Verapamil protects dopaminergic neuron damage through a novel anti-inflammatory mechanism by inhibition of microglial activation

Abstract

Verapamil has been shown to be neuroprotective in several acute neurotoxicity models due to blockade of calcium entry into neurons. However, the potential use of verapamil to treat chronic neurodegenerative diseases has not been reported. Using rat primary mesencephalic neuron/glia cultures, we report that verapamil significantly inhibited LPS-induced dopaminergic neurotoxicity in both pre- and post-treatment experiments. Reconstituted culture studies revealed that the presence of microglia was essential in verapamil-elicited neuroprotection. Mechanistic studies showed that decreased production of inflammatory mediators from LPS-stimulated microglia underlay neuroprotective property of verapamil. Further studies demonstrated that microglial NADPH oxidase (PHOX), the key superoxide-producing enzyme, but not calcium channel in neurons, is the site of action for the neuroprotective effect of verapamil. This conclusion was supported by the following two observations: 1) Verapamil failed to show protective effect on LPS-induced dopaminergic neurotoxicity in PHOX-deficient (deficient in the catalytic subunit of gp91(phox)) neuron/glia cultures; 2) Ligand binding studies showed that the binding of [(3)H]Verapamil onto gp91(phox) transfected COS7 cell membranes was higher than the non-transfected control. The calcium channel-independent neuroprotective property of verapamil was further supported by the finding that R(+)-verapamil, a less active form in blocking calcium channel, showed the same potency in neuroprotection, inhibition of pro-inflammatory factors production and binding capacity to gp91(phox) membranes as R(-)-verapamil, the active isomer of calcium channel blocker. In conclusion, our results demonstrate a new indication of verapamil-mediated neuroprotection through a calcium channel-independent pathway and provide a valuable avenue for the development of therapy for inflammation-related neurodegenerative diseases.

Figure 1

Verapamil protects dopaminergic neurons against LPS-induced neurotoxicity. Rat primary mesencephalic neuron-glia cultures were pretreated with different concentrations of verapamil for 30 min prior to treatment with 5 ng·mL^-1 of LPS. Dopamine (DA) neurotoxicity was measured at 7 days following treatment using the [³H]DA uptake (A) and by counting TH-IR neurons number (B). Immunostaining images show differences in morphology (C). In control cultures, TH-IR neurons show an intricate network of TH-IR dendrites. However, TH-IR neurons in LPS-treated cultures were less numerous, with the remainder exhibiting shortened and damaged dendrites. Significant improvement in the morphology of TH-IR neurons, as judged by the intactness of cell bodies and extensiveness of dendrites, was observed in cultures pretreated with verapamil (1 μmol·L^-1) prior to LPS treatment (5 ng·mL^-1, 7 days). Data is expressed as the percent of the control cultures and are the mean ± SEM from three independent experiments performed with triplicate samples. * p < 0.05, ** p < 0.01, compared to LPS-treated cultures.

Figure 2

Post-treatment with verapamil protects dopamine neurons from LPS-induced neurotoxicity. Verapamil (1 μmol·L^-1) was added 30 min before or at different time points after the addition of LPS (5 ng·mL^-1). As controls, cultures were treated with vehicle alone. Seven days later, [³H]DA uptake was measured. Results are expressed as percentage of control cultures and are mean ± SEM of three experiments performed with triplicate. * p < 0.05, ** p < 0.01, compared to LPS-treated cultures.

Figure 3

Microglia are essential for the neuroprotective effect of verapamil. Reconstituted cultures were prepared by adding 20% (1 × 10⁵/well) astrocyte or microglia to neuron-enriched cultures (5 × 10⁵/well). They were pretreated with verapamil (1 μmol·L^-1) prior to treatment with LPS (5 ng·mL^-1). [³H]DA uptake measurements were performed 7days after the indicated treatments. Results were expressed as percentage of vehicle-treated control cultures and were mean ± SEM from three independent experiments in triplicate. ##P<0.01 compared with the vehicle-treated control cultures and **P<0.001 compared with LPS-treated control cultures.

Figure 4

Verapamil inhibits LPS-induced production of pro-inflammatory factors in rat primary mesencephalic neuron-glia or microglia-enriched cultures. Cultures were pretreated with vehicle and verapamil (1 μmol·L^-1) for 30 min before LPS (5 ng·mL^-1) stimulation. Supernatant from neuron-glia cultures were collected at 3 h for TNF-α assay (A) and at 24 h for nitrite (an indicator of NO) assay (B). Levels of superoxide from microglia-enriched cultures were measured as SOD-inhibitable reduction of WST-1(C). Results were expressed as mean ± SEM from three independent experiments in triplicate. **P<0.01 compared with the LPS-treated cultures and ## compared with control.

Figure 5

PHOX is essential in verapamil-elicited neuroprotection. Wild-type (C57) or NADPH oxidase deficient mice (PHOX^-/-) mesencephalic neuron-glia cultures were pretreated with verapamil (1 μmol·L^-1) for 30 minutes prior to the addition of 10 ng·mL^-1 of LPS. Seven days later, [³H]DA uptake was measured. Results are expressed as percentage of control cultures and are mean ± SEM of three experiments performed with triplicate. ## p < 0.01 compared to control cultures, ** p < 0.01, compared to LPS-treated cultures.

Figure 6

[³H]Verapamil binds to gp91, the catalytic membrane subunit of PHOX. Cell membrane fraction (500 μg protein) of either gp91-transfected COS7 (COS7-gp91) or non-transfected control (COS7-WT) membrane was incubated with 1nM [³H]Verapamil for 2 h at 4°C. The incubation was terminated by adding ice-cold assay buffer following immediate filtration through glass fiber filters. The filters were immediately washed three times with 5 mL of ice-cold assay buffer and dried in an oven at 60°C for 2 h. Radioactivity was counted using a liquid scintillation counter. Results were expressed as mean ±SEM from three independent experiments. **P<0.01 compared with the COS7 wild-type.

Figure 7

Both stereoisomers of verapamil show the same potency in neuroprotection, binding capacity to gp91 protein and inhibition of LPS-induced pro-inflammatory factors release. [³H]DA uptake assay (A); Competitive binding assay (B); and Pro-inflammatory factors (C). Results were expressed as mean ± SEM from nine (A) and three (B and C) independent experiments. **P<0.01 compared with LPS (5 ng·mL^-1) treatment.