Spinal TNF elicits AMPAr trafficking and hyperalgesia

描述（由申请人提供）：AMPA受体（AMPAr）密度和磷酸化的动态变化以及质膜中Ca2+可渗透AMPAr的数量有助于长期增强和突触强化。肿瘤坏死因子（TNF）是一种胶质产物，已被证明可引起海马神经元质膜上Ca2+渗透性AMPAr的插入。在脊髓中，TNF有助于多种损伤后的慢性疼痛。TNF增强背角神经元活性的机制尚不清楚。我认为，在外周炎症发生后，胶质肿瘤坏死因子通过PI3K/Akt通路，在两个不同的位点磷酸化GluR1亚基，导致含有它们的AMPAr插入质膜。增加的AMPAr含有不成比例的Ca2+渗透性，进一步促进突触加强。我将测量P-GluR1， AMPAr插入膜和由于脚掌炎症而具有功能Ca2+可渗透AMPAr的背角细胞的数量。我将使用Western blots，亚细胞分离，kainite诱导钴负载和共聚焦显微镜的组合。在提出的通路的每个步骤中，脊髓给予特定抑制剂将证明这些元素对于疼痛行为和GluR1插入神经元质膜以及事件序列是必要的。最后，我们将在小鼠中使用不磷酸化GluR1单个丝氨酸残基的2株knock。使用这些小鼠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乎逻辑、更成功地设计出治疗慢性疼痛的选择性药物。

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.