PROJECT SUMMARY In the dorsal root ganglia (DRG), specialized sensory neurons known as nociceptors play a crucial role in detecting and transmitting painful stimuli. During inflammation, sensory neurons exhibit an increase in their excitability - a form of plasticity known as peripheral sensitization that is associated with the development of chronic pain. Though several mechanisms contributing to peripheral sensitization have been predominantly studied in rodent DRGs, it remains unknown whether these processes also contributes to hyperexcitability in human sensory neurons. Recently, our lab has developed a novel and scalable protocol that reproducibly generates human pluripotent stem cell (hPSC)-derived sensory neurons. These sensory neurons exhibit key markers found in both rodent and native DRGs, functional ion channels that regulate their excitability, and can be activated in response to inflammatory mediators, all of which demonstrate these cells' utility in uncovering the key signaling pathways involved in peripheral sensitization. I hypothesize that changes in de novo protein synthesis and translation rates contribute to peripheral sensitization in hPSC-derived sensory neurons, facilitating remodeling of the cell surface proteome and neuronal hyperexcitability. The objective of the proposed research is to combine stem cell technology, patch-clamp electrophysiology, multi-electrode array recordings, proximity labeling, and ribosomal and proteomic profiling to demonstrate de novo synthesis (Aim 1) and membrane trafficking of proteins (Aim 2) are important for regulating peripheral sensitization. Furthermore, we will also characterize whether translational rates are altered during sensitization (Aim 1) and the surface proteome of human sensory neurons following inflammation (Aim 2). The significance of this project lies in the potential to identify a distinct proteomic signature associated with peripheral sensitization in human DRGs, which is highly relevant to understanding pain etiology.