance (ANOVA) with Dunnett's test as a post test was used to compare two or more groups, respectively, unless otherwise stated.

Results

Dominant Kv3.1 mRNA expression in adult NPCs is sensitive to gene knockdown

Previous studies have used conventional PCR, pharmacological assays and biophysical comparison of KDR channel current characteristics to identify Kv3.1 as a candidate of functional KDR channel expression in adult NPCs (Liebau et al. 2006; Yasuda et al. 2008). This study used qRT-PCR to confirm dominant expression of the Kv3.1 transcript. The relative concentration of Kv3.1 transcripts was compared with that of other KDR channels (Kv2.1, Kv2.2 and Kv3.2) that were estimated to be minor subtypes in adult NPCs (Yasuda et al. 2008). As shown in Fig. 1, expression levels of Kv3.1 mRNA were approximately 4–40 times higher than those of other KDR channels tested. This reinforces the previous results and implies Kv3.1 has a dominant functional role in adult NPCs (Yasuda et al. 2008).

Figure 1. Kv channel mRNA expression levels in neural precursor cells.

Figure 1

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Neurospheres at the passage between P0 and P2 were harvested for mRNA extraction. Their cDNA was synthesized, and then amplified by qRT-PCR. Relative mRNA concentrations of Kv2.1, Kv2.2, Kv3.1 and Kv3.2 were calculated in comparison with a combined internal standard of three housekeeping gene (succinate dehydrogenase complex, subunit A, ubiquitin C and ribosomal protein L13a) mRNA concentrations (n= 4–8). The gene expression level of Kv3.1 was 3.7, 31.4 and 39.1 times higher than that of Kv2.1 (P < 0.01), Kv2.2 (P < 0.001) and Kv3.2 (P < 0.001), respectively (one-way ANOVA with Dunnett's test as a post test).

To inhibit Kv3.1 channel function specifically in adult NPCs, we used siRNA-mediated Kv3.1 gene knockdown. As negative control, NPCs were transfected with non-targeting siRNA up to 300 nm (siRNA control group). Given that the relative Kv3.1 mRNA expression levels of the siRNA control group were around 100% of that of mock-transfected cells, the non-targeting siRNA was confirmed to have no toxicity to NPCs (Fig. 2). We used two sets of siRNA duplexes (#945 and #946), targeting different sequences of Kv3.1 transcript to make sure the effect of gene knockdown was target gene specific, with no off-target effects. Transfection of siRNAs (#945 and #946) at concentrations of 100 nm and 300 nm showed concentration-dependent inhibition of Kv3.1 mRNA expression in NPCs (Fig. 2). Compared with the siRNA control group, siRNA #945 and #946 at 300 nm reduced Kv3.1 expression by 63.9% and 49.5%, respectively. Both siRNAs did not significantly affect Kv2.1 mRNA expression (control, 111.6 ± 19.7%; #945, 92.3 ± 22.2%; #946, 103.5 ± 9.9% (mean ± SEM, n= 3)), which confirmed their specificity.

Figure 2. Efficiency of Kv3.1 mRNA knockdown by siRNAs in adult NPCs.

Figure 2

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Kv3.1 gene knockdown was tested using two sets of siRNA duplexes (#945 and #946) targeting different sequences of Kv3.1 transcript at concentrations of 100 nm and 300 nm (n= 5). In the siRNA control group, cells were treated with a non-targeting siRNA as negative control. Kv3.1 mRNA expression levels were shown as a percentage of the Kv3.1 expression level of mock control cells (no siRNA was transfected). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding siRNA control group (one-way ANOVA with Dunnett's test as a post test).

