DISCOVERY OF NOVEL TARGETS FOR POST-TRAUMATIC HEADACHE

发现创伤后头痛的新靶标

• 批准号: 10685784
• 负责人: YUQING CAO
• 金额: $ 42.83万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685784
• 关键词: Acute; Adverse effects; Affect; Afferent Neurons; Anatomy; Behavior; Behavioral; Cervical; Chronic; Clinical Research; Colon; Data; Dependovirus; Development; Exhibits; FOS Protein; Face; Female; Fluoro-Gold; Gene Expression; Genes; Genetic Transcription; Goals; Headache; High Prevalence; Human; Hypersensitivity; In Situ Hybridization; Individual; Knowledge; Label; Liver; Lung; Maintenance; Mechanics; Mediating; Membrane Proteins; Migraine; Molecular; Mus; Muscle; NTN1 gene; Neurons; Neuropeptides; Pathogenesis; Pathway interactions; Patients; Peripheral; Persons; Pharmaceutical Preparations; Post-Traumatic Headaches; Posterior Horn Cells; Procedures; Productivity; Proteins; Quality of life; Resolution; Structure of trigeminal ganglion; Therapeutic; Time; Tissues; Tracer; Trauma; Up-Regulation; Voltage-Gated Potassium Channel; abuse liability; axon guidance; base; behavioral sensitization; behavioral study; candidate identification; common symptom; drug discovery; experience; experimental study; follow-up; knock-down; large-conductance calcium-activated potassium channels; male; mild traumatic brain injury; mouse model; netrin receptor; novel; overexpression; pain score; persistent symptom; pituitary adenylate cyclase activating polypeptide; preclinical study; receptor; screening; side effect; single-cell RNA sequencing; small hairpin RNA; targeted treatment; transcriptome

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.

项目摘要/摘要 创伤后头痛（PTH）是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。尽管高度普遍和令人衰弱，但 慢性PTH的理解水平仍然很差，并且治疗方案有限。 临床和临床前研究都表明，PTH可能是由于延长的灵敏度而引起的 外围硬膜传入神经元。识别负责硬膜传入传感器的分子 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诱导的变化 基因转录以公正的方式。我们已经确定了3个基因，这些基因已大大更新7天 MTBI之后：1）O95185，编码Netrin受体UNC5C； 2）Q9NS66，编码 神经肽凤凰受体GPR173; 3）编码KCNMB2的Q9Y691，Ca2+ - 的2亚基 大型电导率（BK通道）的电压门控的K+通道。 所有三个基因编码都以中等至高水平表达的膜蛋白的理解 小鼠和人类三叉神经节（TG）神经元均具有；而它们在其他周围组织中的表达 要低得多。值得注意的是，偏头痛患者的TG组织中的UNC5C和GPR173水平较高。为了 确定这些蛋白是否是外周活动药物治疗PTH的潜在靶标 最小的虐待责任和其他副作用，需要一个关键信息 功能参与PTH发病机理。我们将在本研究中解决这个知识差距。 首先，我们将使用rnascope原位杂交来评估MTBI诱导的空间和临时变化 原发性传入神经元中的UNC5C，GPR173和KCNMB2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； 2）每个候选人的期间 可能有助于PTH的发病机理； 3）它们是否可能是PTH选择性的目标。 其次，我们将在初级传入神经元中敲除UNC5C，GPR173或KCNMB2表达 用腺体相关病毒（AAVS）对单个基因编码shRNA的小鼠转导小鼠。我们将 然后我们会 研究了这些小鼠具有多个终点的MTBI诱导的行为和细胞变化。这些研究 将揭示UNC5C，GPR173或KCNMB2是否调节了PTH-的变化和/或分辨率 相关行为，以及这些蛋白质暴露的治疗益处的过表达或敲低。 这些目标的成功完成将有助于我们确定理解的蛋白质，这些蛋白质对 PTH的开发，维护和/或分辨率是外围作用，非 - 的潜在目标 添加剂PTH处理具有最小的副作用。这将为后续R01提供初步数据 用于进一步深入的机械研究或药物发现项目的倡议。