Humanin Inhibited NMDA-MAPKs Mediated Excitotoxicity

Glutamate is an important neurotransmitter in central nervous system, mediating a variety of excitatory synaptic transmissions. It plays a critical role in the neuronal development, in various excitatory synaptic transmissions in adult brain, and in synaptic plasticity. However, excessive accumulation of glutamate at synaptic clefts would cause neuronal damage even cell death, which is called excitotoxicity. Excitotoxicity has been known to be involved in a variety of pathological processes, such as cerebral ischemic injuries and many neurodegenerative diseases. Studies have shown that during cerebral ischemia and brain trauma, the concentration of glutamate within the brain tissue increases rapidly. Excessive accumulation of glutamate causes overactivation of glutamate receptors on postsynaptic membranes, especially for N-methyl-D-aspartate (N-methyl-D-aspartate, NMDA) receptors. NMDA receptors are ionotropic receptors, activated by glutamate (endogenous neurotransmitter) or NMDA (exogenous agonist), making the coupling Ca2+ channels open, inducing increase of intracellular Ca2+ (i.e. changes of Ca2+ signal), exerting multiple biological effects including physiological and neurotoxic effects. Recently, evidence showed that the influx of Ca2+ induced by activation of NMDA receptors could also cause cell damage through activating mitogen-activated protein kinases (MAPKs) pathways.The excitotoxicity model is the most commonly used (generally recognized) and the most toxic (inducing severe damage even neuronal death) nerve injury model, with the most complicated injury mechanism (involving Ca2+ overload, oxidative stress, mitochondrial dysfunction, cell necrosis and apoptosis, etc.) and the most clinical significance (excitotoxicity is involved in almost all neuropathological processes, such as cerebral ischemia, a variety of neurodegenerative diseases.). MAPKs have multiple signaling pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK pathways, and each pathway varies from each other as for their physiological and pathological significance. Different injuries would cause involvement of different kinds of MAPKs pathways, and activation of each pathway would result in different biological effects. Although some reports have shown that MAPKs pathways indeed involved in NMDA-induced excitotoxicity, the contribution of each pathway and their interactions are still unclear. Therefore, the systematic observation and analysis of the role of each of NMDA-MAPKs pathway in excitotoxicity process would be of significant importance for further understanding the mechanism of excitotoxicity.Humanin (HN) is a 24-amino-acid peptide that was first isolated from brains of patients with Alzheimer's disease (AD). HN is best known for its ability to suppress neuronal cell death induced by AD-related insults such as familial AD proteins and neurotoxic Aβpeptides. Earlier studies focused on the neuroprotection of HN on neuronal cell death induced by AD-related insults. Recently, evidence has revealed that HN might have a broader spectrum of protective activity. For example, HN protects human cerebrovascular smooth muscle cells from Aβ-induced toxicity. It also prevents serum deprivation-induced apoptosis of undifferentiated PC12 cells. The incubation of rat cortical neurons with HN prevented cell death and apoptotic events induced by soluble prion protein fragments. The effect of HN in vivo has been observed as improving the learning and memory impairment caused by scopolamine or Aβin mice. Very recently, we showed that HN rescued cortical neurons from NMDA-induced excitotoxicity in rats.Thus, in the present study, we aimed to (1) Using the selective MAPK inhibitors, systematically investigate the contributions of ERK, JNK and p38 MAPK of the MAPK family to NMDA-mediated excitotoxicity (decrease of cell viability, evoked neuronal damage and cell death) of cortical neurons and observe the neuroprotection of HN against neuronal insults by using WST-8 assay, Calcein-AM staining and LDH levels assay, respectively; (2) Investigate the contribution of apoptosis in NMDA-induced excitotoxicity, characterize the contribution of each MAPKs pathway related to NMDA-induced apoptosis and observe the neuroprotection of HN against apoptosis, as measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining and flow cytometry detection; (3) Based on the above studies, further elucidate the mechanisms or possible targets underlying the HN-mediated neuroprotections. Firstly, observe the effect of HN on NMDA-induced change of intracellular calcium concentration by using calcium fluorescent image, and secondly clarify the activation of the three MAPKs pathways by NMDA and the suppressive effects of HN by Western Blot analysis. This study provided novel insights of NMDA-MAPKs mediated excitotoxicity and further evidence of neuroprotections for HN peptide. Excessive activation of NMDA receptors have been involved in acute and chronic brain injury and also the major contributor to cell