Synergistic Effect of Transient Receptor Potential ... - ACS Publications

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells Andrea Boente-Juncal, Carmen Vale,* Amparo Alfonso, and Luis M. Botana* Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain ABSTRACT: Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results presented here lead to the identification of the TRPC4 channels as contributors to the MTX effects in human neuronal cells. Both, the calcium increase and the cytotoxicity of MTX were either fully (for the calcium increase) or partially (in the case of cytotoxicity) reverted by the blockade of canonical TRPC4 receptors with the selective antagonist ML204. Furthermore, the sodium proton exchanger blocker amiloride also partially inhibited the calcium rise and the cell death elicited by MTX while the combination of amiloride and ML204 fully prevented both the cytotoxicity and the calcium rise elicited by the toxin. KEYWORDS: Maitotoxin, CTX0E16 cell line, neuronal stem cell, cytosolic calcium concentration, neuroprotection, MTT, amiloride, transient potential receptor channel, TRPC4 channels

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the last decades, especially in the Canary Islands11,12 and Madeira.13 Ciguatera food poisoning is a complex human illness and the clinical features of ciguatera can be split into three categories: gastrointestinal, neurological, and cardiovascular. Gastrointestinal acute symptoms begin 6−12 h after fish consumption and include diarrhea, abdominal pain, vomiting, and nausea, but they use to resolve within 1−4 days. Cardiovascular problems such as bradycardia and hypotension may be present during this acute period.8 Neurological symptoms may also emerge and include paresthesias, which spread centrifugally, numbness and tingling of extremities and perioral region, and dysesthesias. Persistent fatigability and weakness are often accompanied by depression, and all of them are common features of chronic ciguatera.8,14 Ciguatoxin and maitotoxin share a part of their chemical structure, with many ether rings forming a ladder shape.1,15 While CTX act mainly through activation of voltage gated sodium channels and blockade of voltage gated potassium channels, the mechanism of action of MTX is not yet completely

INTRODUCTION Maitotoxins (MTX) are among the largest natural nonpolymeric compounds, and MTX-1 is one of the most potent marine toxins identified to date.1,2 These toxins are produced by microscopic algae known as dinoflagellates of the genera Gambierdiscus and Fukuyoa.3,4 Four analogues of MTX named maitotoxin-1 (MTX1), maitotoxin-2 (MTX-2), maitotoxin-3 (MTX-3), and maitotoxin-4 (MTX-4) have been identified from different lineages of these dinoflagellates.3,5,6 In addition to MTX, Gambierdiscus toxicus also produces other toxins such as ciguatoxins (CTX). Both toxins affect human health because they pass from primary producers to commonly consumed fish and based on their common origin7 both families of toxins are assumed to be involved in the origin of a human illness denominated ciguatera fish poisoning (CFP). CFP is one of the most common nonbacterial food borne diseases, which annually affects 10 000 to 50 0000 people worldwide8 although the prevalence of the disease could be underestimated.9 It is often stated that ciguatera is a tropical and subtropical disease, but the earlier map of CFP is now enlarged and the worldwide expansion of CFP is mainly attributed to climate change.10 Although the presence of Gambierdiscus has been reported mainly in tropical areas, CFP intoxications have been identified in Europe during © XXXX American Chemical Society

Received: March 19, 2018 Accepted: May 7, 2018 Published: May 7, 2018 A

DOI: 10.1021/acschemneuro.8b00128 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Concentration and time-dependent effects of MTX on the cell viability of human neuronal stem cells evaluated using the MTT assay. Five days exposure of neuronal cells to MTX, at concentrations ranging from 0.0001 to 1 nM, induced cell death in a concentration dependent manner either in inmature (A) or mature neurons (B). More detailed evaluation of the cytotoxicity of MTX concentrations ranging from 0.1 to 0.5 nM revealed that the cellular toxicity was already evident 24 h (C) and 2 h (D) after toxin addition. Data are mean ± SEM of three or four experiments, each performed in triplicate, from independent cultures.

elucidated.17−22 MTX is a potent activator of Ca2+ influx in a wide variety of cells which, ultimately, leads to cellular toxicity.16,17,20,22,23 However, there is a great controversy regarding the mechanism for the MTX-induced calcium influx and cell death. It has been suggested that MTX could cause the insertion of functional channels into the plasma membrane through a yet unknown mechanism, but it seems clear that MTX acts on plasmalemmal proteins since the toxin did not evoke calcium release from intracellular calcium stores.23,24 Recently, transient receptor potential channels (TRP) were involved in the intracellular calcium increase elicited by MTX in Xenopus laevis oocytes.16 TRP channels are nonselective monovalent cation channels, most of which allow calcium entry into the cell25 and, for a long time, MTX has been proposed to activate non selective calcium channels.19,23 The multimembered superfamily of TRP ion channels is organized into six families: classical or canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), muclopins (TRPML), polycystin (TRPP), and ANKTM1 (TRPA) which are found in many eukaryotic cells from yeast to mammals.26 Since their discovery, there has been considerable interest in the TRPC channel family regarding their possible role as mediators of Ca2+ entry into cells, particularly because TRPC channels mediated both membrane depolarization and sustained increases in intracellular calcium.25,27 Moreover, TRPCs were demonstrated to be involved in the calcium entry elicited by MTX in different cell types.16,19 Several pieces of evidence support the fact that the MTX-induced calcium influx was dependent on extracellular calcium24,28 and it was partially attenuated by modulators of TRPC channels or by silencing TRPC channels.16,23 However, in primary cultures of mice primary cortical neurons, MTX decreased the amplitude of the voltage gated sodium channel currents and caused cellular acidosis.17 In neuroblastoma−glioma hybrid cells and certain

