Neuronal control of CGRP gene expression

描述（由申请方提供）：降钙素基因相关肽（CGRP）由于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，然后恢复正常，同时头痛缓解。然而，尽管偏头痛和其他三叉神经疼痛综合征的流行，调节三叉神经CGRP表达的机制仍然是未知的。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的生物学活性之一是触发细胞因子的释放。我们建议测试的假设，细胞因子创建一个积极的反馈回路突触前受体刺激CGRP基因表达的三叉神经节。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物中，并开始了体内监测启动子活性的研究。在初步数据中，我们已经表明，两种细胞因子，TNF-α和激活素，可以刺激CGRP启动子。在TNF-α的情况下，刺激可以通过结合bHLH-Zip蛋白USF的神经元特异性18-bp增强子的MAP激酶活化来介导。我们建议研究USF在细胞因子激活CGRP基因中的作用。作为减弱这种激活的策略，我们将使用MAP激酶磷酸酶-1的基因转移。该方法将补充文化研究与逆行交付的病毒载体的神经节从小鼠胡须垫。在细胞因子急性和慢性升高后，将通过生物发光测定监测CGRP启动子的调节。以这种方式，病毒基因转移将被用作研究CGRP基因表达的反馈控制和将调节基因靶向神经元的工具。这一建议的意义在于，它将重新建立一个连接细胞因子和神经肽的分子机制，并可能揭示新的治疗策略，以减少神经肽的合成。

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.