Intracellular Calcium Plays a Critical Role in the Microcystin-LR-Elicited Neurotoxicity Through PLC/IP3 Pathway

Abstract

Neurotoxicity of microcystin-leucine-arginine (MCLR) has been widely reported. However, the mechanism is not fully understood. Using primary hippocampal neurons, we tested the hypothesis that MCLR-triggered activation in intracellular free calcium concentration ([Ca²⁺]_i) induces the death of neurons. Microcystin-leucine-arginine inhibited cell viability at a range of 0.1 to 30 μmol/L and caused a dose-dependent increase in [Ca²⁺]_i. This increase in [Ca²⁺]_i was observed in Ca²⁺-free media and blocked by an endoplasmic reticulum Ca²⁺ pump inhibitor, suggesting intracellular Ca²⁺ release. Moreover, pretreatment of hippocampal neurons with intracellular Ca²⁺ chelator (O,O′-bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid, tetraacetoxy-methyl ester) and inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenyl borate) could block both the Ca²⁺ mobilization and the neuronal death following MCLR exposure. In contrast, the ryanodine receptor inhibitor (dantrolene) did not ameliorate the effect of MCLR. In conclusion, MCLR disrupts [Ca²⁺]_i homeostasis in neurons by releasing Ca²⁺ from intracellular stores, and this increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying MCLR-induced neurotoxicity.

Introduction

Microcytins (MCs) are toxic secondary metabolites produced by a range of distantly related cyanobacteria that proliferate in eutrophic freshwater bodies. ^1–2 So far, more than 80 structural-related congeners of MCs have been identified, with microcystin-leucine-arginine (MCLR) considered to be the most common and most toxic variant. ^3–4

The typical toxicological target of MCLR is liver. However, its potential neurotoxicity has been demonstrated in several studies. Microcytins can penetrate blood–brain barrier through organic anion-transporting polypeptides, ^5–6 subsequently accumulating and inducing toxicity in brain cells. Memory loss has been observed in rats after intrahippocampal infusion of MCLR. ⁷ Similarly, we demonstrated that MCLR could induce cognitive impairment, histological and ultrastructural injuries as well as oxidative damage in rats exposed to the same condition. ⁸ Furthermore, we also indicated that treatment with subchronic low dose MCLR results in neuronal degenerative changes and hyperphosphorylation of cytoskeletal-associated protein tau, causing substantial spatial memory retention deficits in rats. ⁹ Moreover, in vitro studies identified several modes of action for MCLR, including inhibition of serine- and threonine-specific protein phosphatases 2A, inducing caspase-dependent apoptosis and microtubule-associated tau protein hyperphosphorylation as well as the inhibition the induction of long-term potential (LTP) in the hippocampus. ^{10 –12} Recently reports suggested maternal exposure to MCLR had adverse effects on neurodevelopment in rat offspring. ^13–14 Although MCLR causes many effects, the biochemical changes in the neuronal system after exposure to MCLR remain largely unknown. In this study, we focused on MCLR-triggered cytotoxicity, as the loss of neurons is one of the most harmful effects of MCLR on the brain. ^8–9

In this study, we investigated the potential role of intracellular calcium ([Ca²⁺]_i) in MCLR-caused cellular injury of primary cultures of hippocampal neurons. The multifunctional role of Ca²⁺ is well recognized, as it is important for fertilization, proliferation, development, learning, and memory. ¹⁵ Maintenance of intracellular Ca²⁺ homeostasis is crucial for cell survival. An early study showed a rapid rise in [Ca²⁺]_i, presumably from intracellular sources, in hepatocytes after exposure to microcystin purified from a cyanobacteria bloom. ¹⁶ Additional studies indicated that the release of Ca²⁺ from the mitochondria induced by MCLR and activation of Ca²⁺/calmodulin-dependent protein kinase are implicated in MCLR-triggered hepatocyte apoptosis. ^17–18 Although early literature indicated that MCLR elevates [Ca²⁺]_i, the detailed mechanisms by which MCLR regulated [Ca²⁺]_i in neurons remain unclear. Therefore, the aim of this study was to investigate whether the neurotoxicity induced by MCLR is associated with calcium homeostasis. Our results found that the release of Ca²⁺ from the endoplasmic reticulum (ER) through activation of inositol 1,4,5-trisphosphate receptor (IP3R) contributes to MCLR-induced cytotoxicity in hippocampal neurons. These data shed new light on the understanding of neurotoxicity of MCLR.

