Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase

Abstract

Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

Figure 1.

Representative fluorescence images of rat cortical neurons transfected with plasmid encoded for silencing of Pyk2 by using a Rat Neuron Nucleofector Kit/Nucleofector program O-03. (a) Rat cortical neurons transfected with plasmid (shRNA construct V2LMM_21947) fused with green fluorescence protein (GFP) and stained by DAPI (4’,6-diamidino-2-phenylindole) fluorescent DNA marker (blue). Transfection efficiency was analyzed by comparing DAPI’s blue to GFP’s green emissions. Images of immunocytochemistry/immunofluorescence–Pyk2 positive control (b) and Pyk2-transfected (c) primary rat cortical neurons. Transfection efficiency was analyzed by comparing the numbers of Pyk2 positive cells in control and Pyk2-transfected cells.

Figure 2.

Mefloquine causes a concentration-dependent decrease in the number of viable control (a) and transfected (b) cells. The quantity of purple formazan product was measured at 570 nm after 24-hour incubations with increasing concentrations of mefloquine. Cytotoxicity is expressed as the percent of MTT activity in control cells (non-treated neurons). Data represent mean ± SEM values from 3 independent experiments with n ≥ 24. *p<0.05 versus control by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 3.

Mefloquine causes a concentration-dependent increase in the number of dead and dying control (a) and transfected (b) cells. Activity of lactate dehydrogenase (LDH) released into the media was measured spectrophotometrically according to the manufacturer’s protocol. Cytotoxicity is expressed as the percent of LDH activity in control cells (non-treated neurons). Data represent mean ± SEM (bars) values from 3 independent experiments with n ≥ 24. *p<0.05 versus control by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 4.

Representative images of apoptotic bodies in Pyk2-transfected cortical neurons exposed to a graded concentration of mefloquine. Apoptotic nuclei, evaluated by using the DeadEnd Colorimetric TUNEL System, are stained dark brown, and control cells are stained blue.

Figure 5.

Percent of apoptotic bodies in control (a) and Pyk2-transfected (b) primary rat cortical neurons following mefloquine exposure. The number of apoptotic neurons was evaluated after 24-hour incubations with graded concentrations of mefloquine using the DeadEnd^™ Colorimetric TUNEL System. Percentages of apoptotic neurons are presented as mean±SEM (bars) values from two experiments with n=18. *p<0.05 versus control by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 6.

Mefloquine causes a concentration-dependent decrease in intracellular GSH levels. The GSH content of control (a) and transfected (b) primary rat cortical neurons was determined after 24-hour incubations with graded concentrations of mefloquine using monochlorobimane, a fluorescent probe for GSH. Data represent mean ± SEM (bars) values from 3 independent experiments with n ≥ 24. *Significant difference between values from control and mefloquine-treated neuronal cells (p<0.05).