pituitary tumor cells MTX-elicited Ca2+ influx was blocked, at least in part, by dihydropyridines that are selective blockers of type L voltage-dependent Ca2+ channels.29 Moreover, MTXinduced Ca2+ influx and intracellular acidification in primary cultured neurons were ameliorated by voltage-gated sodium channel blockers.17 Altogether, in spite of the fact that the mechanism of action of MTX is not yet elucidated, it is clear that MTX induces calcium influx in most cell types and profoundly affects cell functioning causing cell death. During the last two decades, conditionally immortalized human neural progenitor cells (hNPCs) have emerged as a robust source of native neural cells to investigate physiological mechanisms and toxicity both in health and disease.30,31 The conditionally immortalized, cortically derived, human NPC line, CTX0E16, provides a robust source of cortical neurons with functional properties and a glutamatergic phenotype providing an ideal model system to investigate neurodevelopmental mechanisms in native human cells.30 This previous work has also demonstrated that CTX0E16 NPCs express neurotransmitter gated G-protein-coupled receptors and ionotropic receptor subunits, including those belonging to glutamatergic, dopaminergic, serotonergic, and cholinergic receptor families.30,31 Thus, in this work, we aimed to analyze the effect of MTX in differentiated human neuronal stem cells analyzing the effects of the toxin on cellular viability and intracellular calcium levels using calcium imaging and pharmacological blockade of the MTX-induced calcium entry. The data presented here indicate that human neuronal stem cells were sensitive to the MTX-induced cytotoxicity in a similar way to other cellular models including primary cultures of cortical neurons. Furthermore, the effects of several pharmacological approaches on the MTX-induced calcium entry support a prominent role of B


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ACS Chemical Neuroscience TRPC4 channels on the MTX-induced calcium entry in human neuronal stem cells.

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RESULTS MTX Caused a Concentration and Time-Dependent Cell Death in Neuronal Stem Cells. In vitro toxicity of MTX in the CTX0E16 cell line was evaluated using the MTT assay. Initially, the effect of the toxin on cell viability was assessed after 5 days exposure of neuronal stem cells to MTX concentrations ranging from 0.0001 to 1 nM. As indicated in Figure 1, MTX produced a concentration-dependent decrease in cell viability, causing complete cell death at 1 nM while at 0.1 nM MTX no cell death was observed. First, the effect of MTX on cell viability was evaluated in neurons differentiated for 7 days (Figure 1A). In this case, the IC50 for MTX-induced cell death was 2.04 × 10−10 M with a 95% confidence interval (CI) between 1.12 × 10−10 and 3.7 × 10−10 M. Nevertheless, we have previously reported that 24 h exposure of murine cortical neurons to MTX caused a concentration-dependent decrease in cell viability, causing complete cell death at 1 nM.17 Therefore, in order to determine if the low toxicity of MTX could be due to the immaturity of the cells, next we evaluated the effect of the toxin in human neuronal stem cells differentiated for 30 days (Figure 1B). Similarly to that observed in immature cells, in this case, the IC50 for MTXinduced cell death was 2.32 × 10−10 M (95% CI: 1.5 × 10−10−3.6 × 10−10 M). However, insofar as the time of exposure of CTX0E16 cells was much larger than the treatment in cortical neurons, additional experiments were performed to evaluate the toxicity of intermediate MTX concentration with respect to the exposure time. As shown in Figure 1C, 24 h exposure of differentiated CTX0E16 cells to MTX yielded a complete cell death at concentrations of 0.25 nM providing an IC50 of 0.13 nM (95% CI intervals: 1.5 × 10−11−1.1 × 10−9 M). A similar result was found when the differentiated cells were exposed to MTX for 2 h (Figure 1D). Also, in this case a complete absence of cell death was observed at MTX concentrations of 0.1 nM while at 0.25 nM the toxin caused complete cell death. In these conditions the IC50 for the MTX-induced cell death was 1.6 × 10−10 M (95% CI: 1.3 × 10−10−2.0 × 10−10 M). However, even in long-term treatments (14 days) the toxin did not produce any toxicity at concentrations of 0.1 nM. MTX Caused a Concentration Dependent Increase in the Cytosolic Calcium Concentration ([Ca2+]c) That Was Dependent on the Presence of Calcium in the Extracellular Medium. Fluctuations in the neuronal calcium concentration, [Ca++]c, act as signals that regulate numerous cell functions including neuronal excitability, neurotransmitter release and synaptic plasticity and apoptosis.32,33 Previous reports have clearly demonstrated that MTX increased calcium flux in a wide range of cell types.17,19,20,22−24,29 Nevertheless, the mechanisms involved in the intracellular calcium increase induced by MTX are still controversial, and, as far as we know, the effects of the toxin had not yet been evaluated in human neuronal stem cells. Therefore, we first ought to evaluate the effect of MTX on the cytosolic calcium concentration in differentiated CTX0E16 cells. As indicated in Figure 2, MTX at concentrations ranging from 0.1 nM to 5 nM increased [Ca2+]c in a concentration dependent manner, reaching a maximal effect at concentrations of 1 and 5 nM (Figure 2A). Thus, measuring the cytosolic calcium concentration at the time point of 400 s, MTX at 0.5, 1, and 5 nM significantly enhanced the [Ca2+]c by 23.7 ± 5.05% (n = 3; p < 0.01; t = 0.0017), 62.2 ± 13.46% (n = 3; p < 0.01; t = 0.0017), and 64.68 ± 4.36% (n = 3, p < 0.001; t =

Figure 2. Effects of MTX on the cytosolic calcium concentration in human neuronal stem cells. (A) MTX, at concentrations ranging from 0.1 to 5 nM, produced a concentration dependent increase on [Ca2+]c. The arrow indicates toxin addition. (B) Bath application of 1 nM MTX in a Ca2+-free medium (first arrow) did not modify basal calcium levels until calcium was reestablished to the bath solution (second arrow). (C) Elimination of sodium from the bathing medium did not alter the rise in calcium elicited by 1 nM MTX. First arrow indicates toxin application and the second gray arrow represents the reestablishment of sodium to the extracellular media. Data are mean ± SEM from three to five independent experiments, each performed in duplicate.

