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

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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.