DISCOVERY OF NOVEL TARGETS FOR POST-TRAUMATIC HEADACHE

项目摘要/摘要 创伤后头痛(PTH)是继轻度创伤后最常见和持续的症状之一。 脑损伤(MTBI)，每年影响5000万人。尽管这种疾病非常普遍，令人虚弱， 慢性甲状旁腺激素仍然知之甚少，治疗选择有限。 临床和临床前研究都表明甲状旁腺激素可能是由于 外周硬脑膜传入神经元。识别导致硬脑膜传入敏化的分子 PTH，我们进行了硬脑膜传入神经元的单细胞RNA测序，以揭示mTBI引起的 以公正的方式进行基因转录。我们已经确定了3个在7天内显著上调的基因 在mTBI之后：1)O95185，它编码Netrin受体unc5C；2)Q9NS66，它编码 3)编码钙离子-2亚单位KCNMB2的Q9Y691。 和大电导的电压门控K+通道(BK通道)。 这三个基因都编码未被研究的膜蛋白，这些蛋白在 小鼠和人三叉神经节(TG)神经元均有表达；而它们在其他外周组织中的表达 要低得多。值得注意的是，偏头痛患者的甘油三酯组织中UNC5C和GPR173水平较高。按顺序 确定这些蛋白质是否是外周活性药物治疗甲状旁腺激素的潜在靶点 为了将滥用责任和其他副作用降至最低，人们需要一条关键的信息来说明它们是否 在功能上参与甲状旁腺激素的发病。我们将在本研究中解决这一知识差距。 首先，我们将使用RNAScope原位杂交来评估mTBI诱导的神经元的空间和时间变化。 UNC5C、GPR173和KCNMB2在初级传入神经元中的表达。结果将使我们能够预测：1) 由每个候选人调节的硬脑膜传入亚型(S)；2)每个候选人 可能与甲状旁腺激素的发病有关；3)它们是否是甲状旁腺激素的选择性靶点。 其次，我们将通过以下方式抑制初级传入神经元中UNC5C、GPR173或KCNMB2的表达 用编码针对个别基因的shRNA的腺相关病毒(AAVs)转导小鼠。我们会 也通过AAV介导的转导在初级传入神经元中过表达个别蛋白。到时候我们会的 研究mTBI在这些多终点小鼠中诱导的行为和细胞变化。这些研究 将揭示是否unc5C、GPR173或KCNMB2调节甲状旁腺素的时序和/或分辨率- 相关的行为，以及这些蛋白的过度表达或敲除是否显示出治疗益处。 成功完成这些目标将帮助我们确定未被研究的蛋白质，这些蛋白质对 开发、维护和/或解决PTH，并是外围行动、非 甲状旁腺激素成瘾性治疗，副作用最小。这将为后续的R01提供初步数据 应用于进一步深入的机制研究或启动药物发现项目。

Project Summary/Abstract Post-traumatic headache (PTH) is one of the most common and persistent symptoms following mild traumatic brain injury (mTBI), which affects 50 million people every year. Despite being highly prevalent and debilitating, chronic PTH remains poorly understood and with limited treatment options. Both clinical and preclinical studies indicate that PTH may result from the prolonged sensitization of peripheral dural afferent neurons. To identify molecules that are responsible for dural afferent sensitization in PTH, we performed single cell RNA sequencing of dural afferent neurons to reveal mTBI-induced changes of gene transcription in an unbiased manner. We have identified 3 genes that are significantly upregulated 7 days after mTBI: 1) O95185, which encodes the netrin receptor UNC5C; 2) Q9NS66, which encodes the neuropeptide phoenixin receptor GPR173; and 3) Q9Y691, which encodes KCNMB2, the 2 subunit of Ca2+- and voltage-gated K+ channels of large conductance (BK channels). All three genes encode understudied membrane proteins that are expressed at moderate to high levels in both mouse and human trigeminal ganglion (TG) neurons; whereas their expression in other peripheral tissues are much lower. Notably, UNC5C and GPR173 levels are higher in TG tissues from migraine patients. In order to determine whether these proteins are potential targets for peripherally-active drugs to treat PTH with minimal abuse liability and other side effects, one needs a critical piece of information as to whether they are functionally involved in PTH pathogenesis. We will address this knowledge gap in the present study. First, we will use RNAScope in situ hybridization to assess mTBI-induced spatial and temporal changes of UNC5C, GPR173 and KCNMB2 expression in primary afferent neurons. The results will allow us to predict: 1) the dural afferent subtype(s) that are regulated by each candidate; 2) the period during which each candidate likely contributes to the pathogenesis of PTH; and 3) whether they may be targets that are selective for PTH. Secondly, we will knockdown UNC5C, GPR173 or KCNMB2 expression in primary afferent neurons by transducing mice with adeno-associated viruses (AAVs) encoding shRNA against individual genes. We will also overexpress individual proteins in primary afferent neurons via AAV-mediated transduction. We will then investigate mTBI-induced behavioral and cellular changes in these mice with multiple endpoints. These studies will reveal whether UNC5C, GPR173 or KCNMB2 regulate the chronification and/or the resolution of PTH- related behaviors, and whether overexpression or knockdown of these proteins exhibit therapeutic benefit. Successful completion of these aims will help us identify understudied proteins that are critical for the development, maintenance and/or the resolution of PTH and are potential targets for peripherally-acting, non- addictive PTH treatment with minimal side effects. This will provide preliminary data for subsequent R01 application to further in-depth mechanistic studies or for the initiation of a drug discovery project.