Project #1 Single-soma RNA-seq and spatial transcriptomics of human TGs

偏头痛是最常见的原发性头痛疾病之一，每四个美国家庭中就有一个受到影响。这个建筑群 神经功能障碍的部分原因是三叉神经躯体感觉的改变，表现为头/脸-头-面-头-面-头-头-面-面 三叉神经痛和/或三叉神经痛。偏头痛的有效治疗方法仍然有限，我们对 人类三叉神经系统在基线和偏头痛的情况下是稀疏的。响应RFA-NS-22-018，修复 倡议：人类疼痛相关基因和细胞的发现和功能评估，我们建议 成立宾夕法尼亚人类精确疼痛中心(Penn HPPC)，以阐明分子、细胞、表观遗传学和 人三叉神经节(TG)感觉神经元在基线和偏头痛状态下的生理特征。 宾夕法尼亚大学HPPC将由宾夕法尼亚大学和具有多学科专业知识的国际调查人员组成。这个 PI、两个MPI和两个协同IS目前正在合作研究人类背根的单胞体深度RNA序列 神经节(DRG)神经元项目，为这一应用奠定了坚实的基础。宾夕法尼亚大学的HPPC将包含 三个核心，执行三个项目。项目1将由国际和平协会罗博士领导，并与 CoI，Ernfors博士，并得到数据核心PI Li博士和项目2 PI Wu博士的支持。具体地说， 项目1将对来自对照供体的人TGS的神经元和非神经元细胞进行单体体RNAseq 还有那些偏头痛患者。三种互补的方法，一种基于激光捕获显微切割的单核细胞- Ron-Soma Deep RNA-seq(由PI的实验室开发的一种新方法，已成功用于 人背根神经节神经元)、10倍维西姆(一种商业化的空间转录平台)和MERSCOPE (另一个商用的空间转录切割平台)将用于这一目的。目标1将 重点是控制人类TG组织，而AIM 2将专注于来自偏头痛捐赠者的人类TG组织。我们 将通过集成单胞体和10倍Viem数据集来执行细胞类型去卷积，以生成人类TG 正常和非神经细胞中神经元和非神经元细胞的空间定位单胞体转录图谱 偏头痛的情况。我们还将比较对照组和偏头痛捐赠者之间的数据集。总而言之，我们的 预期结果将产生史无前例的人类TGS分子和细胞图谱，以了解正常 与偏头痛相关的三叉神经感觉和异常感觉。我们的结果也可能导致发现 用于偏头痛诊断和/或识别治疗偏头痛的新的潜在药物靶点的新生物标志物- 门槛。

Migraine, one of the most common primary headache disorders, affects 1 in 4 US households. This complex neurologic disorder is mediated in part by alterations in trigeminal somatosensation, which manifests as head/fa- cial pain and/or trigeminal allodynia. Effective treatments for migraine are still limited, and our knowledge about human trigeminal system at baseline and migraine conditions are sparse. In response to RFA-NS-22-018, HEAL Initiative: Discovery and Functional Evaluation of Human Pain-associated Genes & Cells, we propose to form the Penn Human Precision Pain Center (Penn HPPC) to elucidate molecular, cellular, epigenetic, and physiological profiles of human trigeminal ganglion (TG) sensory neurons at baseline and migraine conditions. The Penn HPPC will be composed of Penn and international investigators with multidisciplinary expertise. The PI, two MPIs, and two co-Is are currently collaborating on a single-soma deep RNA-seq of human dorsal root ganglion (DRG) neuron project, which form a strong foundation for this application. The Penn HPPC will contain three cores and perform three projects. The project 1 will be led by the PI, Dr. Luo, in collaboration with the Co-I, Dr. Ernfors, and supported by Dr. Li, PI of the data core, and Dr. Wu, PI of the project 2. Specifically, the project 1 will conduct single-soma RNAseq of neurons and non-neuron cells of human TGs from control donors and those with migraine. Three complementary approaches, a laser capture microdissection based single-neu- ron-soma deep RNA-seq (a novel method developed by the PI’s lab, which has been successfully used with human DRG neurons), 10 x Visium (a commercially available spatial transcriptomics platform), and MERSCOPE (another commercially available spatial transcriptomics platform) will be employed for this purpose. Aim 1 will focus on control human TG tissues, whereas Aim 2 will focus on human TG tissues from “migraine” donors. We will perform cell type deconvolution by integrating single-soma and 10x Visium datasets to generate human TG atlases with spatially-located single-soma transcriptomes of neurons and non-neuronal cells under normal and migraine conditions. We will also compare datasets between control and migraine donors. Taken together, our anticipated results will generate unprecedent molecular and cellular atlas of human TGs to understand normal trigeminal sensations and abnormal sensations associated with migraine. Our results may also lead to discovery of new biomarkers for migraine diagnosis and/or identification of novel potential drug targets for migraine treat- ment.