Date of Award

Summer 8-22-2025

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (Biomedical Science)

Department

Biomedical Sciences

First Committee Advisor

Derek Molliver

Second Committee Member

Ian Meng

Third Committee Member

Robert Burgess

Additional Committee Members

Katherine Motyl

Benjamin Harrison

Abstract

Translating analgesic mechanisms from rodents to humans remains a central challenge in chronic pain research. In an effort to address this gap, we combined transcriptomic profiling with functional signaling assays to evaluate the human dorsal root ganglion (DRG)-derived HD10.6 cell line alongside primary mouse DRG neurons. RNA seq of proliferating versus mature HD10.6 cells revealed transcriptional changes and a strong correlation with primary human nociceptor subtypes identified in single nucleus human DRG datasets, whereas several induced pluripotent stem cell sensory neuron (iPSC SN) lines showed distinct and less uniform correlations. Human DRG-derived HD10.6 cells express a diverse range of ion channels and G protein-coupled receptors (GPCRs), including some that are expressed in human sensory neurons but absent from the rodent genome. This characteristic makes HD10.6 cells a valuable alternative human model for research purposes. Functionally, we profiled several Gαs coupled GPCRs some of which are implicated in nociceptor sensitization. Using phospho substrate immunoblotting and live cell reporters, we mapped protein kinase A (PKA) and RAPGEF (EPAC) signaling to downstream protein kinase C (PKC) and ERK MAP kinase. Beraprost, an agonist for the prostacyclin receptor PTGIR, robustly activated PKC and ERK in both mouse and human cells, whereas TACR2 and DRD1/5 agonists showed species differences in the activation of PKC. P2RY11 was functional in HD10.6 but not mouse cells. Across receptors, we observed species and receptor-specific phospho protein band signatures. ERK activation depended predominantly on EPAC→PKC in both species, but PKA also contributed to ERK activation in HD10.6 cells. Strikingly, acute PKA inhibition potentiated EPAC signaling only in HD10.6 cells, revealing a human-specific negative feedback loop. To investigate cAMP effectors in vivo, we deleted the cAMP binding domain of Rapgef2 in TRPV1 lineage DRG neurons (Rg2ko mice). Male mutants displayed reduced baseline heat sensitivity, yet retained beraprost evoked heat hyperalgesia. This modality and sex-specific phenotype was correlated with a deficit in trafficking of the noxious heat-gated ion channel TRPV1 to the plasma membrane, as well as an elevation of basal pERK, which was dependent on compensatory Rapgef3/4 activity. Collectively, these data (i) highlight fundamental species differences between human and mouse sensory neurons in GαsPCR/cAMP signaling, (ii) position Rapgef2 as a critical regulator that shapes nociceptor responses in a sex and stimulus-dependent manner, and (iii) establish HD10.6 cells as a scalable, human-derived platform for dissecting GPCR pathways and for high throughput analgesic discovery.

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