1.46 × 10−6), respectively. Hence, in human neuronal stem cells, the MTX-induced calcium increase was similar to that previously reported in primary cultures of mice cortical neurons,17 although in this case the MTX concentration needed to produce a significant rise in calcium was higher (0.5 nM in our study and 0.1 nM in primary neurons). In view of these effects, in the rest of the experiments, the toxin was employed at concentrations of 1 nM. Next, and since it has been previously described that the MTX effects on intracellular calcium were strongly dependent on the presence of extracellular calcium ions,19,23,24,28 the effect of the toxin in calcium-free medium was evaluated. As shown in Figure 2B, at 1 nM, the toxin did not elicit a cytosolic calcium increase in a calcium free medium; however, after reestablishment of calcium to the extracellular medium, the toxin evoked a rapid rise in calcium that was faster and bigger than the calcium entry C


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Table 1. Pharmacological Agents Acting on TRP Channels Employed to Analyze the Effect of MTX on the Cytosolic Calcium Concentration in the Human Neuronal Cell Line CTX0E16 channel inhibitor

source

concn

selectivity

SKF96365 lanthanum gadolinium 2-APB clemizole hydrochloride ML204 amiloride

Tocris Sigma-Aldrich Tocris Tocris Tocris Tocris Sigma-Aldrich

30 and 50 μM 1 mM 50 μM 50 μM 10 μM 2, 10, and 50 μM 100 and 500 μM

TRPC1,3,4,5; TRPV2; TRPP1, STIM1, voltage gated Ca2+ T-type channels, K channels TRPC1,3,4,5,6; TRPM3,8; TRPP2, TRPV2,4, 6 TRPC1,3,5; TRPM;·,TRPP2; TRPV4; Nav2.1; ASIC3; Maxi Cl; CaSR TRPM3,7,8; TRPC1,3,4,5,6,7; TRPV1,6 TRPC5,4,3,6,7; TRPM3,8; TRPV1−4, H1-antagonist. TRPC4,5,6 ASIC1, ASIC2, ASIC3, sodium/hydrogen exchanger 1 and 3, ENaC α, β, and γ, TRPP3

effect of this TRP blocker on the MTX-induced calcium influx was evaluated (Figure 3). However, in contrast with the previous

elicited by the toxin in a calcium containing medium. Thus, in the calcium-containing medium, the calcium peak elicited by 1 nM MTX occurred about 180 s after the addition of the toxin while in calcium-free medium, the calcium peak occurred about 50 s after bath application of Ca2+. The lack of effect of MTX in absence of calcium in the extracellular medium is in agreement with previous studies in different cell types.19,24 Furthermore, the effect of the toxin was also evaluated in a sodium free medium (with sodium equimolarly replaced by N-methyl-D-glucamine, NMDG) since the nonselective cation currents evoked by MTX in Xenopus oocytes have been shown to be abolished in the absence of extracellular sodium.23To this end, the effect of 1 nM MTX on the cytosolic calcium concentration was evaluated in normal Locke’s buffer and in sodium-free Locke’s buffer as indicated in Figure 2C. In human neuronal stem cells, substitution of external Na+ by NMDG did not modify the basal calcium levels, but it slightly delayed the calcium rise induced by MTX. Therefore, insofar as it can be ascertained, the effects of MTX on human neuronal stem cells are similar to those previously described in CHO cells and in Xenopus oocytes showing the dependence of the MTX effects on the presence of extracellular calcium and sodium ions.23 Effects of TRP Channel Blockers on the MTX-Induced Calcium Increase in Human Neuronal Stem Cells. In the central nervous system, TRP channels are one of the largest groups of ion channels that act as cellular sensors and translate fluctuations in the external milieu into changes in membrane excitability and second messenger signals, particularly Ca2+. In general, TRP channels are cation channels weakly voltagesensitive and poorly selective.26 So far no reports have evaluated the role of TRP channels in calcium rises in the human neuronal cell line CTX0E16. However, TRP channels, particularly those of the canonical type TRPC1 and TRPC4, are present in human postmitotic neurons where they participate in the generation of robust calcium transients which are particularly sensitive to the canonical TRP antagonist SKF96365.34 Nowadays, and remarkably important for the treatment of the symptomatology associated with ciguatera fish poisoning, TRP blockers are regarded as pharmacological agents potentially useful to alleviate neuropathic pain.35 Previous work has evidenced the role of MTX as activator of TRPC1 channels in both liver cells and Xenopus oocytes.16,19 To evaluate the involvement of TRP channels on the MTX-induced calcium increase, several pharmacological blockers were evaluated (Table 1); however, it should be noted that, besides the compound 4-methyl-2-(1piperidinyl) quinoline (ML204), most of the commercially available blockers of TRP channels are not selective as indicated in Table 1.36,37 Pan-TRP Blockers Did Not Affect the MTX-Induced Calcium Increase. Since SKF96365 was previously reported to inhibit all the MTX effects in human C6 glioma cells,20 first, the

Figure 3. Effect of SKF96365 on the [Ca2+]c increase elicited by 1 nM MTX. (A) Addition of 30 μM SKF96365 to the bath solution (first, gray arrow) failed to significantly affect the MTX-induced calcium increase. (B) Preincubation of CTX0E16 cells with 50 μM SKF96365 15 min before bath application of MTX slightly delayed the rise in [Ca2+] elicited by the toxin. Black arrows indicate toxin addition. Results are expressed as means ± SEM of three to seven independent experiments, each performed in duplicate.