Materials and Methods

Materials

Purified MCLR was purchased from Alexis Biochemicals (Lausen, Switzerland). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), O,O′-bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid, tetraacetoxy-methyl ester (BAPTA/AM), 2-aminoethoxydiphenyl borate (2-APB), dantrolene, carbonylcyanide m-chlorophenylhydrazone (CCCP), xestospongin C, U-73122, U-73343, thapsigargin Hoechst 33258, propidium iodide (PI), and trypsin were purchased from Sigma (St Louis, Missouri). Fura-2/AM was obtained from Biotium (Hayward, California). Dulbecco modified Eagle medium (DMEM)/F-12 supplement (F12) and B27 supplement were obtained from Gibco Invitrogen Corporation (Carlsbad, California). Other general agents were available commercially. Other agents were purchased from commercial suppliers. Agents were prepared as stock solutions and stored at −20°C. They were diluted to the final concentrations before application. The final concentration of dimethyl sulfoxide (DMSO) was <0.05%. No detectable effect of the vehicles was found in our experiments.

Cell Culture

Neonatal Sprague-Dawley rats (day 0-3) of both sexes were obtained from the Center for Disease Control of Hubei Province, China. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Neurons were isolated as previously described with some modification. ¹⁹ Briefly, hippocampi of newborn rats were dissected and rinsed in ice-cold dissection buffer. Blood vessels and white matter were removed, and tissues were incubated in 0.125% trypsin for 25 minutes at 37°C. Neurons were collected by centrifugation and resuspended in DMEM/F-12 (1:1; Gibco Invitrogen corporation) with 10% fetal bovine serum (heat-inactivated, Hyclone, Logan, UT, USA), 2 mmol/L l-glutamine (Sigma), and penicillin (100 U/mL)–streptomycin (100 U/mL). Cells were plated at a density of 10⁴ to 10⁵ per 35 mm² on coverslides precoated with poly-l-lysine and kept at a 37°C in a 5% CO₂ incubator. After 24 hours, the culture medium was changed to DMEM containing 2% B27 and 2 mmol/L glutamine. Astrocytes were minimized by treating the culture with cytarabine (10 μmol/L) on day 3. The medium was replaced with fresh medium every 3 days. Experiments were performed on days 5 to 7.

Cell Viability and Apoptosis Assay

The integrity of mitochondrial enzymes in viable neurons was evaluated with a colorimetric assay using MTT levels. Forty-eight hours after MCLR exposure, the cultures were incubated with MTT solution (0.5 mg/mL) for 4 hours at 37°C. The medium was discarded, and DMSO was added to solubilize the reaction product formazan by shaking for 10 minutes. Absorbance at 490 nm was measured with a microplate reader. Cell viability of control groups exposed to no MCLR was defined as 100%.

Fluorescent microscopic studies were performed to distinguish between the apoptotic and necrotic neurons using Hoechst dye (33258) and PI labeling. Apoptotic cells were identified on the basis of morphological changes in their nuclear assembly by observing chromatin condensation and fragment staining by the Hoechst dye. Necrotic cells were identified based on positive staining with PI and apoptotic nuclear morphology with Hoechst dye. The experiments were repeated for 3 times, and in each case, at least 4 microscopic fields were photographed randomly to assess and quantify the living and dead cells.

Calcium Imaging Experiment

Digital calcium imaging was performed as described by our previous study. ¹⁹ Hippocampal neurons were washed 3 times with 1 μmol/L Fura-2/AM in artificial cerebrospinal fluid (ACSF, containing 140 mmol/L NaCl, 5 mmol/L KCl, 1mmol/L MgCl₂, 2 mmol/L CaCl₂, 10 mmol/L glucose, and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.3), then incubated in the same solution for 30 minutes at 37°C. In calcium-free experiments, ethylene glycol tetraacetic acid (100 μmol/L) was substituted for CaCl₂. Before each experiment, the coverslides were mounted on a chamber positioned on the movable stage of an inverted Olympus IX-70 microscope equipped with a calcium imaging system (TILL Photonics GmbH, Gräfelfing, Germany) and superfused by ACSF for 10 minutes. Fura-2/AM loaded cells were illuminated at 340 nm for 150 ms and 380 nm for 50 ms at 1 second intervals using a TILL Polychrome monochromator (TILL Polychrome monochromator, Gräfelfing, Germany). Fura-2 fluorescence emission was imaged at 510 nm by an intensified cooled charge coupled device (TILL Photonic GmbH) through an X-70 fluor oil immersion lens (Olympus) and a 460 nm long-pass barrier filter. F340/F380 fluorescence ratios were generated by TILL vision 4.0 software. Paired F340/F380 fluorescence ratio images were acquired every second for [Ca²⁺]_i. The [Ca²⁺]_i is presented as the ratio of the fluorescence signals obtained (340/380 nm). All experiments were repeated at least 3 times using different batches of cells.