Kv3.1 is responsible for KDR outward current in adult NPCs

In response to step depolarization pulses from −80 to +60 mV, from a holding potential of −80 mV, NPCs exhibited non-inactivating delayed rectifier outward currents that had a high-threshold (∼−20 mV) for activation and rapid activation and deactivation kinetics. KDR current properties in adult NPCs were very similar to those of Kv3.1 (Rudy & McBain, 2001). As described in a previous report (Yasuda et al. 2008), currents were inhibited by the non-specific KDR channel inhibitor TEA in a concentration-dependent manner (Fig. 3A). Both siRNAs targeting Kv3.1, siRNA #945 and #946, inhibited the KDR outward currents in NPCs at a concentration of 300 nm (Fig. 3B). When compared to an siRNA control group, inhibition of the outward currents by siRNA #945 and #946 at a voltage of +50 mV was 52.2% (P < 0.001) and 31.5% (P < 0.01), respectively. Under the same conditions, neither TEA nor Kv3.1 knockdown affected passive electrical membrane properties, including the resting membrane potential (Tables 2 and 3). Taking into account that about 25% of voltage-dependent outward currents are a TEA-insensitive unidentified component (Yasuda et al. 2008), 52% inhibition by #945 is equivalent to ∼70% of the TEA-sensitive component. The degree of inhibition (∼70%) of KDR currents fits closely with that of Kv3.1 mRNA expression by 300 nm of #945 (64%). Therefore, these results indicate that the siRNAs worked effectively to knockdown functional Kv3.1 channel protein expression, and more importantly, that Kv3.1 is the dominant KDR channel subtype expressed in adult NPCs.

Figure 3. Effect of Kv3.1 channel knockdown on TEA-sensitive KDR channel outward currents in adult NPCs.

Figure 3

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Current traces represent non-inactivating outward KDR channel currents in response to step depolarization from −80 to +60 mV, from a holding potential of −80 mV. Peak current amplitude was plotted as a function of test potential (mV), which is shown on the right of each set of representative current traces. A, TEA-induced KDR channel current suppression in a concentration-dependent manner (n= 7). Cells were cultured in the absence (control) or presence of TEA (0.2 or 2 mm) for 5–10 min before testing KDR channel outward currents. B, cells transfected with siRNA (#945 or #946) at a concentration of 300 nm for Kv3.1 knockdown exhibited smaller outward currents than the siRNA control (negative control) group, in which non-targeting siRNA was transfected (n= 17–18).

Table 2.

Effect of TEA on passive membrane electrical properties of adult NPCs

Control 0.2 mm TEA 2 mm TEA
VR (mV) −77.1 ± 4.1 (6) −79.7 ± 5.3 (6) −78.2 ± 3.0 (6)
Rin (MΩ) 238.8 ± 83.4 (6) 148.2 ± 59.7 (6) 312.2 ± 103.1 (6)
Cm (pF) 24.3 ± 3.6 (6) 26.4 ± 3.9 (6) 28.5 ± 2.5 (6)
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Data represent mean ± SEM and the number of cells is indicated in parentheses. There is no significant difference between the control and each TEA treatment group (P > 0.05).

Table 3.

Effect of Kv3.1 knockdown on passive membrane electrical properties of adult NPCs

siRNA control siRNA #945 siRNA #946
VR (mV) −77.9 ± 1.2 (12) −79.0 ± 1.7 (13) −78.9 ± 1.9 (13)
Rin (MΩ) 214.6 ± 44.4 (12) 211.2 ± 48.5 (13) 186.7 ± 45.5 (13)
Cm (pF) 22.8 ± 1.9 (12) 21.9 ± 1.5 (13) 24.5 ± 1.5 (13)
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Data represent mean ± SEM and the number of cells is indicated in parentheses. There is no significant difference between the siRNA control and each Kv1.3 knockdown siRNA transfection group (P > 0.05).

Kv3.1 is involved in NPC proliferation and neuronal differentiation

Using the validated Kv3.1 gene knockdown, we next evaluated the physiological role of Kv3.1 channels through various cell-based functional assays. Consistent with previous results (Yasuda et al. 2008), TEA at a concentration of 2 mm, which remarkably suppressed KDR channel current, significantly inhibited cell growth for 4 days by 66.6% (Fig. 4A). Likewise, 300 nm of siRNAs #945 and #946 significantly reduced cell growth by 52.9% and 50.5%, respectively (Fig. 4B). This suggests that Kv3.1 channel activation plays a critical role in NPC growth.

Figure 4. Effects of KDR channel inhibition and Kv3.1 knockdown on adult NPC growth.

