Spinal TNF elicits AMPAr trafficking and hyperalgesia

描述（由申请人提供）：AMPA受体（AMPAR）密度和磷酸化以及质膜中Ca 2+可渗透AMPAR数量的动态变化有助于长时程增强和突触强化。肿瘤坏死因子（TNF）是一种神经胶质产物，可引起海马神经元质膜上钙离子渗透性AMPAR的插入。在脊髓中，TNF导致各种损伤后的慢性疼痛。TNF增强背角神经元活动的机制尚不清楚。我建议，外周炎症后，胶质细胞TNF，通过PI 3 K/Akt通路，磷酸化GluR 1亚基在2个不同的网站，导致AMPAR包含它们被插入到质膜。增加的AMPAR含有不成比例的Ca 2+渗透性，进一步促进突触强化。我将测量P-GluR 1、AMPAR插入膜中以及由于爪炎症而具有功能性Ca 2+可渗透AMPAR的背角细胞的数量。我将使用蛋白质印迹，亚细胞分级分离，钾盐镁矾诱导的钴负载和共聚焦显微镜的组合。脊髓给药的具体抑制剂的每一个步骤中提出的途径将证明，这些元素是必要的疼痛行为和GluR 1插入到神经元质膜以及事件的顺序。最后，我们将使用2株敲入小鼠，其在单个丝氨酸残基处不磷酸化GluR 1。这些小鼠的使用将进一步描绘途径并指定多用途激酶的必要作用点。这些实验将进一步加深我们对脊髓基本机制的理解。最终，它将使我们能够更合理和成功地设计选择性药物来治疗慢性疼痛。 公共卫生相关性：组织炎症诱导脊髓释放TNF、脊髓神经元致敏和疼痛行为。TNF致敏疼痛传递神经元的机制尚不清楚。该提案探讨了TNF通过几个确定的步骤导致越来越多的活性谷氨酸受体插入神经元膜使其更易兴奋的可能性。

DESCRIPTION (provided by applicant): Dynamic changes in AMPA receptor (AMPAr) density and phosphorylation as well as number of Ca2+ permeable AMPAr in plasma membrane contribute to long term potentiation and synaptic strengthening. Tumor necrosis factor (TNF), a glial product, has been shown to elicit insertion of Ca2+ permeable AMPAr in plasma membrane of hippocampal neurons. In spinal cord, TNF contributes to chronic pain following a variety of injuries. The mechanism by which TNF enhances dorsal horn neuronal activity is unknown. I propose that following peripheral inflammation, glial TNF, working through a PI3K/Akt pathway, phosphorylates GluR1 subunits at 2 distinct sites, causing the AMPAr containing them to be inserted into plasma membranes. The increased AMPAr contain a disproportionate number that are Ca2+permeable, further contributing to synaptic strengthening. I will measure P-GluR1, insertion of AMPAr into the membrane and number of dorsal horn cells with functional Ca2+permeable AMPAr as a result of paw inflammation. I will use a combination of Western blots, subcellular fractionation, kainite induced cobalt loading and confocal microscopy. Spinal administration of specific inhibitors to every step in the proposed pathway will demonstrate that these elements are necessary for pain behavior and GluR1 insertion into neuronal plasma membranes as well as the sequence of events. Finally we will use 2 strains of knock in mice that do not phosphorylated GluR1 at individual serine residues. Use of these mice will further delineate the pathway and specify the necessary point of action of multi-purpose kinases. These experiments will further our understanding of fundamental spinal cord mechanisms. Ultimately, it will allow us to more logically and successfully design selective agents to treat chronic pain. PUBLIC HEALTH RELEVANCE: Tissue inflammation induces spinal cord release of TNF, sensitization of spinal neurons, and pain behavior. The mechanism by which TNF sensitizes pain transmission neurons is unknown. The proposal explores the possibility that TNF, via several identified steps, causes insertion of more and more active glutamate receptors into neuronal membranes to make them more excitable.