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Published on: November 6, 2017
Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons
Xiao-Rong Yang1, Ping Sun, Hua-Ping Qin
1Department of Physiology; Department of Nutrition of the First Hospital, Shanxi Medical University, Taiyuan 030001, China.
Abstract:
NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.
Insights
N-methyl-D-aspartate (NMDA) induces neuronal apoptosis and necrosis. The p38 mitogen-activated protein kinase (MAPK) pathway is involved in NMDA-induced apoptosis, offering a potential neuroprotective target.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- NMDA receptor overactivation leads to excitotoxicity, causing significant neuronal damage.
- Understanding the molecular mechanisms of NMDA-induced neuronal death is crucial for developing neuroprotective strategies.
Purpose of the Study:
- To quantify NMDA-induced apoptosis in rat cortical neurons.
- To elucidate the specific mitogen-activated protein kinase (MAPK) pathways involved in NMDA-induced neuronal apoptosis and necrosis.
Main Methods:
- Primary rat cortical neurons were treated with NMDA.
- Apoptosis was assessed using caspase-3 activity, TUNEL, and Annexin V staining.
- Neurons were pretreated with inhibitors of ERK, JNK, and p38 MAPK pathways.
Main Results:
- NMDA induced significant caspase-dependent apoptosis (22.49%).
- Inhibition of p38 MAPK (SB203580) reduced NMDA-induced caspase-3 activity and apoptosis.
- JNK inhibition (SP600125) decreased late-stage apoptosis/necrosis, while ERK inhibition had no significant effect.
Conclusions:
- NMDA triggers both apoptosis and necrosis in cortical neurons.
- The p38 MAPK pathway plays a critical role in NMDA-induced neuronal apoptosis.
- Targeting the p38 MAPK pathway demonstrates neuroprotective potential against NMDA excitotoxicity.
