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.

项目总结/摘要 创伤后头痛（Post-traumatic headache，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。尽管这种疾病非常普遍而且使人衰弱， 慢性PTH仍然知之甚少，治疗选择有限。 临床和临床前研究表明，PTH可能是由于长期致敏的 外周硬膜传入神经元为了确定负责硬脑膜传入敏化的分子， 我们对硬膜传入神经元进行了单细胞RNA测序，以揭示mTBI诱导的PTH变化。 基因转录以无偏见的方式。我们已经确定了3个基因，在7天内显著上调， mTBI后：1）O 95185，其编码netrin受体UNC 5C; 2）Q9 NS 66，其编码 神经肽phoenixin受体GPR 173;和3）Q9 Y 691，其编码KCNMB 2，Ca 2 +-Ca 2+的KCNMB 2亚基。 和大电导的电压门控K+通道（BK通道）。 所有这三个基因都编码未被充分研究的膜蛋白，这些膜蛋白在大肠杆菌中以中等至高水平表达。 小鼠和人三叉神经节（TG）神经元;而它们在其他外周组织中的表达 要低得多。值得注意的是，在偏头痛患者的TG组织中，UNC 5C和GPR 173水平较高。为了 以确定这些蛋白质是否是外周活性药物治疗PTH的潜在靶点， 最小的滥用责任和其他副作用，一个人需要一个关键的信息，以确定他们是否是 在功能上参与PTH发病机制。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诱导的空间和时间变化， 初级传入神经元中的UNC 5C、GPR 173和KCNMB 2表达。结果将使我们能够预测：1） 由每个候选者调节的硬脑膜传入亚型; 2）每个候选者 可能有助于PTH的发病机制;和3）它们是否可能是PTH的选择性靶点。 其次，我们将通过以下方法敲低初级传入神经元中的UNC 5C、GPR 173或KCNMB 2表达： 用编码针对单个基因的shRNA的腺相关病毒（AAV）转导小鼠。我们将 还通过AAV介导的转导在初级传入神经元中过表达单个蛋白质。然后我们将 在这些小鼠中研究mTBI诱导的行为和细胞变化，具有多个终点。这些研究 将揭示是否UNC 5C，GPR 173或KCNMB 2调节PTH的慢性化和/或解决。 相关行为，以及这些蛋白质的过表达或敲低是否表现出治疗益处。 这些目标的成功完成将有助于我们识别出研究不足的蛋白质，这些蛋白质对蛋白质的合成至关重要。 发展，维护和/或PTH的决议，是潜在的目标，外周作用，非 副作用最小的PTH成瘾治疗。这将为后续R 01提供初步数据 申请进一步深入的机制研究或启动药物发现项目。