Figure 4

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A, effects of TEA on cell growth. Dissociated neurosphere cells (2.5 × 105 cells per group) were cultured for 4 days in the absence (control) or presence of TEA (n= 4). B, effects of siRNA (#945 and #946)-mediated Kv3.1 channel knockdown on cell growth. Dissociated neurosphere cells (5 × 105 cells per group) were transfected with siRNAs at a concentration of 300 nm and cultured for 5 days (n= 5 for #945, n= 6 for #946). In the siRNA control group, cells were transfected with non-targeting siRNA as negative control. *P < 0.05, **P < 0.01 compared with each control group (one-way ANOVA with Dunnett's test as a post test for A, paired t test for B).

Given that cell growth is a balance between cell proliferation and cell death, we tested the effect of TEA-induced KDR channel inhibition on cell death. For this purpose, imaging assays for apoptotic and necrotic cells were used. Cells were stained with YO-PRO-1 as an apoptotic cell marker and PI as a dying cell marker for necrotic and late apoptotic cells. Staurosporine (1 μm), which is a positive control because it causes apoptosis in various mammalian cells, significantly increased the number of late apoptotic cells, especially YO-PRO-1 and PI double-positive late apoptotic cells, after 18 h incubation (Fig. 5A and B). Adding a high concentration of TEA (3 mm) to the same conditions did not affect the number of apoptotic or necrotic cells (Fig. 5C), confirming that the decline of cell growth by KDR channel inhibition was independent of cell death. Next, the contribution of Kv3.1 to cell proliferation was examined using a BrdU incorporation assay. As shown in Fig. 6, both TEA and Kv3.1 knockdown by siRNAs significantly inhibited cell proliferation to the same extent (60.4% inhibition by 2 mm TEA and 59.4% inhibition by 300 nm siRNA #945). In combination, these results clearly show that Kv3.1 channel activation enhances NPC growth by stimulating cell proliferation, but not by inhibiting cell death.

Figure 5. KDR channel inhibition does not induce cell apoptosis in adult NPCs.

Figure 5

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Dissociated neurosphere cells were plated into a 96-well plate at a cell density of 4 × 103 cells per well and cultured for 18 h in the absence (control) or presence of drugs in NSA media supplemented with growth factors. A, representative images of staurosporine (1 μm)-treated NPCs stained with Hoechst 33342 (Hoe), YO-PRO-1 (YO) and propidium iodide (PI). In the analysed cell image, green cells are Hoe+ve, YO−ve and PI−ve live cells; blue cells are Hoe+ve, YO+ve and PI−ve early apoptotic cells; pink cells are Hoe+ve, YO+ve and PI+ve late apoptotic cells; and red cells are Hoe+ve, YO−ve and PI+ve necrotic cells. B, images of staurosporine-induced cell apoptosis were summarized as a positive control experiment (4 independent experiments (n= 4)). Staurosporine (1 μm) significantly increased the number of late apoptotic cells. C, effect of KDR channel inhibition by TEA (3 mm) on cell apoptosis. In comparing the control group, TEA did not affect cell apoptosis or necrosis. NPCs (5 independent experiments (n= 5)). ***P < 0.01 compared with DMSO control group (one-way ANOVA with Dunnett's test as a post test).

Figure 6. Effects of KDR channel inhibition and Kv3.1 knockdown on adult NPC proliferation.

Figure 6

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Adult NPC proliferation was evaluated with BrdU incorporation for 4 h. A, effect of TEA on NPC proliferation. Dissociated neurosphere cells (5 × 104 cells per group) were cultured for 3 days in the absence or presence of TEA before BrdU incubation. Images show that TEA (2 mm) decreased the number of BrdU-incorporated cells (BrdU+ve cells; red). Nuclei were counterstained with DAPI (blue) to count the total number of cells. Summarized data of the effect of TEA on NPC proliferation are shown in the bar graph (4 independent experiments (n= 4)). A high concentration of TEA (2 mm) significantly inhibits NPC proliferation (a population of BrdU+ve cells). B, effect of Kv3.1 channel knockdown on NPC proliferation. Dissociated neurosphere cells (3 × 105 cells per group) were transfected with 300 nm siRNAs and cultured overnight. Then, cells were passaged with a short enzymatic digestion and cultured for another 3 days before BrdU treatment. Images show that siRNA (#945) targeting Kv3.1 decreased the number of BrdU-incorporated cells (red). Nuclei were counterstained with DAPI (blue). The bar graph shows summarized data of 6 independent experiments (n= 6). Kv3.1 channel knockdown by siRNA (#945 and #946) inhibits NPC proliferation. In the siRNA control group, cells were transfected with non-targeting siRNA as a negative control. *P < 0.05, **P < 0.01 compared with each control group (one-way ANOVA with Dunnett's test as a post test).

