Neuronal control of CGRP gene expression

描述（由申请人提供）：降钙素基因相关肽（CGRP）由于其在三叉神经节中的表达以及在神经源性炎症和伤害感受中的作用而与颅面疼痛和头痛有关。特别是，CGRP水平在偏头痛中升高，然后在头痛缓解的同时恢复正常。然而，尽管偏头痛和其他三叉神经痛综合征普遍存在，但调节三叉神经痛CGRP表达的机制仍不清楚。偏头痛的持续疼痛表明存在维持CGRP水平升高的反馈机制。CGRP的生物活性之一是触发细胞因子的释放。我们建议测试细胞因子在突触前受体上产生正反馈回路以刺激三叉神经节中CGRP基因表达的假设。我们已经将报告基因引入大鼠三叉神经节神经元的原代培养中，并开始了监测体内启动子活性的研究。在初步数据中，我们已经表明两种细胞因子，TNF-a和激活素，可以刺激CGRP启动子。在TNF-a的情况下，这种刺激可能是通过MAP激酶激活神经元特异性的18bp增强子介导的，该增强子结合bHLH-Zip蛋白USF。我们拟研究USF在细胞因子激活CGRP基因中的作用。作为削弱这种激活的策略，我们将使用MAP激酶磷酸酶-1的基因转移。该方法将通过将病毒载体从小鼠须垫逆行递送到神经节来补充培养研究。CGRP启动子的调节将通过急性和慢性细胞因子升高后的生物发光测定来监测。通过这种方式，病毒基因转移将作为研究CGRP基因表达的反馈控制和调控基因靶向神经元的工具。这一建议的意义在于，它将建立一个连接细胞因子和神经肽的分子机制，并有可能揭示新的治疗策略来减少神经肽的合成。

DESCRIPTION (provided by applicant): Calcitonin gene-related peptide (CGRP) has been implicated in craniofacial pain and headache by virtue of its expression in the trigeminal ganglion and roles in neurogenic inflammation and nociception. In particular, CGRP levels are elevated in migraine, then returned to normal coincident with headache relief. However, despite the prevalence of migraine and other trigeminal pain syndromes, the mechanisms that regulate trigeminal CGRP expression remain mostly unknown. The sustained pain of migraine suggests the existence of feedback mechanisms that maintain elevated CGRP levels. One of the biological activities of CGRP is to trigger the release of cytokines. We propose to test the hypothesis that cytokines create a positive feedback loop onto presynaptic receptors to stimulate CGRP gene expression in the trigeminal ganglion. We have introduced reporter genes into primary cultures of rat trigeminal ganglia neurons and have begun studies monitoring promoter activity in vivo. In preliminary data we have shown that two cytokines, TNF-a and activin, can stimulate the CGRP promoter. In the case of TNF-a, the stimulation may be mediated by MAP kinase activation of the neuron-specific 18-bp enhancer that binds the bHLH-Zip protein USF. We propose to study the role of USF in CGRP gene activation by cytokines. As a strategy to attenuate this activation, we will use gene transfer of MAP kinase phosphatase-1. The approach will be to complement culture studies with retrograde delivery of viral vectors to the ganglia from the mouse whisker pad. Regulation of the CGRP promoter will be monitored by bioluminescence assays following acute and chronic elevation of cytokines. In this manner, viral gene transfer will be used as a tool for studying feedback control of CGRP gene expression and for targeting regulatory genes to neurons. The significance of this proposal is that it will restablish a molecular mechanism linking cytokines and neuropeptides and potentially reveal new therapeutic (strategies to attenuate neuropeptide synthesis.