data in glioma cells in our cellular model, SKF96365 at 30 μM failed to block the MTX-induced calcium increase as shown in Figure 3A. In view of this result, CTX0E16 cells were preincubated for 10 min in the presence of 50 μM SKF96365 (Figure 3B). In this case, although the magnitude of the calcium increase elicited by the toxin was not modified by the channel blocker, the rise in calcium was delayed by about 80 s when the toxin was added to the cells previously treated with 50 μM SKF96365. Next, the blocking effects of the trivalent cations lanthanum (La+++) and gadolinium (Gd+++) on the MTX-induced calcium increase were evaluated. Both La+++ and Gd+++ have been reported to inhibit the currents induced by low MTX concentrations in Xenopus oocytes but not the currents elicited by MTX concentrations higher than 700 pM38,39 and are also non selective blockers of TRP channels (Table 1). As shown in Figure 4 none of the compounds blocked the calcium rise elicited by 1 nM MTX in human neuronal cells. Figure 4A shows the D


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Figure 5. TRPC5 channel blockers had little effect on the MTX-induced calcium increase. Neither 50 μM 2-APB nor 10 μM clemizole affected the calcium increase evoked by 1 nM MTX. The gray arrows indicate solvent or blocker addition and the black arrows represent solvent or MTX addition to the bath solution. Results are mean ± SEM of at least three independent experiments, each performed in duplicate.

Figure 4. Effect of the trivalent cations lanthanum and gadolinium on the MTX- induced calcium increase. (A) Gadolinium, at 50 μM, did not modify the MTX elicited calcium entry. (B) Lanthanum, at 1 mM, elicited a small delay in the calcium increase observed after bath application of MTX without affecting the peak calcium rise elicited by the toxin. Gray arrows indicate solvent or cation addition and black arrows indicate bath application of maitotoxin. Results are mean ± SEM of three to six independent experiments, each performed in duplicate.

compound alone induced a small cytosolic calcium increase, a fact that was previously demonstrated in glomerular cells.44 Figure 6A, shows that at 2 μM ML204 alone caused a small calcium increase and not fully prevented the rise in calcium elicited by MTX, although the plateau calcium increase elicited by MTX in the presence of 2 μM ML204 was decreased by 28.17 ± 13.03% (n = 5; t = 2.163; df = 9) when measured 10 min after baseline, although this decrease was not statistically significant. Similarly, after bath application of 10 μM ML204 (Figure 6B) a rapid increase in the cytosolic calcium concentration was observed, but further application of 1 nM MTX did not modify the cytosolic calcium concentration. In this case, the [Ca2+]c in the presence of both ML204 and MTX was 59.30 ± 7.01%, lower than that evoked by MTX alone (n = 3; t = 8.456; df = 6; p = 0.0001). A similar situation was observed after bath addition of 50 μM ML204 and the subsequent application of 1 nM MTX (Figure 6C). In this case, at the time point of 600 s, the cytosolic calcium increase evoked by MTX was decreased by 61.59 ± 6.69% (n = 3; t = 9.21; df = 6; p < 0.001), in the presence of 50 μM ML204 versus the [Ca2+]c increase elicited by MTX alone. A summary of the effects of the different concentrations of ML204 on the MTX-induced calcium influx is shown in Figure 6D, measuring the 340/380 ratio at the time point of 600 s. As shown in the figure, 1 nM MTX increased the [Ca2+]c by 74.18 ± 4.52% versus control cells, while in cells treated with 2, 10, and 50 μM ML204 the calcium increment was reduced to 46.00 ± 16.05%, 14.88 ± 4.89%, and 12.58 ± 3.94%, respectively. Largely, in view of the failure of 1 nM MTX to elicit further calcium increases in the presence of different concentrations of ML204, these results suggest that the cytosolic calcium increase evoked by maitotoxin in human neuronal stem cells is primarily mediated by activation of TRPC4 receptors. MTX Increased the Expression of TRPC4 Channels in Human Neuronal Stem Cells. A relevant physiological characteristic of TRPC channels is that their expression is regulated by changes in intracellular calcium levels.45 Hence,

effect of 50 μM Gd+++ on the MTX elicited calcium entry. In contrast with the previously described effect of gadolinium partially inhibiting the MTX-induced currents in Xenopus oocytes with an EC50 of 158 μM39 no effect of Gd+++ was observed on the calcium influx caused by MTX in differentiated CTX0E16 cells. Next, the effect of lanthanum was also evaluated. La+++ has been shown to block the MTX induced current by about 75% in Xenopus oocytes39 and it is also an unspecific blocker of TRP channels. As shown in Figure 4B, at 1 mM, La+++ did not modify the maximum rise in cytosolic calcium elicited by MTX but it delayed more than 100 s the calcium increase elicited by the toxin. TRPC5 Channel Blockers Had Little Effect on the MTXInduced Calcium Increase. Next, the effect of 2-aminoethoxydiphenyl borate (2-APB) on the MTX induced calcium influx was evaluated. 2-APB at micromolar concentrations blocks heterologously expressed TRP channels, such as TRPC1, TRPC3, TRPC5, TRPV6, TRPM8, and TRPV1−3, showing high affinity for the TRPC5 channel while acting as an agonist at TRPV1, TRPV2, and TRPV3 channels.40,41 As shown in Figure 5A, at 50 μM, 2-APB did not affect the calcium rise evoked by MTX and only delayed the toxin-induced rise in calcium by about 50 s. A similar situation was found with the other potent TRPC5 antagonist clemizole,42 which at 10 μM did not affect the MTXinduced calcium influx (Figure 5B). TRPC4 Blockers Decreased the MTX-Induced Calcium Increase. Finally, the effect of ML204, a compound known as a selective inhibitor of TRPC4 channels37 was evaluated. ML204 selectively blocks TRPC4 with an IC50 value of 0.96 μM and exhibited 19-fold selectivity against TRPC6 channel activation and modest selectivity against TRPC3 and TRPC5 (9-fold) channels.43 As shown in Figure 6, different ML204 concentrations affected the calcium response elicited by MTX. At the three ML204 concentrations evaluated (2, 10, and 50 μM), the E


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Figure 6. TRPC4 antagonist ML204 decreased the [Ca2+]c increase produced by 1 nM MTX in a concentration dependent manner. (A) Effect of 2 μM Ml204. (B) Bath application of ML204 at 10 μM increased the basal calcium level of CTX0E16 cells and preventer any further rise in calcium after bath application of MTX. (C) A similar situation was observed in the presence of 50 μM ML204. Gray arrows represent solvent or ML204 addition and black arrows indicate solvent or MTX bath application. (D) Pooled results representing the mean increase, at the time point of 600 s, in the calcium concentration elicited by MTX in the absence or presence of the different ML204 concentrations with respect to control values. Values are means ± SEM of three to six independent experiments, each performed in duplicate. ***p < 0.001.