NPCs give rise to neurons and glial cells, such as astrocytes and oligodendrocytes. We evaluated the role of Kv3.1 in NPC differentiation into neurons using NPCs derived from the SVZ of DCX-GFP mice. DCX is a specific marker for newly generated neurons, i.e. neuroblasts (Gleeson et al. 1999). We established an imaging assay to detect GFP-positive DCX-expressing neuroblasts. After the differentiation period of 5 days, most (>90%) differentiated cells were astrocytes, with neuroblasts making 5–10% of the total cell population. Under these conditions, TEA (3 mm) significantly decreased the DCX-expressing neuroblast population by 59.1% (Fig. 7A). No significant effect on neuronal differentiation was detected with 1 mm TEA, whereas 3 mm TEA effectively inhibited differentiation. Given that 1 mm TEA has been shown to inhibit Kv3.1 currents by >50% (IC50= 630 μm; Yasuda et al. 2008), Kv3.1 inhibition may be only partly involved in differentiation. As well as inhibiting Kv3.1, the high TEA concentration (3 mm) may affect some other component.

Figure 7. Effects of KDR channel inhibition and Kv3.1 knockdown on adult NPC neuronal differentiation.

Figure 7

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Dissociated neurosphere cells were subjected to differentiating conditions for 5 days. A, effect of TEA on neuronal differentiation. Differentiating NPCs were exposed to TEA for the last 4 days of the differentiation process. Images show that TEA (2 mm) decreased the number of DCX-GFP+ve cells, namely DCX-expressing neuroblasts (green). Nuclei were counterstained with DAPI (blue). The bar graph shows summarized data of the percentage of the GFP+ve neuroblast number in the total cell number (5 independent experiments (n= 5)). TEA significantly inhibits neuronal differentiation of adult NPCs. B, effect of Kv3.1 channel knockdown on neuronal differentiation. Dissociated neurosphere cells (4 × 105 cells per group) were transfected with 300 nm siRNAs and cultured for 2 days, then subjected to differentiating conditions for 5 days. Images show that siRNA (#945) targeting Kv3.1 decreased the number of GFP+ve neuroblasts (green). Nuclei were counterstained in blue. Summarized data of 7 independent experiments (n= 7) is shown in the bar graph. Kv3.1 channel knockdown by siRNA (#945 and #946) inhibited neuroblast generation, i.e. neuronal differentiation. In the siRNA control group, cells were transfected with non-targeting siRNA as a negative control. **P < 0.01 compared with each control group (one-way ANOVA with Dunnett's test as a post test).

Kv3.1 knockdown also significantly inhibited neuronal differentiation. Neuronal differentiation was inhibited by siRNA #945 and #946 by 31.4% and 34.8%, respectively (Fig. 7B). Although TEA and siRNAs are likely to be less effective in this neuronal differentiation assay than in the proliferation assay, Kv3.1 was shown to be involved in NPC differentiation into neurons, i.e. neuronal lineage commitment, at least in part.

