Department of Neurology, San Francisco Veterans Affairs Medical Center, University of California, San Francisco, CA 94121, USA.
Cell Death Dis. 2013 Apr 4;4(4):e580. doi: 10.1038/cddis.2013.111.
Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKCζ), which in turn phosphorylates the p47(phox) organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKCζ. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor.
神经元 N-甲基-D-天冬氨酸（NMDA）型谷氨酸受体的持续激活会导致中风、创伤和神经退行性疾病中的兴奋性细胞死亡。兴奋性细胞死亡部分是由于神经元 NADPH 氧化酶（NOX2）产生的超氧化物引起的，但 NMDA 受体如何与神经元 NOX2 激活偶联尚不清楚。在这里，我们确定了一条将 NMDA 受体激活与原代神经元培养物中的 NOX2 激活偶联的信号通路。NMDA 受体的 NR2B 亚基通过钙内流导致磷酯酰肌醇 3-激酶（PI3K）的激活。PI3K 形成磷脂酰肌醇（3,4,5）-三磷酸（PI（3,4,5）P3），激活非典型蛋白激酶 C，PKCζ（PKCζ），其反过来磷酸化神经元 NOX2 的 p47（phox）组织亚基。NR2B 包含的 NMDA 受体通过钙内流引发线粒体去极化、NOX2 激活、超氧化物形成和细胞死亡。然而，尽管引起线粒体去极化，离子霉素诱导的等效幅度钙升高不会诱导 NOX2 激活或神经元死亡。PI3K 抑制剂wortmannin 阻止 NMDA 诱导的 NOX2 激活和细胞死亡，而不会阻止细胞肿胀、钙升高或线粒体去极化。wortmannin 的作用可以通过外源性提供 PI3K 产物 PI（3,4,5）P3 和转染蛋白激酶 M（PKCζ 的一种组成激活形式）来规避。这些发现表明，超氧化物形成和兴奋性细胞死亡可以与线粒体去极化分离，并确定 PI3K 在该细胞死亡途径中的新作用。该途径的扰动可能会增加或减少 NMDA 受体激活后的超氧化物产生，从而影响兴奋性毒性是致病因素的神经退行性疾病。
