Mapacalcine protects mouse neurons against hypoxia by blocking cell calcium overload

Abstract

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.

Figure 1. Glutamate and Oxygen Glucose Deprivation

(OGD) protocols. Schematic representation of protocols used for internal calcium measures following 100 µM glutamate stimulation (A) or following OGD experiments (B). Cell survival counting protocols are also depicted (C). In these experiments, the vehicle was water. CCM, Cell Calcium Medium, Mapa, mapacalcine, PFA, 4% ParaFormAldehyde.

Figure 2. Dose-response effects of mapacalcine on calcium currents recorded in cortical neurons

(n = 10 for each dose). The holding potential was −80 mV. Calcium currents were recorded from −70 to +50 mV. (A) Calcium currents were recorded in control condition and in the presence of either 0.1 µM (A), or 1 µM (B) or 10 µM (C) of mapacalcine. In each condition, typical current traces and current densities (pA/pF) as a function of membrane potential (mV) are shown (D). Histograms of current densities measured at different potentials in control condition (black bars), in the presence of 1 µM of mapacalcine (white bars) and after washout (grey bars). Bars represent the SEM values. *, p<0.05.

Figure 3. Effects of mapacalcine on electrophysiological recordings in cortical neurons in the presence of calcium channel blockers.

(A) Effects of 1 µM of mapacalcine on remaining calcium current after application of 1 µM of ω-conotoxine GVI A, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (B) Effects of 1 µM of mapacalcine on remaining calcium current after application of 250 nM SNX 482, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (C) Effects of 1 µM of mapacalcine on remaining calcium current after application of 75 nM of calcicludine, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (D) % of calcium current inhibition measured at −10 mV by mapacalcine when applied alone (control) or after previous application of the different toxine. Bars represent the SEM values. *, p<0.05, **, p<0.01. n = 10 for each experiment.

Figure 4. Effects of mapacalcine on electrophysiological recordings in cortical neurons or HEK-293 transfected cells.

(A) Cortical neurons, effects of 1 µM of mapacalcine on remaining calcium current after application of 1 µM of nifedipine, (a) I(pA/pF)  =  f(mV) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (B) Cortical neurons, effects of 1 µM of nifedipine on remaining calcium current after application of 1 µM of mapacalcine, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (C) Comparaison of the % of inhibition of the calcium current measured at −10 mV by mapacalcine when it was applied after or before nifedipine. (D) HEK-293 transfected cells, effects of 1 µM of mapacalcine on Ca_V 1.2 calcium channels, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces. (E) HEK-293 transfected cells, effects of 1 µM of mapacalcine on Ca_V 1.3 calcium channels, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces. (F) Cortical neurons, effects of a delayed stimulation protocol on mapacalcine calcium channel inhibition. Each circle represent a stimulation cycle consisting in a 0.5 ms pulse from −80 to +10 mV, time between two pulses, 0.5 s. Bars represent the SEM values. *, p<0.05, **, p<0.01. n = 10 for each experiment.

Figure 5. Calcium and electrophysiological measures after glutamate stimulation in cortical neurons.

(A) The increase of intracellular calcium was triggered by application of 100 µM of glutamate. The different conditions are summarized under the histogram. Mapa acute, application of 1 µM of mapacalcine immediately before the measure. Mapa pre-incub, application of 1 µM of mapacalcine for 45 min before the measure. Washout, cells were perfused without glutamate. (B–H) Electrophysiological recording of glutamate currents (n = 6 per condition). (B) Typical current trace in control condition and after 100 µM of glutamate application. (C) Typical current trace in control condition and after 100 µM of glutamate application in the presence of 1 µM of mapacalcine. (D) Corresponding histogram of the current density values measured at the glutamate peak. (E) Typical current trace in control condition and after 100 µM of NMDA application. (F) Typical current trace in control condition and after 100 µM of NMDA application in the presence of 1 µM of mapacalcine. (G) Corresponding histogram of the current density values measured at the NMDA peak. (H) Typical current trace in control condition and after 100 µM of glutamate application in the presence of 10 µM of APV and 50 µM of CNQX inhibitors NMDA and AMPA/Kainate receptors, respectively (n = 10). Bars represent the SEM values. ***, p<0.001.

Figure 6. OGD on cortical neurons: cell survival.

(A, B) Cell survival was determined after two hours OGD followed by two hours post treatment. (A) Histogram showing cell survival in the absence (control OGD) or the presence of 1 µM of mapacalcine (mapacalcine OGD). The number of cell survival was determinated after two hour OGD followed by two hour treatment with either vehicle (control post OGD) or 1 µM of mapacalcine (mapacalcine post OGD). (B) Typical picture of each condition obtained after Hoescht labeling of nuclei (scale bar  = 50 µM). (C–D) Cell survival was analyzed after two hours OGD followed by twenty four hours post treatment. (C) Histogram showing cell survival determined by counting, in control condition or in the presence of 1 µM of mapacalcine or 1 µM of nifedipine. (D) Histogram showing cell survival determined by LactateDesHydrogenase/AquaCellTiter ratio evaluations (LDH/ACT), in control condition and in the presence of 1 µM of mapacalcine or 1 µM of nifedipine. Values are the mean ± SEM (bars). *, p<0.05, **, p<0.01, *** p<0.001.

Figure 7. OGD on cortical neurons: calcium measures.

Typical fluorescence ratio (340nm/380nm) obtained in different conditions. (A) Vehicle without OGD (V), (B) 1 µM mapacalcine without OGD (M), (C) Vehicle immediately after OGD (VaO), (D) Vehicle post-OGD (VpO), (E) Mapacalcine applied for two hours after OGD (MpO), (F) Corresponding histograms. Values are the mean ± SEM (bars). ***, p<0.001.