Figure 7. Long-term treatment of differentiated CTX0E16 cells with very low concentrations of MTX increased the expression of TPRC4 proteins. The left panel shows representative experiment showing Western blot bands indicating the level of expresion of TPRC4 in control neurons and in neurons treated for 14 days with 0.01 or 0.1 nM MTX. The corresponding band intensities quantifications are shown on the right. Both concentrations of MTX significantly increased the expression of TRPC4 channels. Values are means ± SEM of three to four independent experiments and triplicate wells were used in each condition. **p < 0.01.

band intensity was 100.0 ± 5.7 (n = 4), while in the presence of 0.01 nM MTX TRPC4 band intensity was 135.3 ± 7.5 (n = 4; t = 3.77; df = 6; p = 0.009). In cells maintained with 0.1 nM MTX, the corresponding TRPC4 band intensity was 137.1 ± 4.98 (n = 3; t = 4.70; df = 5; p = 0.005). Thus, the fact that the specific TRPC4 blocker inhibited the MTX induced calcium increase, together with the upregulation of TRPC4 expression after longterm exposition of human neuronal stem cells to MTX, suggest that TRPC4 receptors are involved in the MTX-induced calcium rise in human neuronal stem cells. The Combination of Amiloride and ML204 Fully Blocked the MTX-Induced Rise in [Ca2+]c. As mentioned above, to date, the MTX-elicited calcium influx has been attributed to a wide variety of mechanisms, including nonselective cation channels,19,38,46,47 TRPC1,16,19,48 receptoroperated Ca2+ channels,20 and also to the sodium/calcium

decreasing the [Ca2+]c will decrease the expression of TRPC channels and vice versa. In view of the ability of the selective TRPC4 blocker ML204 to abolish the calcium rise elicited by MTX, we sought to demonstrate the expression of the TRPC4 protein in these cells and its modulation by MTX. To this end, next, the effect of chronic exposure of the cells to low, nontoxic, concentrations of MTX on TRPC4 protein levels was evaluated. To do this, CTX0E16 cells differentiated for 30 days were treated with MTX concentrations of 0.01 and 0.1 nM for 14 days in the culture medium. At the MTX concentrations chosen for the chronic treatment, the cells remained healthy without showing signs of toxicity. Figure 7 shows representative Western blot bands for TRPC4 protein expression in human neuronal stem cells, either in the absence or in the presence of the different concentrations of MTX on the left. The corresponding band quantifications are shown on the right. In control cells, TRPC4 F


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Figure 8. Combined application of amiloride and ML204 reverted the calcium increase elicited by 1 nM MTX to basal levels. (A) 500 μM amiloride significantly decreased the MTX-elicited calcium influx. (B) The combination of 100 μM amiloride and 2 μM ML204 completely avoided the calcium rise evoked by 1 nM MTX. Results are expressed as mean ± SEM of at least three independent experiments. **p < 0.01 versus MTX alone.

Figure 9. Combination of amiloride and ML204 fully reverted the toxic effect of MTX in human neuronal stem cells. (A) Amiloride, at 500 μM but not at 100 μM, partially decreased the cytotoxic effect of MTX. (B) ML204 at 50 μM and 100 μM decreased the cell death elicited by MTX. (C) 100 μM amiloride in combination with 50 μM ML204 only partially prevented the MTX-induced toxicity but this effect was complete in the presence of 100 μM amiloride and 100 μM ML204. (D) In the simultaneous presence of 500 μM amiloride and either 50 or 100 μM ML204, an almost complete prevention of the MTX induced cell death was observed. Data are means ± SEM of four to six independent experiments performed in triplicate wells for each experimental condition. Cell viability was assessed by the MTT assay.

exchanger operating in reverse mode49 or to the activation of the sodium hydrogen exchanger in cortical neurons.50 In this way,

MTX has been shown to cause intracellular acidification in primary cultured neurons17 and blockade of NHE by amiloride G


