MODULATION OF MIGRAINE CIRCUIT BY SEROTONIN 1D RECEPTOR-ASSOCIATED PROTEINS

描述（由申请人提供）：偏头痛是最常见的神经系统疾病之一，是卫生系统的一大负担。目前治疗偏头痛的方法往往不足。三叉神经节（TG）神经元的中枢末梢上的5-羟色胺1D受体（5-HT 1DR）是曲坦类抗偏头痛药物的主要靶点。曲坦类药物与5-HT 1DR结合后激活多种Gi/o偶联的信号转导途径，主要是抑制电压门控性钙通道（VGCC）活性，减少TG末端神经递质/神经肽的释放。开发新的偏头痛治疗剂的策略之一是选择性地增强TG神经元中5-HT 1DR的合成、质膜运输和/或细胞内信号传导。与人5-HT 1DR的第三胞内环相互作用的蛋白质已经通过酵母双杂交筛选鉴定。PLEKHB 1是一种含有普列克底物蛋白同源性（PH）结构域的膜整合蛋白，它优先与5-HT 1DR相互作用，而不与其他11种5-HT受体亚型相互作用。内源性PLEKHB 1和5-HT 1DR蛋白的表达在TG神经元中基本重叠。免疫共沉淀实验进一步证实了内源性PLEKHB 1与5-HT 1DR在TG组织中的相互作用。过表达PLEKHB 1可降低HEK细胞和培养的TG神经元中5-HT 1DR的表达。重要的是，PLEKHB 1显著降低HEK细胞中5-HT 1DR介导的VGCC电流抑制。该提案的研究目的是检验PLEKHB 1调节5-HT 1DR的代谢和信号传导，从而调节TG神经元的活性以及偏头痛相关神经元回路的增益的假设。首先，我们将研究PLEKHB 1是否调节TG神经元中5-HT 1DR蛋白水平和质膜表达。其次，我们将测试PLEKHB 1是否通过5-HT 1DR调节TG神经元中的信号传导，重点是PLEKHB 1对5-HT 1DR介导的VGCC电流抑制以及TG末端的突触传递的影响。最后，我们将研究PLEKHB 1是否调节疼痛反应行为以及头痛小鼠模型中的活动依赖性基因表达。特别地，我们将测试PLEKHB 1是否影响曲坦类药物在体内的功效和/或效力。 头痛的老鼠模型。总之，该项目的结果不仅将为PLEKHB 1在头痛产生和调节中的功能意义提供有价值的见解，而且还为开发基于机制的新偏头痛治疗方法奠定了重要的基础工作。

DESCRIPTION (provided by applicant): Migraine is one of the most common neurological disorders and a big burden of health system. Current treatments for migraine headache often fall short. The serotonin 1D receptor (5-HT1DR) on the central terminals of the trigeminal ganglion (TG) neurons is the prime target of the triptan family of anti-migraine drugs. Triptans bind to 5-HT1DR and activate multiple Gi/o-coupled signal transduction pathways, mainly the inhibition of voltage-gated Ca2+ channel (VGCC) activity and the subsequent reduction of neurotransmitters/neuropeptides release from TG terminals. One of the strategies to develop new migraine therapeutics is to selectively enhance the synthesis, plasma membrane trafficking and/or intracellular signaling of 5-HT1DR in TG neurons. Proteins that interact with the third intracellular loop of human 5-HT1DR has been identified through yeast two- hybrid screening. PLEKHB1, a pleckstrin homology (PH) domain-containing integral membrane protein, preferentially interacts with 5-HT1DR but not the other 11 subtypes of 5-HT receptors. The expression of endogenous PLEKHB1 and 5-HT1DR proteins overlaps substantially in TG neurons. Furthermore, the interaction between endogenous PLEKHB1 and 5-HT1DR in TG tissue is confirmed by co-immunoprecipitation experiment. Over-expression of PLEKHB1 decreases the expression of 5-HT1DR in HEK cells as well as cultured TG neurons. Importantly, PLEKHB1 significantly reduces 5-HT1DR-mediated inhibition of VGCC currents in HEK cells. The research objective of this proposal is to test the hypothesis that PLEKHB1 modulates the metabolism and signaling of 5-HT1DR, thereby regulating the activity of TG neurons as well as the gain of the neuronal circuit underlying migraine headache. First, we will investigate whether PLEKHB1 modulates 5-HT1DR protein level and plasma membrane expression in TG neurons. Second, we will test if PLEKHB1 modulates signaling through 5-HT1DR in TG neurons, focusing on the effects of PLEKHB1 on 5-HT1DR-mediated inhibition of VGCC currents as well as synaptic transmission at TG terminals. Finally, we will investigate whether PLEKHB1 regulates the nocifensive behavior as well as the activity-dependent gene expression in a mouse model of headache. In particular, we will test if PLEKHB1 affects the efficacy and/or potency of triptans in the mouse models of headache. Together, the outcome of this project will not only offer valuable insights into the functional significance of PLEKHB1 in headache generation and modulation, but also lay important ground work for the development of mechanism-based screening of new migraine therapeutics.