Discussion

The Kv3.1 subtype of Kv channels is widely distributed in the central nervous system and plays a pivotal role in high-frequency repetitive firing of mammalian neurons (Gan & Kaczmarek, 1998; Rudy & McBain, 2001; Song, 2002). Kv3.1 channel currents are characterized by a high-voltage threshold for activation, and quick activation and deactivation kinetics. These biophysical properties are responsible for transient inactivation of voltage-gated Na+ and Ca2+ channels during neuronal excitation. Kv3.1 channels are also expressed in pulmonary arteries (Osipenko et al. 2000) and carotid bodies (Pérez-García et al. 2004), where they are suggested to act as arterial oxygen sensors by decreasing channel currents in hypoxic conditions (Patel & Honoré, 2001). Notably, Kv3.1 channels were identified not only in excitable cells, but also in electrically inexcitable cells, such as T-lymphocytes (Grissmer et al. 1992; Deutsch & Chen, 1993), oligodendrocyte precursor cells (Tiwari-Woodruff et al. 2006) and NPCs (Cai et al. 2004; Liebau et al. 2006; Yasuda et al. 2008; Schaarschmidt et al. 2009; Prüss et al. 2011). The functional role of Kv3.1 in T-lymphocytes is still unclear. Although KDR channels are essential for regulatory volume decrease in response to hypotonic shock in T-lymphocytes, this response is attributed to activation of a low-voltage-gated KDR channel subtype, Kv1.3, not Kv3.1 (Deutsch & Chen, 1993).

A functional relevance of Kv3.1 in inexcitable cells was first demonstrated in oligodendrocyte precursor cells. Using TEA, anti-Kv3.1 antibody and Kv3.1 knockout mice, Kv3.1 has been shown to be involved in oligodendrocyte precursor cell proliferation and migration (Tiwari-Woodruff et al. 2006). Gene and protein expressions of Kv3.1 in NPCs were confirmed in human and rat embryos (Cai et al. 2004; Liebau et al. 2006; Schaarschmidt et al. 2009) and adult mice (Yasuda et al. 2008; Prüss et al. 2011). A physiological role for Kv3.1 in NPCs is controversial. In embryonic rats, Kv3.1 was suggested to negatively regulate NPC proliferation (Liebau et al. 2006). In contrast, the KDR channel was found to be essential for NPC proliferation, and Kv3.1 emerged as a KDR channel candidate based on a pharmacological assay, conventional PCR and immunostaining (Yasuda et al. 2008). However, in the previous approaches, non-selective KDR channel inhibitor TEA was used to inhibit Kv3.1 channel function. Therefore, for conclusive evidence that Kv3.1 is physiologically relevant in NPCs, functional assays with specific Kv3.1 inhibition need to be implemented.

In this study, we confirmed that Kv3.1 expression in adult NPCs was more dominant than the expression of other Kv channels examined (Kv2.1, Kv2.2 and Kv3.2). In agreement with this result, Kv2.1, Kv2.2 and Kv3.2 expression in embryonic human and rat, and adult mouse, NPCs was either not detected or reported to be minor (Liebau et al. 2006; Schaarschmidt et al. 2009; Prüss et al. 2011). Controversially, significant Kv2.1 gene expression and channel currents were detected in embryonic rat NPCs (Cai et al. 2004; Smith et al. 2008). Although minor Kv2.1 mRNA expression was detected in this study, expression was not enough to cause significant KDR channel currents, because the currents were not inhibited by stromatoxin-1, a selective inhibitor (Yasuda et al. 2008). NPCs in the SVZ are known to be glial fibrillary acidic protein (GFAP)-positive astrocytic cells and share most electrophysiological properties with astrocytes (Liu et al. 2006; Yasuda et al. 2008). Interestingly, to our knowledge, there is no report that Kv3.1 is expressed in astrocytes. In gerbils, Kv3.1-expressing hippocampal astrocytes were found only in seizure-sensitive animals, but not in seizure-resistant ones (Lee et al. 2009). Therefore, in normal adult animals, Kv3.1 could be a selective cell surface marker to distinguish NPCs from astrocytes.

We used two different sets of siRNA in the knockdown experiments, because gene knockdown often causes non-specific off-target effects. Both siRNA sets exhibited a decrease in Kv3.1, but not Kv2.1, mRNA expression of approximately 50%. This indicates their specific and sufficient gene knockdown effect. As a result of Kv3.1 knockdown, KDR currents significantly decreased. Moreover, taking into account the existence of the non-KDR currents, the degree of Kv3.1 knockdown-induced current inhibition was almost the same as the Kv3.1 mRNA expression suppression level. Therefore, we conclude that Kv3.1 is a major functional KDR channel subtype expressed in adult NPCs. Similar to TEA, Kv3.1 knockdown significantly inhibited NPC growth. NPC growth inhibition was attributed to decreased cell proliferation. This is the first clear evidence that adult NPC proliferation is regulated by Kv3.1 channel expression and/or activity.

