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

描述(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。位于三叉神经节(TG)神经元中央终末的5-羟色胺1D受体(5-HT1DR)是Triptan家族抗偏头痛药物的主要靶点。Triptans与5-HT1DR结合，激活多个GI/O偶联信号转导通路，主要是抑制电压门控钙通道(VGCC)的活性，从而减少从TG末端释放的神经递质/神经肽。选择性增强三叉神经节神经元5-HT1DR的合成、质膜转运和/或细胞内信号转导是开发偏头痛新疗法的策略之一。通过酵母双杂交筛选，已经鉴定出与人5-HT1DR的第三个胞内环相互作用的蛋白质。PLEKHB1是一种含有Pleckstrin Homology(PH)结构域的完整膜蛋白，它优先与5-HT1DR相互作用，而不与其他11种亚型的5-HT1受体相互作用。内源性PLEKHB1和5-HT1DR蛋白在三叉神经节神经元中的表达基本重叠。免疫共沉淀实验证实了内源性PLEKHB1与5-HT1DR在TG组织中的相互作用。PLEKHB1的过表达降低了HEK细胞和培养的TG神经元中5-HT1DR的表达。重要的是，PLEKHB1显著降低5-HT1DR对HEK细胞VGCC电流的抑制作用。该方案的研究目的是验证PLEKHB1调节5-HT1DR的代谢和信号传递，从而调节TG神经元的活动以及偏头痛相关神经元回路增益的假说。首先，我们将研究PLEKHB1是否调节TG神经元的5-HT1DR蛋白水平和质膜表达。其次，我们将测试PLEKHB1是否通过5-HT1DR在TG神经元中调制信号，重点研究PLEKHB1对5-HT1DR介导的VGCC电流的抑制以及TG终末的突触传递的影响。最后，我们将研究PLEKHB1是否调节头痛小鼠模型中的明示行为以及活动依赖的基因表达。特别是，我们将测试PLEKHB1是否影响Triptans在 头痛的小鼠模型。综上所述，该项目的结果不仅将为PLEKHB1在头痛发生和调节中的功能意义提供有价值的见解，也将为开发基于机制的偏头痛新疗法的筛选奠定重要的基础。 公共卫生相关性：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。5-羟色胺1D受体(5-HT1DR)是曲普坦家族抗偏头痛药物的主要靶点。在这里，我们建议检验5-HT1DR相互作用的蛋白质调节5-HT1DR的代谢和信号，进而控制偏头痛潜在的神经元回路的假设。该项目的成果将为基于机制的偏头痛新疗法筛查的发展奠定重要的基础工作。

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. PUBLIC HEALTH RELEVANCE: 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) is the prime target of the triptan family of anti-migraine drugs. Here we propose to test the hypothesis that 5-HT1DR-interacting proteins modulate the metabolism and signaling of 5-HT1DR, and, in turn, control the neuronal circuit underlying migraine headache. The outcome of this project will lay important ground work for the development of mechanism-based screening of new migraine therapeutics.