Genetic Evidence for Adenylyl Cyclase 1 as a Target for Preventing Neuronal Excitotoxicity Mediated by N-Methyl-D-aspartate Receptors

Hansen Wang,Bo Gong,Kunjumon I Vadakkan,Hiroki Toyoda,Bong-Kiun Kaang,Min Zhuo

doi:10.1074/jbc.m607291200

Hansen Wang, Bo Gong + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m607291200

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Abstract

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.

Highlights

The cAMP signaling pathway has been shown to act downstream of NMDA receptor activation in synaptic plasticity (18 – 22)
We report that Adenylyl cyclases 1 (AC1) is involved in neuronal excitotoxicity mediated by NMDA receptors; genetic deletion of AC1 can protect against glutamate-induced cell death in neuronal cultures and attenuate cortical lesions caused by NMDA injection
Genetic Deletion of AC1 Protects against Neuronal Death Induced by Glutamate—First, the effect of genetic deletion of AC1 or AC8 on glutamate-induced cell death was evaluated by measuring cell viability in cultured cortical neurons after treatment with glutamate

Summary

EXPERIMENTAL PROCEDURES

Animals and Primary Culture of Cortical Neurons—Mutant mice lacking AC1 or AC8 and wild-type mice were generated and maintained as previously described [19, 28, 31]. Cells were rinsed in PBS and incubated with streptavidin-horseradish peroxidase for 30 min at 37 °C. Western Blot Analysis—The cultured cortical neurons were harvested and homogenized in lysis buffer (10 mM Na2HPO4, pH 7.2, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VaO3, 20 mM NaF, 0.1% SDS) containing proteinase inhibitor mixture (Sigma). The membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibody diluted at 1:3000 for 1 h followed by enhanced chemiluminescence detection of the proteins with Western lightning chemiluminescence reagent plus (PerkinElmer Life Sciences) according to the manufacturer’s instructions. Luciferase activity was assayed in the cell lysate by using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol. The cerebral cortex of the injected side were collected and homogenized in the cell lysis buffer

RESULTS

NMDA Receptors in Cultured

DISCUSSION