Statistical Analysis

The analysis of the changes in [Ca²⁺]_i was done on responding cells. Increases in intracellular Ca²⁺ evoked by stimulus were calculated as ΔF/F₀, where ΔF is the difference between resting [Ca²⁺]_i measured prior to the respective stimulation and the stimulus-induced [Ca²⁺]_i peak (Δ ratio [340/380 nm]), F₀ is the resting F340/F380 ratio. Data are presented as mean ± standard error of the mean. Data were analyzed using SPSS 10.0 software. Student t test or 1-way analysis of variance was used to test for significance. Difference were considered significant at P < 0.05.

Results

Microcystin-Leucine-Arginine-Induced Neuronal Injury in a Concentration-Dependent Manner

The viability of hippocampal neurons was significantly reduced after 24 hours of exposure to MCLR in a dose-dependent manner at the range from 0.1 to 30 μmol/L. Cell viability decreased to 71.25%±5.36% following the exposure to MCLR at 1 μmol/L (Figure 1A, n = 3, P < 0.05 vs control). This loss of cell viability occurs because cell death is induced by MCLR. As shown in Figure 1B, 1 μmol/L MCLR increased the number of apoptotic and necrotic neurons by staining with Hoechst 33258 and PI. Statistical analysis also confirmed that 1 μmol/L MCLR increased hippocampal neuronal death rate from 6.29% ± 0.92% to 30.51% ± 2.82% (Figure 1B, n = 3, P < 0.05 vs control). This concentration was chosen for the subsequent studies.

Figure 1.

Microcystin-leucine-arginine (MCLR) inhibited cell viability in hippocampal neurons. A, Neurons were treated with MCLR at a dose range from 0.1 to 30 μmol/L for 24 hours, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B, MCLR triggered apoptotic and necrotic neurons by staining with Hoechst 33258 and propidium iodide (PI). Cell apoptosis and necrosis were assessed by Hoechst/PI double staining technique. Normal cells showed uniform blue fluorescence. Apoptotic cells were seen as bright blue fluorescent spots and necrotic nuclei were identified by staining with PI, which showed purple fluorescence the image. Cell death rate was calculated from the ratio of dead cells to total cell. Arrows pointed to dead cells (n = 3). *P < 0.05 compared with control.

Microcystin-Leucine-Arginine Exposure Caused a Rapid and Sustained Rise in [Ca²⁺]_i

We employed the Ca²⁺ imaging technique to study the dynamic alteration in intracellular Ca²⁺ mediated by MCLR in hippocampal neurons. As shown in Figure 2A, 6 different concentrations of MCLR at 0.1, 0.3, 1, 3, 10, and 30 μmol/L were separately added into ACSF, and a concentration-dependent response was evident. Calcium mobilization included an immediate rise with a sustained plateau at 200 seconds, and the increase in [Ca²⁺]_i lasted at least 360 seconds. From the result, the effect of MCLR was significant at 1 µmol/L (Figure 2B, P < 0.05, n = 31). Repeated 1 µmol/L MCLR stimulation produced equal response, and this response was fully reversed after washout with ACSF (Figure 2C). The response were detected in 57.6% (38 of 66 cells tested) of neurons, resulting in a 91.5% ± 9.4% increase over basal levels. As shown in Figure 2D, MCLR could induce a significant increase of basal [Ca²⁺]_i in Ca²⁺-free medium, and the [Ca²⁺]_i levels were similar to ACSF superfusion (89.4% ± 10.6% increase over basal levels, P > 0.05 compared with Ca²⁺-containing medium). A membrane-permeable intracellular Ca²⁺ chelator, BAPTA/AM blocked MCLR-induced Ca²⁺ mobilization in a Ca²⁺-containing medium (Figure 2E).

Figure 2.

Microcystin-leucine-arginine (MCLR) induced increase in intracellular calcium levels in hippocampal neurons. A, Representative real-time imaging of F340/F380 ratio in response to various concentrations of MCLR. B, Summary data of increase in [Ca²⁺]_i for each dose of MCLR. *P < 0.05 versus corresponding value in control cultures. C, Hippocampal neurons were treated with 1 µmol/L MCLR for designated time. The arrows indicated the time of MCLR application or washout. D, MCLR increased in [Ca²⁺]_i while Ca²⁺-free solution was applied. E, O,O′-Bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid, tetraacetoxy-methyl ester (BAPTA/AM), a membrane-permeable Ca²⁺ chelator, eliminated the MCLR triggered rise in [Ca²⁺]_i. F, 1 µmol/L thapsigargin completely blocked MCLR-induced [Ca²⁺]_i increase. G, 2 µmol/L CCCP, a mitochondrial uncoupler, did not affect MCLR-stimulated [Ca²⁺]_i elevation.

