TNF alpha Signaling: Effects on AMPA Receptor Location

描述(申请人提供)：这项计划将研究肿瘤坏死因子-α信号级联及其对突触活性的快速影响。已有研究表明，肿瘤坏死因子-α可增加非病变神经元的突触活性，并可在疾病或创伤时引起神经元的兴奋性毒性。兴奋性神经元中表达的NMDARs和AMPA型谷氨酸受体(NMDARs和AMPARs)在调节突触活动中起重要作用。因此，这些受体可能是肿瘤坏死因子-α信号调节的靶点。特别是，AMPAR被发现是突触后密度的一个高度调节和移动的成分，并被认为通过快速定位含NMDAR的突触来增加突触增强。最近我们发现，快速(15分钟)向培养的大鼠海马神经元供应肿瘤坏死因子-α可增加突触的活性和AMPAR对突触的定位，但不改变NMDAR的定位。这背后的信号机制尚不清楚。 这项建议将研究可能与肿瘤坏死因子-α诱导的AMPAR定位和突触活性增加有关的肿瘤坏死因子-α信号通路。在海马区和脊髓区这两个中枢神经系统区域的培养神经元中，经过不同处理后，将检测AMPAR的亚细胞定位。分析将利用细胞生物学、免疫组织化学、生化和电生理技术。这项建议的前三个目标将研究培养的大鼠和小鼠海马神经元中的肿瘤坏死因子-α信号。特异性目标1将确定来自两个肿瘤坏死因子-α受体--TNFR1、TNFR2或两者之一的信号是否有助于AMPAR表面定位的增加。 TNFR1和TNFR2均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将验证CaMKII和钙介导的途径对这种活动负责的假设。目的3将研究肿瘤坏死因子-α信号转导后出现在神经元表面的AMPAR的细胞内来源。最后，目标4将询问肿瘤坏死因子-α是否能够导致中枢神经系统不同区域、皮质和脊髓的神经元表面AMPAR定位增加。如果是这样的话，在AIMS 1至3中对海马神经元进行评估时，将评估TNF-α在运动神经元和皮质神经元中AMPAR定位的信号的细节。 这些目标勾勒出合乎逻辑的下一步，扩展了我们的初步数据，表明肿瘤坏死因子-α增加了突触AMPAR的本地化。这一结果将显著增加我们对健康和疾病中中枢神经系统突触功能的理解。在中风或脊髓损伤后，以及在阿尔茨海默氏症、帕金森氏症和肌萎缩侧索硬化症等疾病期间，经历兴奋性毒性细胞死亡的中枢神经系统神经元供应过量的肿瘤坏死因子-α。最近将肿瘤坏死因子-α信号与突触活性增加联系起来的数据表明，供过于求的肿瘤坏死因子-α可能通过将过多的AMPAR转移到突触来杀死神经元。因此，了解肿瘤坏死因子-α信号如何增加AMPAR在特定类型中枢神经系统神经元中的表面定位，可能使我们能够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.