RESUMEN
NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Muerte Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/toxicidad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/toxicidad , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo I/fisiología , Péptidos/toxicidad , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Regulation of N-methyl-d-aspartate receptor (NMDAR) activity by desensitization is important in physiological and pathological states; NMDAR desensitization contributes in shaping synaptic responses and may be protective by limiting calcium influx during sustained glutamate insults. We previously reported that glycine-independent desensitization decreases during hippocampal neuronal development, correlating with NMDAR synaptic localization and association with postsynaptic density 95 (PSD-95). PSD-95/Discs large/zona occludens (PDZ)-1,2 domains of PSD-95 bind to the C-terminus of NMDAR NR2 subunits. The role of PSD-95 in anchoring signaling proteins near NMDARs is well documented. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in human embryonic kidney 293 (HEK293) cells and neurons. In HEK cells, wild-type PSD-95 significantly reduced wild-type NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. The PSD-95 N-terminus truncated after the PDZ1-2 domains was sufficient for this effect in neurons with low endogenous PSD-95 levels; in NMDAR-expressing HEK cells, the effect persisted when PSD-95 multimerization was eliminated. Moreover other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through protein kinase C (PKC) activation increased desensitization to levels found in immature neurons, and this effect was not due to PKC direct regulation of NMDAR activity. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure in neuronal and nonneuronal cells.