death in stroke/ischemia, and certain neurodegenerative disorders. In order to investigate the involvement of MAPK signaling pathways in NMDA-induced excitotoxicity, we systematically clarified the contributions of the MAPK family [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK] to NMDA-mediated excitotoxicity in cortical neurons using the selective MAPK inhibitors.The findings showed that: (1) Cell viability assay showed that NMDA induced obvious decrease of cell viability (WST-8 assay), however, JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 elicited dose-dependent inhibitory effects on the decrease of NMDA-induced cell viability with IC50 values of 18.75μmol/L and 7.97μmol/L, respectively. In contrast, NMDA-induced cell damage was not affected by ERK inhibitor PD98059 in the tested concentration range (10~50μmol/L); Pretreatment of SP600125 (20μmol/L) or SB203580 (10μmol/L) alone or a combination of them or HN (10μmol/L) showed that cell viability was increased by 10.36% (P < 0.05), 20.96% (P < 0.05), 27.07% (P < 0.05) and 37.99% (P < 0.05), respectively, as compared with NMDA-treated group. However, PD98059 (20μmol/L), an inhibitor of ERK, did not work; (2) LDH assay showed that administration of 20μmol/L SP600125 or 10μmol/L SB203580 alone or combination of them or 10μmol/L HN significantly reduced NMDA-induced LDH release by about 23.08% (P < 0.05), 32.90% (P < 0.05), 42.06% (P < 0.05) and 49.90% (P < 0.05), respectively, whereas 20μmol/L PD98059 had no effect; (3) Living cells measured by Calcein-AM staining showed that pretreatment of SP600125 or SB203580 alone or a combination of them or HN, but not ERK inhibitor, increased the number of living cells by 9.52% (P < 0.05), 18.10% (P < 0.05), 25.19% (P < 0.05) and 29.88% (P < 0.05), respectively, as compared with the NMDA-treated group; (4) Measurement of reactive oxygen species (ROS) release showed that NMDA induced ROS increase with a peak response at 12 h after NMDA challenge; Administration of SP600125 or SB203580 or both of them or HN significantly inhibited the NMDA-induced production of ROS by 17.94% (P < 0.05), 33.68% (P < 0.05), 45.33% (P < 0.05) and 75.47% (P < 0.05), respectively, whereas ERK inhibitor had no effect. Meanwhile, the above results revealed that p38 MAPK inhibitor was more effective than JNK inhibitor, and simultaneous administration of two inhibitors achieved better neuronal protective effects, as compared with that by using either alone.These findings indicated that: (1) The JNK and p38 MAPK pathways, but not the ERK pathway, contributed to NMDA-induced cortical neuronal cell death; (2) p38 MAPK showed a more significant role than JNK and furthermore the two pathways might act synergistically in NMDA-mediated excitotoxicity; (3) ROS production increased greatly through activation of JNK and p38 MAPK by NMDA, thus induced excitotoxic neuronal death; (4) HN exhibited more significant protection from excitatory neurotoxicity than single or combination administration of MAPK inhibitors.It is well known that NMDA-induced excitotoxicity cause severe neuronal damage including necrosis. Recently studies showed that NMDA also induced neuronal apoptosis. Using the caspase inhibitors and different measurements of detection of apoptosis, the role of apoptosis or the proportion of apoptotic cell death was evaluated in the cellular model of NMDA-mediated neurotoxicity (100μmol/L, 2h). To further investigate the involvement of MAPK signaling pathways in NMDA-induced apoptosis of cortical neurons, we treated NMDA with different inhibitors in the cortical neuron cultures, as measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining and flow cytometry detection.The results showed that: (1) Exposure of cortical neurons to NMDA (100μmol/L, 2h) resulted in a significant increase of the number of death cells and the proportion of living cells decreased by 22.49% according to caspase inhibitor experiment suggesting caspase-dependent apoptosis with 22.49% of total neuronal death; (2) The activity of caspase-3 by NMDA was significantly decreased by 30.43% (P < 0.05) in p38 MAPK inhibitor SB200358 (10μmol/L)-treated and 32.20% (P < 0.05) in HN (10μmol/L)-treated cells compared with NMDA group. In contrast, administration of ERK inhibitor PD98059 (20μmol/L) or JNK inhibitor SP600125 (10μmol/L) could not inhibit the apoptotic cell death; (3) TUNEL staining showed that NMDA-induced apoptotic rate of 33.63%; pretreatment of SB203580 or HN significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05) and 33.99% (P < 0.05) respectively. However, PD98059 (20μmol/L) and SP600125 (10μmol/L) did not work; (4) An evaluation of flow cytometric assay showed that the apoptotic rate was 30.33% in NMDA-treated group; pretreatment of SB203580 or HN significantly reduced NMDA-induced apoptosis by 45.14% (P < 0.05) and 49.84% (P < 0.05) respectively, whereas PD98059 and SP600125 had no effect.These findings indicated that: (1) NMDA induced neuronal apoptosis (mainly caspase-dependent apoptosis) besides necrosis in cultured cortical