Spinal TNF elicits AMPAr trafficking and hyperalgesia

描述(申请人提供)：AMPA受体(AMPAR)密度和磷酸化的动态变化以及质膜上可透过钙的AMPAR的数量有助于长时程增强和突触增强。肿瘤坏死因子(TNF)是一种胶质细胞产物，可诱导海马神经细胞质膜上钙离子通透性Ampar的插入。在脊髓，肿瘤坏死因子参与了各种损伤后的慢性疼痛。肿瘤坏死因子增强背角神经元活性的机制尚不清楚。我认为，在外周炎症之后，胶质细胞肿瘤坏死因子通过PI3K/Akt途径，在两个不同的位置磷酸化GluR1亚基，导致含有它们的Ampar插入质膜。增加的Ampar含有不成比例的钙离子通透性，进一步促进了突触的加强。我将测量P-GluR1，Ampar插入细胞膜，以及因爪部炎症而具有功能性钙离子通透性Ampar的背角细胞的数量。我将使用免疫印迹、亚细胞分离、海人藻酸钾诱导的钴负载和共聚焦显微镜的组合。在所提出的通路中的每一步，脊髓注射特定的抑制剂将证明这些元素对于疼痛行为和GluR1插入神经细胞膜以及事件的顺序是必要的。最后，我们将在小鼠中使用两种敲打菌株，它们不会在个别丝氨酸残基上磷酸化GluR1。这些小鼠的使用将进一步描绘出这条途径，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、更成功地设计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.