Ethanol and ERK/PKB Signaling in Brain

大脑中的乙醇和 ERK/PKB 信号传导

• 批准号: 7061590
• 负责人: L Judson Chandler
• 金额: $ 32.08万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061590
• 关键词: NMDA receptors; alcoholism /alcohol abuse; biological signal transduction; brain metabolism; cAMP response element binding protein; confocal scanning microscopy; ethanol; immunocytochemistry; laboratory rat; neural plasticity; neurons; synapses; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): NMDA receptors play a central role in synaptic plasticity and neuropathology, and are known targets of ethanol. Two signal transduction systems that regulate plasticity and survival are the ERK and PKB cascades, and we have recently shown that NMDA receptors control ERK and PKB activity through a common signaling module. Furthermore, this control is bidirectional, and involves activation of opposing stimulatory and inhibitory pathways. While the components of the stimulatory pathway have been substantially defined, the nature of the inhibitory pathway remains obscure. An important down-stream effector of ERK/PKB signaling is CREB, and it was recently reported that NMDA receptors show a similar bidirectional control of CREB activity. Importantly, synaptic NMDA receptors promote survival signaling through CREB, whereas extrasynaptic NMDA receptors activate a CREB shut-off pathway and promote neuronal death. We now hypothesize that the ERK/PKB activation pathway is coupled to synaptic NMDA receptors, while the inhibitory pathway is coupled to extrasynaptic NMDA receptors. Furthermore, preliminary data is presented showing that chronic ethanol induces marked changes in the distribution of NMDA receptors between synaptic and extrasynaptic sites. It is proposed that these changes will have profound effects on ERK, PKB, and CREB (EPC) signaling by altering the balance between the stimulatory and inhibitory pathways. The over-arching goal of this proposal is to determine the effects of ethanol on NMDA receptor trafficking between synaptic and non-synaptic sites, and upon regulation of EPC signaling by NMDA receptors. This project will utilize a well-defined neuronal cell culture model and employ various experimental techniques including whole-cell patch-clamp electrophysiology, biochemical and molecular assays, confocal microscopy, and fluorescence immunohistochemistry. The Specific Aims are to: (1) Determine the effects of acute ethanol on the modulation of CREB activity by synaptic and extrasynaptic NMDA receptors; (2) Test the hypothesis that inhibition of CREB by extrasynaptic NMDA receptors involves upstream inhibition of ERK/PKB; (3) Test the hypothesis that chronic ethanol induces a redistribution of NMDA receptors from extrasynaptic to synaptic sites and leads to corresponding changes in EPC signaling; and (4) Test the hypothesis that ethanol-induced changes in NMDA receptor localization and EPC signaling lead to changes in neuronal plasticity and survival. Our preliminary data showing chronic ethanol-induced change in the trafficking of NMDA receptors together with compartmentalization of discrete signaling complexes have important implications for understanding how ethanol leads to enduring changes in the brain.

描述（由申请人提供）：NMDA 受体在突触可塑性和神经病理学中发挥核心作用，并且是乙醇的已知靶点。调节可塑性和存活的两个信号转导系统是 ERK 和 PKB 级联，我们最近表明 NMDA 受体通过共同的信号模块控制 ERK 和 PKB 活性。此外，这种控制是双向的，涉及激活相反的刺激和抑制途径。虽然刺激途径的组成部分已经基本确定，但抑制途径的性质仍然不清楚。 ERK/PKB信号传导的重要下游效应器是CREB，最近报道NMDA受体对CREB活性表现出类似的双向控制。重要的是，突触 NMDA 受体通过 CREB ​​促进生存信号传导，而突触外 NMDA 受体则激活 CREB ​​关闭途径并促进神经元死亡。我们现在假设 ERK/PKB 激活途径与突触 NMDA 受体偶联，而抑制途径与突触外 NMDA 受体偶联。此外，初步数据表明，长期乙醇会引起突触和突触外位点之间 NMDA 受体分布的显着变化。有人提出，这些变化将通过改变刺激和抑制途径之间的平衡，对 ERK、PKB 和 CREB ​​(EPC) 信号传导产生深远影响。该提案的首要目标是确定乙醇对突触和非突触位点之间 NMDA 受体运输的影响，以及 NMDA 受体对 EPC 信号传导的调节。该项目将利用明确的神经元细胞培养模型，并采用各种实验技术，包括全细胞膜片钳电生理学、生化和分子测定、共聚焦显微镜和荧光免疫组织化学。具体目标是： (1) 确定急性乙醇对突触和突触外 NMDA 受体调节 CREB ​​活性的影响； (2)检验突触外NMDA受体抑制CREB涉及上游ERK/PKB抑制的假设； (3) 检验慢性乙醇诱导NMDA受体从突触外重新分布到突触位点并导致EPC信号发生相应变化的假设； (4) 检验乙醇诱导的 NMDA 受体定位和 EPC 信号传导变化导致神经元可塑性和存活变化的假设。我们的初步数据显示，乙醇诱导的 NMDA 受体运输的慢性变化以及离散信号复合物的划分对于理解乙醇如何导致大脑的持久变化具有重要意义。