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and NHE blockers against the MTX elicited calcium increase in human neuronal stem cells. Nowadays, there is broad evidence indicating that MTX modulates several ion channels and many cellular functions could therefore be altered as a consequence of its effect on intracellular calcium levels. For instance, we have described previously that, in cortical neurons, MTX affected voltage gated sodium channels decreasing peak sodium current amplitude, increased the cytosolic calcium concentration and elicited intracellular acidification.17 Although so far, multiple evidence have confirmed the massive calcium influx caused by the toxin which is presumed to lead to cell death, there is a wide controversy regarding the cellular pathways involved in these effects. Therefore, up to date, the MTX-induced Ca2+ influx has been attributed to a very wide range of targets such as nonselective cation channels,19,51 voltage-gated calcium channels,22,52 receptor-operated Ca2+ channels, to the sodium/calcium exchanger operating in reverse mode49 or to activation of the sodium hydrogen exchanger (NHE) in cortical neurons.50 Recently, TRPC1, but not TRPC4, channels were also revealed as responsible for the MTX-induced currents in Xenopus oocytes.16 Thus, in this recent work, it has been shown that picomolar concentrations of MTX selectively activated TRPC1 channels; however, in this case TRPC4 blockade did not alter the MTX effect but the expression of the TRPC4 protein was decreased by the silencing of the TRPC4 gene, a fact that could hinder to observe the effect of the toxin on TRPC4 channels.16 In this work, the effect of the toxin on human neuronal cells was evaluated for the first time. We have demonstrated recently that neuronal stem cells differentiated from the CTX0E16 cell line provide a human, nontumor, cellular model useful to evaluate the cytotoxic effect of marine compounds.31 The results presented here indicate that, as expected, maitotoxin decreased cell viability in a concentration dependent manner, showing a fast effect on cell viability at concentrations higher than 0.1 nM. However, toxin concentrations up to 0.1 nM did not affect cell viability even after 7 or 14 days of exposure of the cells to the toxin. Moreover, the toxin also had a concentration-dependent effect on the cytosolic calcium concentration. However, even at concentrations that caused only a small rise in the cytosolic calcium concentration (i.e., 0.5 nM), the toxin caused a complete cell death after 2 h exposure of the cells to the toxin. The suitability of differentiated human neuronal stem cells to evaluate the cytotoxicity of MTX was supported by the fact that the magnitude of the calcium increase elicited by MTX in this cellular model was similar to that previously reported in other neuronal preparations including primary cultures of cortical neurons17 and showed a strong dependency on the presence of calcium and sodium in the extracellular bathing solution as previously reported.19,23,24 Pharmacological approaches to evaluate the mechanisms for the MTX elicited calcium increase and cell death have not yet analyzed the involvement of TRP channels, although MTX has been proposed for a long time to activate non specific cation channels.19,46,51 In this work the assessment of the mechanisms involved in the calcium increase elicited by MTX in human neuronal stem cells revealed that the TRPC4 channel subtype was involved in the effects of MTX in differentiated human neuronal stem cells. In our experimental conditions, MTX induced a rapid rise in the cytosolic calcium concentration that was blocked by the selective TRPC4 receptor antagonist ML204 in a concentration dependent manner. Although, so far, no studies have evaluated the expression of TRP receptors in the CTX0E16 cell line, the TRPC4 subtype was reported to be the

derivatives reduced the calcium increase and the cell death elicited by MTX in the same cellular model.50 Moreover, amiloride, in the micromolar range, has been shown to block TRPP3 channels.36 Therefore, in the next set of experiments, the effect of 500 μM amiloride over the MTX-induced calcium increase was evaluated. As shown in Figure 8, at 500 μM, amiloride significantly decreased the MTX-elicited calcium influx (Figure 8A) by 63.04 ± 12.60% (n = 5; t = 5.001; df = 10; p = 0.0005), a result that was in agreement with that previously reported in cortical neurons.50 In view of the individual effects of both amiloride and ML204 on the MTX-induced calcium increase, in the following experiments, the effect of the combination of both compounds was evaluated. As shown in Figure 8B, bath application of 100 μM amiloride and 2 μM ML204 fully prevented the MTX-induced calcium increase in human neuronal stem cells, decreasing the MTX-elicited calcium influx by about 69.50 ± 12.11% (n = 3; t = 5.74; df = 4; p = 0,005) and returning the cytosolic calcium concentrations to control values. The Combination of Amiloride and ML204 Fully Prevented the MTX-Induced Toxicity. Finally, we tried to determine if both TRPC4 receptors and the sodium hydrogen exchanger were involved in the toxic effect of MTX in human neuronal cells. In order to do this, we evaluate the ability of these compounds to block the toxicity of maitotoxin in this preparation. With this purpose, cells were pretreated with either amiloride at 100 or 500 μM, ML204 at 50 or 100 μM or the combinations of both compounds at the two concentrations for 30 min before addition of 0.25 nM MTX for 1 h (Figure 9). As shown in Figure 9A, at 500 μM, amiloride increased the cell viability in cells treated with 0.25 nM MTX from 25.8 ± 6.5% (n = 6) in cells treated with MTX alone to 58.6 ± 8.5 (n = 4) in cells treated with the combination of 0.25 nM MTX and 500 μM amiloride (t = 3.1; df = 8; p < 0.05). However, no significant differences were found in the presence of 100 μM amiloride alone. Interestingly and accordingly with the proposed interaction of MTX with TRPC4 channels, the TRPC4 antagonist ML204 at 50 and 100 μM significantly decreased the cell death elicited by MTX. As displayed in Figure 9B, the mitochondrial function was 25.8 ± 6.5% of control (n = 6) in the presence of 0.25 nM MTX alone, 57.7 ± 1.7% (n = 6) in the presence of 0.25 nM MTX + 50 μM ML204 (t = 4.7; df = 10; p < 0.001) and 72.7 ± 3.7% (n = 4) when the cells were preincubated with 100 μM ML204 before MTX addition (t = 5.4; df = 8; p < 0.001). Furthermore, the combination of 100 μM amiloride with 50 and 100 μM ML204 (Figure 9C) further decreased the cell death elicited by MTX from 34.2 ± 5.5% (n = 4) of control in cells treated with MTX only, to 71.3 ± 6.4% (n = 4) for cells kept in the presence of 100 μM amiloride and 50 μM ML204 (t = 4.4; df = 6; p < 0.005) and to 108.8 ± 4.1% (n = 4) when 100 μM amiloride and 100 μM ML204 were added before the MTX treatment (t = 10.9; df = 6; p < 0.0001). However, the combined effect achieved in the presence of 500 μM amiloride and 50 or 100 μM ML204 was lower (Figure 9D). In this case, for MTX alone, cell viability was 34.2 ± 5.5% of control values (n = 4) and the mitochondrial function was significantly increased to 82.0 ± 4.2% (n = 4; t = 6.9; df = 6; p = 0.0005) in cells treated with MTX after preincubation with 500 μM amiloride and 50 μM ML204 and to 88.8 ± 9.9% (n = 4; t = 4.8; df = 6; p = 0.0030) in cells treated with MTX after pretreatment with the combination of 500 μM amiloride and 100 μM ML204. Consequently, these results confirm the synergistic effect of both TRPC4 antagonists H