neurons; (2) NMDA-induced apoptosis of cortical neurons might involve the activation of p38 MAPK pathway, but not the JNK and ERK pathway, and inhibition of the apoptotic signaling pathway corresponded to this neuroprotection; (3) JNK pathway might be involved in NMDA-induced cortical neuronal necrosis rather than apoptosis; (4) HN might exert its neuroprotection through protecting neurons from NMDA-induced apoptosis.Although NMDA-induced excitotoxicity has been studied extensively, neuroprotection of HN against NMDA toxicity has less been explored. The two parts of above experiments have shown that HN exhibited more effective antagonism against neuronal toxicity than single or combined use of MAPK inhibitors; HN could protect neurons from NMDA-MAPKs induced damage including neuronal apoptosis. The present study further investigated the activation of MAPKs pathways by NMDA and the antagonism of HN. We first examined the effects of HN on NMDA-induced intracellular Ca2+ response, and next analyzed the activation of MAPKs by NMDA and the antagonistic effects of HN in order to determine whether HN exerts its effects through affecting NMDA receptors or their downstream molecules.The results showed that: (1) Exposure of cortical neurons to NMDA resulted in elevation of intracellular Ca2+ by 162.81%, as compared with the control (P < 0.05) by using calcium fluorescent image, while pretreatment with HN did not reduce NMDA-induced Ca2+ influx; (2) Western Blot analysis showed that the maximal response of p-JNK1 in NMDA-treated cells appeared at 6 h, still sustained high level at 12 h, within 24 h of NMDA treatment (P < 0.05), however, the activation of JNK2 was only occurrence at 12 h after exposed to NMDA (P < 0.05). The total protein levels of JNK1/2 were not significantly changed at these time points after NMDA treatment. Pretreatment of HN significantly reduced NMDA-induced JNK1 phosphorylation by 32.10% at 6 h and 11.79% at 12 h (P < 0.05), and also inhibited activation of JNK2 by 19.24% at 12 h after NMDA challenge (P < 0.05); (3) The level of p-p38 MAPK in NMDA-treated cells became elevated at 3, 6, and 12 h after NMDA treatment (P < 0.05), with a peak response at 6 h. Total levels of p38 MAPK were virtually unchanged under all of these experimental conditions. When pretreated with HN, p-p38 MAPK activity was suppressed by 16.98% at 3 h, 35.98% at 6 h and 12.36% at 12 h after NMDA exposure, with the maximal inhibitory effects at 6 h (P < 0.05). NMDA had no effect on the expression of ERK or p-ERK.These findings indicated that: (1) NMDA induced JNK and p38 MAPK activation, but not ERK activation, which consistented with the results of the first and second parts of experiments; (2) NMDA-induced activation of JNK and p38 MAPK pathways (6 h) was earlier than the production of ROS (12 h ), therefore activation of JNK and p38 MAPK might promote ROS production, which mediated the neuronal damage; (3) The target of HN against NMDA-MAPKs system might not be NMDA receptors but its downstream molecules, resulting in consequent inhibition of MAPK signaling pathways; (4) Based on the fact that HN exhibited more significant protection from excitatory neurotoxicity than single or combination administration of MAPK inhibitors, HN could also produce cellular protection by interaction of JNK or p38 MAPK, and with concomitant of other signaling pathways (e.g., PI3K/Akt pathway).Conclusions: (1) Under the same experimental condition of NMDA-induced neurotoxicity, the present study systematically investigated and analyzed NMDA-induced activation of MAPK signaling pathways and the contributions of different signaling pathways, which provided novel insights into the neuronal mechanisms of NMDA-induced excitotoxicity; (2) The JNK and p38 MAPK pathways, but not the ERK pathway, contributed to NMDA-induced cortical neuronal insults; p38 MAPK showed a more significant role than JNK and furthermore the two pathways might act synergistically in NMDA-mediated excitotoxicity; (3) ROS induction through JNK and p38 MAPK activation might mediate consequent excitotoxic neuronal death; (4) The activation of p38 MAPK pathway, but not the JNK and ERK pathways was involved in NMDA-induced apoptosis of cortical neurons; JNK pathway might involve in NMDA-induced cortical neuronal necrosis; (5) HN exerted its neuroprotection through inhibiting the activation of NMDA-JNK and NMDA-p38 MAPK signaling pathways; The target of HN against NMDA-MAPKs system might not be NMDA receptors but its downstream molecules, resulting in consequent inhibition of MAPK signaling pathways; (6) One of the neuroprotective mechanisms of HN might be down-regulation of ROS production through inhibiting the activation of JNK and p38 MAPK pathways; (7) Based on the fact that HN exhibited more significant protection from excitatory neurotoxicity than single or combination administration of MAPK inhibitors, there is the hypothesis that HN could exert protection by inactivation of JNK or p38 MAPK, and with concomitant of other signaling pathways (e.g., PI3K/Akt pathway).