To determine the possible mechanisms of the calcium mobilization triggered by MCLR, several inhibitors of intracellular Ca²⁺ stores were used. As shown in Figure 2F, after preincubation with 1 µmol/L thapsigargin to deplete the intracellular Ca²⁺ store in neurons, MCLR had no effect on [Ca²⁺]_i (Figure 2F). However, after depleting the mitochondrial Ca²⁺ store with CCCP (2 μmol/L), a mitochondrial uncoupler, addition of 1 μmol/L MCLR induced a [Ca²⁺]_i elevation that was indistinguishable from the control response shown in Figure 2A.

Involvement of the IP3R Signaling Pathway in MCLR-Triggered Calcium Mobilization

To explore which intracellular signaling pathway is activated in MCLR-induced [Ca²⁺]_i increase, an IP3R inhibitor (2-APB, xestospongin C) and ryanodine receptor (RyR) inhibitor (dantrolene) were preincubated to cells, respectively. As shown in Figure 3B and C, pretreatment with 2-APB and xestospongin C could block MCLR-induced increase in intracellular Ca²⁺ release completely (P < 0.05). In the presence of dantrolene, 1µmol/L MCLR was able to induce a significant increase of basal [Ca²⁺]_i (Figure 3D, 78.9% ± 6.5% over unstimulated level), and it is not significantly different than that induced by MCLR alone (P > 0.05, n = 25).

Figure 3.

Activation of PLC/inositol 1,4,5-trisphosphate (IP3) signaling pathways mediated by microcystin-leucine-arginine (MCLR) stimulated [Ca²⁺]_i increase. A, Neurons loaded with Fura-2/tetraacetoxy-methyl ester (AM) and stimulated with 1 µmol/L MCLR. B, 2 µmol/L 2-APB, an IP3R inhibitor, blocked MCLR-stimulated [Ca²⁺]_i elevation. C, 1 µmol/L xestospongin C blocked MCLR-stimulated [Ca²⁺]_i elevation. D, 2 µmol/L dantrolene did not affect MCLR-stimulated [Ca²⁺]_i elevation. E, 10 µmol/L U73122 blocked MCLR-stimulated [Ca²⁺]_i elevation. F, 10 µmol/L U73343 did not alter MCLR-stimulated [Ca²⁺]_i elevation. G, Summary data of the results with 2-aminoethoxydiphenyl borate (2-APB), xestospongin C, dantrolene, U73122, and U73343 preincubation on MCLR-mediated elevation in hippocampal neurons. The data were presented as mean and SD of 3 independent experiments. *P < 0.05 compared with MCLR alone.

Activation of phopholipase C (PLC) is known to stimulate PI hydrolysis, IP3, and 1,2-diacylglycerol production. Subsequently, IP3 stimulates the Ca²⁺ release from ER stores. U73122, an inhibitor of PLC, blocked MCLR-mediated increase in [Ca²⁺]_i (P < 0.05, n = 26), whereas U73343, an inactive analog of U73122 (without inhibitory activity), did not alter MCLR-stimulated [Ca²⁺]_i elevation (Figure 3E-G, n = 23, P > 0.05).

Inhibition of Intracellular Ca²⁺ Release Eliminated the Neuronal Death Caused by MCLR in Hippocampal Neurons

We next tested the hypothesis that preventing MCLR-induced increase in [Ca²⁺]_i ameliorates the cell death caused by MCLR. BAPTA/AM, an intracellular Ca²⁺ chelator, prevented MCLR exposure-induced cell death. The IP3R antagonist (2-APB, 2 µmol/L) also inhibited cell death after exposure to MCLR (Figure 4A). As shown in Figure 4B, the preapplication of 1 µmol/L BAPTA/AM, 2 µmol/L 2-APB, and 10 µmol/L U73122 significantly prevented the loss of cell viability caused by MCLR (P < 0.05 compared with MCLR alone). However, the antagonist of RyRs, dantrolene (2 µmol/L), and the inactive analog of PLC, U73343 (10 µmol/L) did not ameliorate the effect of MCLR. Notably, BAPTA/AM, 2-APB, dantrolene, U73122, and U73343 alone did not affect cell viability.