Research Article


bicarbonate (Sigma) supplemented with 0.03% human serum albumin (Sigma), 100 μg/mL apotransferrin (Scipac Ltd., Kent, U.K.), 16.2 μg/ mL putrescine (Sigma), 5 μg/mL human insulin (Sigma), 60 ng/mL progesterone (Sigma), 2 mM L-glutamine (Sigma), and 40 ng/mL of sodium selenite (Sigma). Under proliferative conditions, cells were cultured in the presence of 10 ng/mL human fibroblast growth factor (FGF2), 20 ng/mL human epidermal growth factor (EGF), both from PeproTech (Rocky Hill, NJ), and 100 nM hydroxy tamoxifen (4-OHT, Sigma). CTX0E16 hNPCs were seeded onto poly-D-lysine (PDL, 5 μg/ cm2, Sigma,) and laminin-coated (1 μg/cm2; Sigma) tissue culture flasks, with full media changes occurring every 2−3 days. Cells were passaged once they reached 70−80% confluence using accutase (Sigma) and maintained between 25 and 30 passages; all experiments were carried out using cells from passages 12 to 30. Neural Progenitor Cell Differentiation. For differentiation, CTX0E16 cultures were washed twice with nonsupplemented DMEM:F12 medium and passaged onto PDL and laminin-coated tissue culture plates or glass coverslips at a density of 50 000 cells/mL. Cells were then washed in warm Dulbecco’s phosphate-buffered saline (DPBS; Thermofisher) and maintained in neuronal differentiation media (NDM: Neurobasal Medium (Thermofisher) supplemented with human serum albumin, apotransferrin, putrescine, human insulin, progesterone, L-glutamine, and sodium selenite at the concentrations used for proliferation and 1 × B27 serum-free supplement (Thermofisher); half medium changes were performed every 2−3 days and cultures were differentiated for up to 40−50 days. Toxins and Drugs Used. MTX-1 was obtained from Wako and dissolved at a concentration of 10 μM in DMSO. Following dilutions were performed in deonized water and Locke’s buffer solution. The toxin had purity higher than 99%. The final concentration of compound solvent (DMSO) was less than 0.01%. All other chemicals were of reagent grade and purchased from Sigma and Tocris. Determination of Cellular Viability. Cell viability was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The assay was performed in cultures grown in 48 well plates and exposed to MTX-1 concentrations ranging from 0.0001 to 1 nM added to the culture medium. Initial experiments were performed in cultures maintained in the presence of the toxin at 37 °C in a humidified 5% CO2/95% air atmosphere for 5 days. After the exposure time, cells were rinsed and incubated for 1 h with a solution of MTT (500 μg/mL) dissolved in NDM as previously described.17,31 After washing off excess MTT, cells were disaggregated with 5% sodium dodecyl sulfate, and the absorbance of the colored formazan salt was measured at 595 nm in a spectrophotometer plate reader Synergy 2 (BioTek Instruments). Saponin or DMSO at 10% were used as death control and its absorbance was subtracted from the rest of the data. Western Blot. Cell cultures were maintained for 14 days in the presence of maitotoxin. Afterward, the cells were washed three times with cold PBS and cell lysates were prepared in RIPA lysis buffer (Thermofisher), containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40 (nonyl phenoxypoliethoxylethanol), 1% sodium deoxycholate and 0.1% SDS) supplemented with commercial phosphatase and protease inhibitors mixtures (Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail Kit, respectively, Thermofisher), and stored at −20 °C when needed. The total protein concentration for each lysate was determined in triplicate by the Bradford assay, using bovine serum albumin (BSA) as standard. Cell lysates samples containing 25 μg of total protein were denaturalized in commercial 4x Laemmli sample buffer (Biorad, containing 277.8 mM Tris-HCl, pH 6.8, 4.4% SDS, 44.4% glycerol, and 0.02% bromophenol blue, supplemented with 2.5% 2-mercaptoethanol), resolved by SDS-PAGE electrophoresis in 10% polyacryamide gels at a constant voltage of 200 V for 38 min. Afterward, proteins were transferred from the gel to PVDF membranes at a voltage of 10 V for 30 min using a semidry transfer cell (Biorad). The membranes were blocked for 1 h with 5% BSA following the antibody manufacturer’s recommendations and incubated overnight at 4 °C with a primary antibody against goat TPRC4 (Sigma) at a final concentration of 1 μg/μL. Immunoreactive bands were detected using the Supersignal West Pico chemiluminescent substrate (Pierce) and the Diversity 4 gel documentation and analysis system (Syngene, Cambridge, UK).

