TNF alpha Signaling: Effects on AMPA Receptor Location

描述（由申请人提供）：本提案将研究肿瘤坏死因子α（TNF-α）信号级联及其对突触活性的快速影响。TNF-α已显示出增加非患病神经元中的突触活性，并且可在疾病或创伤期间诱导神经元中的兴奋性毒性。已知在兴奋性神经元中表达的NMDA型和AMPA型谷氨酸受体（NMDARs和AMPAs）在调节突触活动中具有主要作用。因此，这些受体可能是TNF-α信号调节的靶点。特别地，已经发现AMPAR是突触后密度的高度调节的和移动的组分，并且被认为通过快速定位于含NMDAR的突触来增加突触增强。最近，我们已经表明，TNF-α提供给大鼠海马神经元培养迅速（15分钟）增加突触活动和AMPAR的突触定位，但不改变NMDARs的定位。这背后的信号机制是未知的。 该提案将研究TNF-α信号通路可能负责TNF-α诱导的AMPAR定位和突触活性的增加。在来自两个CNS区域（海马和脊髓）的培养的神经元中进行各种处理后，将检查亚细胞AMPAR定位。分析将利用细胞生物学、免疫组织化学、生物化学和电生理学技术。本提案的前三个目标将研究培养的大鼠和小鼠海马神经元中的TNF-α信号。具体目标1将确定来自两种TNF-α受体之一-TNFR 1、TNFR 2或两者的信号传导是否有助于增加AMPAR表面定位。 TNFR 1和TNFR 2都在海马神经元中表达，并引起重叠但不同的信号通路。具体目标2将检验CamKII和钙介导的途径负责该活性的假设。目的3将研究TNF-α信号转导后出现在神经元表面的AMPAR的细胞内起源。最后，目标4将询问TNF-α是否能够引起CNS、皮质和脊髓的不同区域的神经元上的表面AMPAR定位的增加。如果是，则将评估TNF-α信号传导对运动神经元和皮质神经元中AMPAR定位的细节，如在目标1至3中评估的海马神经元。 这些目标概述了逻辑的下一步扩展我们的初步数据显示，TNF-α增加突触AMPAR定位。 这些结果将大大增加我们对健康和疾病中CNS突触功能的理解。经历兴奋性毒性细胞死亡的CNS神经元在中风或脊髓损伤后以及在诸如阿尔茨海默氏症、帕金森氏症和ALS的疾病期间过量供应TNF-α。最近将TNF-α信号传导与突触活动增加联系起来的数据表明，过量的TNF-α可能通过将过多数量的AMPAR移动到突触来杀死神经元。因此，TNF-α信号传导如何增加特定类别的CNS神经元中的AMPAR表面定位的具体知识可以使我们更有效地预防一系列疾病和创伤中的兴奋性毒性。

DESCRIPTION (provided by applicant): This proposal will study the tumor necrosis factor alpha (TNF-alpha) signaling cascade and its rapid effects on synaptic activity. TNF-alpha has been shown to increase synaptic activity in non-diseased neurons, and can induce excitotoxicity in neurons during disease or trauma. NMDA-type and AMPA-type glutamate receptors (NMDARs and AMPARs) expressed in excitatory neurons are known to have major roles in modulating synaptic activity. These receptors therefore are possible targets of TNF-alpha signaling regulation. In particular, AMPARs have been found to be a highly regulated and mobile component of the postsynaptic density and are thought to increase synaptic potentiation via rapid localization to NMDAR-containing synapses. Recently we have shown that TNF-alpha supplied to rat hippocampal neurons in culture rapidly (15 minutes) increased synaptic activity and AMPAR localization to synapses, but did not alter the localization of NMDARs. The signaling mechanisms underlying this are unknown. This proposal will examine TNF-alpha signaling pathways potentially responsible for the TNF-alpha-induced increase in AMPAR localization and synaptic activity. Subcellular AMPAR localization will be examined after various treatments in cultured neurons from two CNS regions, the hippocampus and spinal cord. The analysis will utilize cell biological, immunohistochemical, biochemical and electrophysiological techniques. The first three aims in this proposal will examine TNF-alpha signaling in cultured rat and mouse hippocampal neurons. Specific aim 1 will determine if signaling from one of the two TNF-alpha receptors- TNFR1, TNFR2, or both contribute to increased AMPAR surface localization. TNFR1 and TNFR2 are both expressed in hippocampal neurons and elicit overlapping yet distinct signaling pathways. Specific aim 2 will test the hypothesis that CamKII and a calcium mediated pathway are responsible for this activity. Aim 3 will investigate the intracellular origin of the AMPARs that appear on the neuron surface after TNF-alpha signaling. Finally, Aim 4 will ask if TNF-alpha is able to cause increases in surface AMPAR localization on neurons of different regions of the CNS, the cortex and spinal cord. If so, the details of TNF-alpha's signaling on AMPAR localization in motorneurons and cortical neurons will be assessed as hippocampal neurons were assessed in aims 1 through 3. These aims outline the logical next step extending our preliminary data showing that TNF-alpha increases synaptic AMPAR localization. The results will significantly increase our understanding of synaptic function in the CNS in both health and disease. CNS neurons that undergo excitotoxic cell death are oversupplied with TNF-alpha after stroke or spinal cord injury as well as during diseases such as Alzheimer's, Parkinson's, and ALS. Recent data linking TNF-alpha signaling to increased synaptic activity suggests that an oversupply TNF-alpha may kill neurons by moving excessive numbers of AMPARs to synapses. Thus, specific knowledge of how TNF-alpha signaling increases AMPAR surface localization in particular classes of CNS neurons may allow us to more effectively prevent excitotoxicity in a range of diseases and trauma.