Figure 4.

O,O′-Bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid, tetraacetoxy-methyl ester (BAPTA/AM), 2-aminoethoxydiphenyl borate (2-APB) and U73122 rescued neurons from MCLR-induced cell loss. A, BAPTA/AM, U73122, and 2-APB prevented apoptotic and necrotic neuronal death triggered by MCLR. B, MCLR-induced decrease of cell viability was ameliorated by BAPTA/AM, 2-APB, and U73122. Data were presented as mean and standard deviation (SD) for 3 independent experiments with triplicate determination. Arrows pointed to dead cells (n = 3). *P < 0.05 compared with control, ^# P < 0.05 compared with MCLR alone.

Discussion

In the present study, we found that MCLR treatment of hippocampal neurons disrupted [Ca²⁺]_i levels by release of Ca²⁺ from intracellular stores. Prevention of the increase in [Ca²⁺]_i by cell-permeable chelator or specific inhibitor of the IP3R ameliorated the MCLR-triggered neuronal death, which suggests a strong association between these 2 effects.

In vitro studies identified regulation of the intracellular [Ca²⁺]_i as one of the critical parameters affected by MCs. ^{16 –18,20–21} This is of significance for the neurotoxic potential of MCs as Ca²⁺ plays a crucial role in numerous cellular processes. ^{22 –24} Our result was consisted with the previous reports that MCLR could cause [Ca²⁺]_i mobilization. ^17–18 The disruption of Ca²⁺ homeostasis may result from influx of Ca²⁺ from the extracellular environment and release from intracellular organelles. ²⁵ The present data showed the ER store appears to play a dominant role because the MCLR-induced Ca²⁺ release was significantly inhibited by depletion of ER store with thapsigargin. The Ca²⁺ store in the mitochondria did not appear to play a significant role since depletion of mitochondrial Ca²⁺ with CCCP did not affect MCLR-induced Ca²⁺ release.

The activation of PLC can hydrolyze phosphatidylinositol 4,5-bisphosphate to generate IP3 that bind to IP3R, resulting in Ca²⁺ release from the ER. ^26–27 In our research, inhibitor of PLC (U73122) caused blockage of the MCLR-mediated increase in [Ca²⁺]_i, whereas U73343, an inactive analog of U73122, did not alter MCLR-stimulated [Ca²⁺]_i elevation, indicating that MCLR-stimulated Ca²⁺ elevation is mediated via a PLC/IP3-dependent pathway. The present study explored the possibility that the increase in [Ca²⁺]_i by MCLR is linked to its neurotoxicity. Chelation of Ca²⁺ with BAPTA/AM or inhibition of the internal reservoir in the ER with 2-APB prevented MCLR-induced neuronal death, which indicated that the intracellular Ca²⁺ elevation from ER plays a key role in the onset of cell death in MCLR-treated neurons. Inconsistent with this, the previous study had found MCLR could induce apoptosis with unprecedented rapidity, independently of new gene transcription or protein synthesis through increase mitochondrial Ca²⁺ level in hepatocytes. ^{17–18,20,28} The discrepancy above may due to the difference in the cell types. It is well known that the release of Ca²⁺ from ER and consequent increase in [Ca²⁺]_i can play pivotal roles in regulating cell survival and apoptosis in a variety of cell types including neurons. ²⁹ The Ca²⁺ release from the ER is mediated through ER-resident IP3Rs and RyRs. ^30–31 2-Aminoethoxydiphenyl borate, the inhibitor against IP3R, completely blocked the elevation of MCLR on [Ca²⁺]_i, whereas the RyR agonist dantrolene failed to prevent this [Ca²⁺]_i response, suggesting that the increase in [Ca²⁺]_i was attributed to the calcium mobilization via IP3R, rather than the RyR. In accordance with this, blocking the function of IP3R enhanced cell survival of neurons upon MCLR exposure. Therefore, Ca²⁺ release from IP3R of ER may be a primary cause of MCLR-induced neuronal death. While the present study provided in vitro data regarding the neurotoxicity of MCLR, in vivo evidence supporting these findings is still required.

Conclusion

Our study provided the clear evidence that MCLR-induced increase in [Ca²⁺]_i is a key determinant for neuronal death in hippocampal neurons, as chelation of Ca²⁺ with BAPTA/AM or inhibition of the IP3R with 2-APB prevented this cell death. Intervention aimed at alleviating the MCLR-caused disruption of [Ca²⁺]_i homeostasis may potentially lessen neuronal loss, one of the most damaging effects of neurotoxicity of MCLR.