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.

Project Narrative Chronic post-traumatic headache is highly debilitating, poorly understood and difficult to treat. In this application, we aim to identify understudied membrane proteins in dural afferent neurons that regulate the chronification and resolution of post-traumatic headache. This will provide potential targets for the development of novel, peripherally-active drugs to treat PTH with minimal abuse liability and other adverse effects.