Ginsenoside Rd Protects Against Neuronal Excitotoxicity Via Suppression Of NMDA Receptor Activity

NMDA receptor (NMDAR) is a specific type of ionotropic glutamate receptor, whichis characterized by high calcium permeability. A large number of basic/preclinical studieshave provided evidence that the suppression or blockage of NMDAR is effective in theprevention of pathology in various models of neurological diseases, especially in ischemicstroke. However, clinical trials for NMDAR antagonists have all ended in a failurebecause of intolerable side effects. Extensive researches in the past decade havesignificantly advanced the understanding of the potential mechanisms underlyingNMDAR structure and function in physiological and pathophysiological conditions,leading to the development of promising novel therapeutics based on subunit selectivemodulation of NMDAR. Our preclinical studies and clinical trials have provided strong evidence of theneuroprotection of ginsenoside Rd (GSRd) against acute ischemic stroke, which suggeststhat it may be a promising neuroprotectant. However, the underlying mechanisms ofGSRd neuroprotection remain unknown. In this study, we explored the relationshipbetween GSRd neuroprotection and NMDAR in cultured hippocampal neurons in vitro byusing electrophysiological and molecular biological techniques to reveal the potentialmechanisms underlying the neuroprotection of GSRd.Part1Effects of GSRd on NMDAR-mediated currentsObjectives: Our preliminary studies indicated that NMDAR may be involved in theneuroprotection of GSRd. In the first part of this study, we explored whether NMDAR wasa potential target of GSRd.Methods: Rat hippocampal neurons were primary cultured for10-14d in vitro (DIV10-14). The NMDA receptor-mediated currents from hippocampal neurons in Mg2+-freeextracellular solution (ECS) were measured to exogenous NMDA by using whole-cellpatch recording and “Y-tube” perfusion system. We examined the effects of GSRd onneuronal NMDAR-mediated currents under normal and excitotoxic injury conditions.Results:1. In rat hippocampal neurons, rapid and brief application of NMDA evokedpartially desensitizing inward currents. NMDAR antagonists AP-5and MK-801remarkably inhibited the inward currents, indicating that the evoked currents were typicalNMDAR-mediated currents.2. GSRd reversibly reduced the peak values of NMDAR-mediated currents in adose-dependent manner and the EC50was7.7μM.3. After excitotoxic injury, the peak values of NMDAR-mediated currents weresignificantly increased, compared with the control group, while bath application of GSRdattenuated the increased NMDAR-mediated currents.Conclusions: GSRd depresses the activity of NMDAR channels moderately andattenuates the overactive NMDAR channels after excitotoxic injury. Part2Selective action of GSRd on NMDAR SubunitObjectives: Functional NMDARs are tetrameric complexes consisting of two essentialsubunits NR1and two regulatory subunits NR2A or NR2B. Recent studies suggest thatNR2A and NR2B subunits have distinct location and function in synapses andextrasynaptic sites. Based on these facts, we examined whether GSRd can modulateNMDAR activity in a subunit selective manner.Methods: Rat hippocampal neurons were primary cultured for DIV10-14. We recordedNMDAR currents by using whole-cell patch recording and “Y-tube” perfusion system.After that, we applied NR2A-specific antagonist NVP-AAM077and NR2B-specificantagonist ifenprodil to examine whether these drugs blocked the inhibitory impact ofGSRd on NMDAR-mediated currents. We measured NMDAR-mEPSC to reflect theactivity of synaptic NMDAR, and recorded the extrasynaptic NMDA receptor-mediatedcurrents (eNMDAc) according to MK-801trapping protocol. Furthermore, we expressedrecombinant NR1/NR2A or NR1/NR2B receptors in HEK293cells, and recorded theNR1/NR2A receptor currents (INR1/NR2A) and NR1/NR2B receptor currents (INR1/NR2B), andobserved the effects of GSRd on these currents. At last, we performed the radioligandbinding experiment using the antagonist radioligand [3H]CGP39653to test whether GSRdwas able to bind to NMDAR directly.Results:1. NVP-AAM077did not affect the inhibitory effect of GSRd on NMDAR-mediatedcurrents while ifenprodil eliminated the GSRd-induced decrease of NMDAR-mediated currents.2. There was no significant difference in the amplitude and frequency ofNMDAR-mEPSC before and after application of GSRd. While GSRd significantlydecreased the peak amplitude of eNMDAc.3. GSRd did not affect INR1/NR2Aand INR1/NR2Bin HEK293cells. Meanwhile, theinhibitory effect of GSRd on [3H]CGP39653specific binding was very weak, indicatingthat GSRd has a low affinity binding to NMDAR.Conclusions: GSRd depresses the activity of NMDAR channels through affecting NR2Bsubunit. However, the negative results in transfection and binding experiments indicatethat GSRd may act on NMDAR in an indirect way but not a direct way. Part3The phosphorylation of NR2B subunit is involved in GSRdneuroprotection against excitotoxicity.Objectives: NR2B subunit has long intracellular C-terminal domain containing severalphosphorylation sites which are essential for the functional regulation of NMDAR. Wefurther investigated whether the phosphorylation of NMDAR is involved in theneuroprotection of GSRd against neuronal excitotoxicity.Methods: Rat hippocampal neurons were primary cultured for DIV10-14and were usedto record NMDAR currents by using whole-cell patch recording and “Y-tube” perfusionsystem. Various kinase inhibitors were applied to test their effects on the inhibitoryregulation of NMDAR by GSRd. Using western blot, we examined the expression andphosphorylation levels of NR2B subunit after excitotoxic injury and GSRd treatment. Inaddition, the expression of cleaved caspase-3and the activation of Akt and ERK wereexamined.Results:1. The selective PKC inhibitor G6983eliminated the GSRd-induced decrease ofNMDAR currents while both Src inhibitor PP2and CaMKII inhibitor failed to blockGSRd-elicited inhibitory effects on NMDAR currents.2. GSRd did not affect the total NR1, NR2A and NR2B subunit expression. Afterexcitotoxic injury and GSRd treatment, the levels of phosphorylated Tyr1472and Ser1480were not affected. However, levels of phosphorylated Ser1303were obviously elevatedafter excitotoxic injury while GSRd treatment significantly depressed the elevated NR2Bsubunit phosphorylation at Ser1303.3. GSRd reduced the elevation of cleaved caspase-3expression induced byexcitotoxic injury. Although there was no significant difference of total Akt and ERKexpression among all these groups, GSRd increased the levels of P-Akt and P-ERK, whichwere depressed by excitotoxic injury.Conclusions: GSRd may reduce the activity of NMDAR channels through depressing theNR2B subunit phosphorylation at Ser1303. This may be an important mechanismunderlying the neuroprotection of GSRd against excitotoxicity.