The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro

Abstract

Background: There is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis.

Methods: Laser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin.

Results: At physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro.

Conclusions: Mefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro.

Figure 1

Effects of mefloquine on ER calcium homeostasis. The effects of mefloquine on cytoplasmic calcium levels in rat neurons were investigated using confocal microscopy. Neurons were loaded with the calcium-sensitive dye Fluo-3 that was replaced with low-calcium Locke's buffer after 1 h. The neurons were scanned at 10 s intervals to measure baseline fluorescence prior to the addition of DMSO (0.2%) or mefloquine (Mef, 80 μM) followed by thapsigargin (Thaps, 1 μM). Arrows indicate additions after scans 2 and 37, respectively. Mefloquine increased cytoplasmic calcium and antagonized the pharmacological action of thapsigargin, suggesting that the drug mobilizes the ER calcium store. The lack of a subsequent glutamate response (Glu, 1 μM after scan 63) demonstrates the external medium was substantially devoid of free calcium, whilst the presence of a subsequent CaCl₂ (1.6 mM after scan 67) response indicates that the neurons remain viable at the termination of the experiment.

Figure 2

Effects of low calcium Locke's buffer on neuronal viability. Neurons were exposed to MEM (controls), Locke's buffer with and without Ca²⁺ and/or 1 mM EGTA for 15 min or to a low calcium Locke's buffer (LCLB) containing 920 μM Ca²⁺ and 1 mM EGTA for 2.5, 5, 7.5, 10, 12.5 and 15 min. Viability was assessed using the colorimetric MTT assay, where a loss of viability is reflected in a decrease in absorbance at λ = 540 (A₅₄₀). The substitution of normal Locke's buffer for one containing no added calcium results in a loss of neuronal viability (27%, Welch's test, P < 0.005, n = 8, indicated by a superscript a). This solution was not used for the mefloquine studies because control experiments showed that glutamate elicited a calcium response in neurons exposed to this buffer, presumably because the rinsing procedure did not remove all residual free calcium. A 10 min exposure to LCLB was required to reduce viability below that of the calcium-free Locke's buffer (one way ANOVA and Dunnett's test, P < 0.05, n = 44, * designates a significant change). Use of this buffer does not elicit a glutamate response, and therefore should not contain substantial amounts of free calcium. Experiments that utilized this buffer to investigate the effect of mefloquine on neuronal calcium homeostasis were performed within this ten-minute window.

Figure 3

Dose-response curves for mefloquine's effects on neuronal cell viability and mobilization of ER calcium stores. Neurons were exposed to mefloquine (0.75–200 μM) for 5 min, 20 min or 24 h. Viability was assessed using the colorimetric MTT assay. Dose-response data is presented as mean viability (% ± SEM compared to appropriate DMSO controls) and represents data from at least three pooled experiments. For the ER-calcium experiments, changes in neuronal intracellular calcium concentrations induced in response to different mefloquine treatments were monitored in real time using confocal microscopy. Dose-response effects are expressed as a percentage of the maximal elevation of cytoplasmic calcium occurring at 200 μM mefloquine. The dose-response curves for mefloquine's effects on neuronal viability (5 min exposure) and ER calcium store mobilization are superimposable, as are the 20 min and 24 h exposure curves. At a concentration of 50 μM, mefloquine exhibited greater toxicity after 20 min and 24 h exposure as compared to 5 min exposure (one way ANOVA and Dunnett's t-test, P < 0.0001, n = 12, see asterixed points on graph). Therefore, it appears that the neurotoxic effects of mefloquine occur within 20 min of exposure, but cannot be accounted for solely by the disruptive effect of mefloquine on ER calcium homeostasis.

Figure 4

Effects of mefloquine on whole cell calcium homeostasis and their antagonisation by thapsigargin. The effects of mefloquine on neuronal whole cell calcium homeostasis were investigated using confocal microscopy and Locke's buffer containing physiological calcium levels (2.3 mM). Neurons were loaded with the calcium-sensitive dye Fluo-3 that was replaced with normal Locke's buffer after 1 h. Neurons were scanned at 15 s intervals. Mefloquine (MEF80, 80 μM), thapsigargin (THAPS, 4 μM) or DMSO (0.4%) were added at scan 13 (as indicated by the first arrow). Subsequently (second arrow at scan 28), DMSO (0.2%) or mefloquine (80 μM) were added. Mefloquine (but not DMSO) caused an immediate and sustained rise in the cytosolic calcium concentration. This elevation is due to an influx across the plasma membrane as it was not observed in neurons bathed in low calcium Locke's buffer. The effect was antagonized by prior treatment with thapsigargin. As this agent is a specific inhibitor of the ER calcium pump, the data suggest that the effects of mefloquine on calcium homeostasis are mediated at the level of the ER in rat neurons.

Figure 5

Effects of mefloquine and thapsigargin treatment on viability of rat neurons in vitro. A: The effect of mefloquine (Mef, 10 or 80 μM for 5 min), before and after pretreatment with thapsigargin (Thaps, 1 μM or DMSO, 0.4% for 5 min), on the viability of rat neurons was assessed using the MTT assay. Data shown are from eight replicate experiments. There was a significant interaction between mefloquine and thapsigargin (two way ANOVA, P < 0.01, n = 48). Thapsigargin had a neuroprotective effect against the toxic action of mefloquine, as indicated by the partial prevention (by 60%) of the reduction in viability (A₅₄₀ units) caused by mefloquine.

Figure 6

Effects of mefloquine on the transcription of ER stress response genes. Changes in the mRNA levels of tubulin, GADD153, GRP78 and PERK induced in rat neurons after exposure to mefloquine (5 min treatment followed by 24 h washout) were quantitated using Affymetrix U34 Rat Genome arrays. Mefloquine (80 μM) upregulated the transcription of GADD153, GRP78 and PERK but not tubulin (one way ANOVA, P < 0.05, n = 20), suggesting that the drug induces an ER stress response at this concentration.