How Kv3.1 channels induce NPC growth is still unclear. In various cells, such as T-lymphocytes and cancer cells, it has been suggested that KDR channel inhibition induces membrane depolarization, decreasing the driving force for Ca2+ influx and inhibiting Ca2+-dependent events that are essential for cell growth (Wonderlin & Strobl, 1996; Sundelacruz et al. 2009). However, this is not the case in adult NPCs, because neither TEA nor Kv3.1 knockdown causes membrane depolarization, as shown in this study. In adult NPCs, the resting membrane potential is almost solely controlled by Ba2+-sensitive Kir channels (Liu et al. 2006; Yasuda et al. 2008). Moreover, slight membrane depolarization by Kir channel inhibition potentiates cell growth in adult NPCs (Yasuda et al. 2008). Under physiological and pathophysiological conditions, K+ and Cl− flux affect cell volume, i.e. cell size. Dubois and Rouzaire-Dubois proposed the ‘volume theory’, in which the rate of cell proliferation is a bell-shape function of the cell size (Rouzaire-Dubois et al. 2004; Dubois & Rouzaire-Dubois, 2012). Therefore, Kv3.1 impairment by TEA or gene knockdown may disturb an appropriate volume regulation necessary for adult NPC proliferation. Further investigation is needed to prove this hypothesis in adult NPCs.

In addition, one may ask how high-voltage-gated K+ channels, like Kv3.1 channels, are activated in inexcitable cells. Kv3.1 is a well-recognized, critical KDR channel activated by membrane depolarization beyond ∼−20 mV in various neurons. Dominant expression of inward rectifier K+ (Kir4.1) channels in NPCs makes the resting membrane potential approximately −80 mV (Liu et al. 2006; Yasuda et al. 2008). This resting membrane potential is similar to glial cells or some neurons, but more negative than T-lymphocytes or cancer cells (Levin, 2012). In T-lymphocytes, a low-voltage-activated K+ channel subtype, Kv1.3, is supposed to be in steady-state activation at a resting membrane potential of between −50 mV and −70 mV (Panyi, 2005). In adult NPCs, the activation threshold of Kv3.1 is ∼−20 mV, so there is a gap of ∼60 mV between the threshold and resting membrane potential. Given that adult NPCs have a very low input resistance of 30–200 MΩ (Liu et al. 2006; Yasuda et al. 2008), the membrane will not be depolarized easily by cation influx or anion efflux. GABA released from neuroblasts has been shown to cause a depolarizing current of 5–30 pA in ambient NPCs (Liu et al. 2005). However, considering the very low input resistance of NPCs, this depolarizing current must have limited impact on the resting membrane potential. At most, the impact is probably around +5 mV. Membrane potential has been shown to dynamically change during the cell cycle in astrocytes (MacFarlane & Sontheimer, 2000). Although electrophysiological properties of adult NPCs are similar to astrocytes (Liu et al. 2006; Yasuda et al. 2008), it is not clear if a similar, but more prominent change in the membrane potential occurs in NPCs. It should be noted that there are some NPCs (∼10%) with a more depolarized membrane potential of between −35 mV and −70 mV (Yasuda et al. 2008). Although there is still a voltage gap between the high-voltage activation threshold and resting membrane potential, these depolarized NPCs might be responsible for Kv3.1 activation and proliferation. It is suggested that the high-voltage activation threshold of Kv3.1 is maintained by constitutive channel phosphorylation by casein kinase II, so it is interesting that channel dephosphorylation can cause a negative shift of the activation by 20 mV (Macica & Kaczmarek, 2001). If this negative shift happens in NPCs with a relatively positive resting membrane potential, it could trigger Kv3.1 channel activation, and therefore cell proliferation and differentiation.