most prevalent protein of this group of channels expressed in neuronal human stem cells of 21 days of differentiation although both TRPC1 and TRPC4 were the primary subunits expressed in early human forebrain neurons.34 Accordingly with the proposed role of TRPC4 channels on the MTX-induced calcium influx, we demonstrated here, for the first time, the expression of such channels in the CTX0E16 cell line and its upregulation after chronic toxin treatment, an observation that is in agreement with the role of intracellular calcium on the regulation of these channels.45 Altogether the data presented here indicate that TRPC4 channels contribute to the calcium increase elicited by MTX in human neuronal stem cells and extend a previous report pointing to the role of TRPC1 channels in the MTX-induced currents.16 Although the relative expression of the different TRPC subunits was not evaluated in this work, the results presented here confirmed the importance of these channels for the calcium increase elicited by MTX. Furthermore, the combination of the specific TRPC4 inhibitor ML204 with amiloride, a known blocker of the NHE exchanger, which also blocks TRPP channels, fully blocked the calcium entry and the toxicity elicited by MTX thus confirming that several cellular functions are altered by this potent marine toxin. The effect of TRPC4 antagonists against the MTX-induced toxicity in human neuronal cells points out to a new player on the maitotoxin effects in mammalian cells. Both TRPC4 and TRPC5 are activated in a phospholipase C-dependent manner and are potentiated by micromolar concentrations of La+++ and Gd+++,53 a fact that will explain the lack of effect of these cations against the MTX elicited calcium increase demonstrated here. Interestingly, TRPC4 and TRPC5 respond to changes in extracellular pH54 and the currents through these channels are increased with extracellular acidification, and thus these channels were proposed to act as sensors of pH linking decreases in extracellular pH to calcium entry and depolarization.54 In this sense, it is noteworthy that we have previously reported that MTX induces intracellular acidification in cortical neurons, and recently the proton-sensing receptor involved in pH homeostasis named ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68), which activates with intracellular acidification, has been shown to promote the expression of TRPC4 in cerebellar granule cells.55 This reported linkage between intracellular pH and TRPC4 channels is likely the reason for the synergy between amiloride and ML204 against the MTX effects in human neurons. Moreover, our results could suggest that the intracellular acidification caused by MTX may be related to the increase in TRPC4 expression after long-term exposure of neuronal cells to low MTX concentrations. Although further studies are necessary to fully elucidate the mechanism of action of MTX, the data presented here constitute the first report indicating the involvement of the TRPC4 channels on the MTXinduced calcium increase and toxicity in a human neuronal cell line and point out to a relationship between the intracellular acidification reported earlier for the toxin and the elicited calcium increase and cell death.

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METHODS

Cell Line. Use of the immortalized human neural progenitor cells CTX0E16 cell line was kindly granted by ReNeuron Limited (Guildford, Surrey, U.K.) under a material transfer agreement. CTX0E16 cells were obtained from the developing embryonic cortex of a 12 week gestation fetus and conditionally immortalized by ectopic expression of the cMycERTAM transgene.30 Human neural progenitor cells CTX0E16 were cultured following the provider instructions as previously reported.30 Proliferating cells were maintained in reduced modified medium (RMM) containing DMEM:F12 with 15 mM HEPES and sodium I



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Chemiluminescence was measured with the Diversity GeneSnap software (Syngene). β-Actin (Millipore) was used as control for lane loading and to normalize chemiluminescence values. Each condition was analyzed in triplicate per each experiment. Determination of Cytosolic Calcium Concentration ([Ca2+]c). Mature neurons seeded onto 18 mm diameter glass coverslips were loaded with the cell permeant calcium sensitive dye Fura-2 acetoxymethyl ester (Fura-2 AM) at a final concentration of 2.5 μM for 30 min at 37 °C in Locke’s buffer containing in mM: 154 NaCl, 5.6 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, and 5.6 glucose (pH 7.4), supplemented with 0.1% BSA. After incubation, loaded cells were washed three times with cold Locke’s buffer. Experiments performed in calcium free medium were realized in nominally calcium free medium, without added calcium chelating agents. To evaluate the effects of the toxin in a sodium free medium, sodium was equimolarly replaced by Nmethyl-D-glucamine. The glass coverslips were then inserted into a thermostated chamber at 37 °C (Life Science Resources), and cells were viewed with a Nikon Diaphot 200 microscope, equipped with epifluorescence optics (Nikon 40× immersion UV-Fluor objective). The cytosolic calcium ratio was obtained from the images collected by fluorescence equipment (Lambda-DG4, Sutter Instruments). The light source was a xenon arc bulb, and the different wavelengths used were chosen with filters. The excitation wavelengths for Fura-2 were 340 and 380 nm, and emission was collected at 510 nm as previously described.56 Channel inhibitors were added before the addition of MTX and data were analyzed with the Metafluor software (Molecular Devices, LLC, Sunnyvale CA) and expressed as 340/380 ratio. The experiments were carried out in duplicate coverslips obtained from at least three independent cultures. Statistical Analysis. All data are expressed as means ± SEM of n determinations. Statistical comparison was by Student’s t test. P values < 0.05 were considered statistically significant.

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Research Article

ACKNOWLEDGMENTS

The authors thank ReNeuron Limited and researchers of the King College of London (Anderson GW, Williams BP, Srivastava DP) for kindly providing the CTX0E16 cell line and their culture conditions under a material transfer agreement.

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ABBREVIATIONS TRP, transient receptor potential channels; TRPC4, transient receptor potential channel classical or canonical type 4; MTX, maitotoxin; CTX, ciguatoxin; CFP, ciguatera fish poisoning; hNPC, human neural progenitor cells; dd, days of differentiation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 2-APB, 2-aminoethoxydiphenyl borate; ML204, 4-methyl2-(1-piperidinyl) quinoline; NHE, sodium−hydrogen exchanger; NDM, neuronal differentiation medium; RMM, reduced modified medium; PBS, phosphate buffered saline

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Mailing address: Departamento de Farmacologia,́ Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002, Lugo, Spain. Tel/Fax:34-982822233. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Carmen Vale: 0000-0002-9842-6223 Author Contributions

A.B.-J. performed the experiments, analyzed the data, prepared the figures, and wrote the manuscript with the contribution of C.V. L.M.B., A.A., and C.V. developed the idea, designed experiments for the study, and revised the manuscript. All authors participated in discussion and proofreading the manuscript. Funding

The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economia,́ Industria y Competitividad, AGL2014-58210-R, AGL201678728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC2016-5507-2. From CDTI under ISIP Programme, Spain, IDI20130304 APTAFOOD and ITC-20161072. From European Union POCTEP 0161-Nanoeaters −1-E-1, and Interreg AlertoxNet EAPA-317-2016. ABJ is recipient of a predoctoral fellowship from the Spanish Ministry of Education. Notes

The authors declare no competing financial interest. J


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