To evaluate NPC neuronal differentiation, we established an imaging assay selectively detecting newly generated neurons, i.e. neuroblasts, using NPCs from adult DCX-GFP mice. DCX is a specific marker for neuroblasts and selectively expressed in neurogenic regions, such as the SVZ, rostral migratory stream and dentate gyrus (Gleeson et al. 1999; Walker et al. 2007). We previously reported that electrophysiological properties of DCX-positive neuroblasts in the SVZ are different from those of NPCs, except for them both exhibiting remarkable TEA-sensitive KDR channel currents (Walker et al. 2007). However, the KDR current density of DCX-positive neuroblasts (∼30 pA pF−1 at +40 mV) was almost one-third that of NPCs (∼100 pA pF−1 at +40 mV; Liu et al. 2006; Walker et al. 2007; Yasuda et al. 2008). Similarly, the total outward current size of DCX-positive neuroblasts was about 7–10 times less than that of GFAP-expressing NPCs (Lai et al. 2010). In this context, it has been speculated that KDR channels are more relevant in early stages of adult neurogenesis, where NPCs expand and give rise to neuroblasts. In agreement with this, our neuronal differentiation assay showed that TEA treatment and Kv3.1 knockdown reduces neuroblast generation. This indicates that Kv3.1, at least in part, plays an important role in NPC differentiation into neuroblasts.

Our findings demonstrate that Kv3.1 is functionally relevant in adult NPC expansion and neuronal lineage commitment. The functional roles of Kv3.1 in adult neurogenesis need to be confirmed in future in vivo studies. Under normal physiological conditions, adult mouse NPCs in the SVZ are primarily involved in replacing olfactory neurons (Imayoshi et al. 2008; Whitman & Greer, 2009). Neuroblasts derived from NPCs in the SVZ migrate into the olfactory bulb and differentiate into granule cells or periglomerular cells, which are integrated into the olfactory system. Under pathological conditions, the SVZ can supply neuroblasts to the damaged or neurodegenerative area in the cerebral cortex and striatum (Curtis et al. 2012). Pathological conditions that affect neurogenesis in the SVZ include ischaemic injury/stroke (Jin et al. 2001, 2003; Yamashita et al. 2006; Leker et al. 2007) and neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease and Parkinson's disease (Jin et al. 2004; Ziabreva et al. 2006; Rodríguez et al. 2009; Cherubini et al. 2010; Hamilton et al. 2010; Curtis et al. 2012). Therefore, it is important to investigate the role of Kv3.1 expressed in NPCs under not only normal physiological conditions but also pathological conditions.

Ion channels are well recognized as potential drug targets for the treatment of various diseases. Although the activation mechanism of Kv3.1 is unclear, our data suggest that selective activation of Kv3.1 in adult NPCs may be a new therapeutic approach to treating neurodegenerative diseases.

Acknowledgments

This work was supported by an NHMRC Project Grant (511091) awarded to D.J.A. D.J.A. is an Australian Research Council (ARC) Australian Professorial Fellow. We thank Henry Simila and Dr Linette Tan for their valuable help with tissue culture and functional assays of neurospheres.

Glossary

BrdU

5-bromo-2-deoxyuridine

DAPI

4′,6-diamidino-2-phenylindole

DCX

doublecortin

EGF

epidermal growth factor

FGF-2

fibroblast growth factor 2

GFAP

glial fibrillary acidic protein

GFP

green fluorescent protein

KA

rapidly inactivating, A-type K+ channel

KDR

voltage-gated delayed rectifier K+ channel

NPC

neural stem/precursor cell

NSA

neurosphere assay

PI

propidium iodide

qRT-PCR

real-time quantitative RT-PCR

siRNA

small interfering RNA

SVZ

subventricular zone

Author contributions

This work was carried out at the Queensland Brain Institute, University of Queensland, and the Health Innovations Research Institute, RMIT University. T.Y. and D.J.A. designed the experiments. T.Y., H.C. and D.J.A. collected, analysed and interpreted data. T.Y., H.C. and D.J.A. drafted and revised the article.

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