Hi, everyone. My name is Christopher Detroya. I'm a third year medical student at Unicom, and this is our project on cell signaling in sensory neurons. Since 1999, the world has been fighting a rapidly progressing opioid epidemic due to an increasing number of people struggling with acute and chronic pain. The number of people who died from a drug overdose in 2021 was greater than six times the number in 1999. Additionally, over 75% of the 107,000 drug overdose deaths in 2022 involved opioids. Opioids continue to be used because they are highly effective analgesics. However, with risk factors such as respiratory depression, addiction, and life-threatening constipation, there has been a major effort to identify new analgesics that are as effective and without these adverse effects, embracing the osteopathic philosophy that mind and body are interrelated. Prior studies have shown that mitochondrial uncoupling drugs, such as DNP and BAM-15, have analgesic effects in diverse models of inflammatory, neuropathic, and opioid-induced hyperalgesia in rats and mice, in addition to having potential for treatment of disorders ranging from diabetes, obesity, and non-alcoholic steatohepatitis to neurodegenerative disease. While current research on cell signaling and sensory neurons has been promising, it has largely been limited to rodent cell lines. To determine whether signaling channels expressed in rodent sensory neurons are expressed in human sensory neurons, we tested IHC-stained mouse pain models in vivo and by patch-clamp electrophysiology in acutely isolated DRG neurons in vitro, as well as in human DRG-derived cell lines. The major channel we tested was MUOC1, a kinase related to AMPK that has not been previously investigated in sensory neurons. It is activated by LKB1, a kinase that acts as a master stress sensor, and is known to activate AMPK. AMPK activates metabolic pathways in response to stress that conserve energy and promote energy production in response to stress or starvation. Cells obtained from HD10.6 human DRGs were incubated and stimulated with BAM-15 for 10 minutes and fixed in PFA for 15 minutes. Control HD10.6 cells and injury model mouse lumbar DRGs were not stimulated with BAM-15 and only fixed in PFA. The cells were then stained via IHC with MUOC1, rabbit polyclonal antibody diluted 1 to 250 in blocking buffer, and TRIBV1, a goat polyclonal antibody diluted 1 to 1,000 in blocking buffer, and sat covered at room temperature overnight. Secondary antibodies were added after 24 hours, alpaca anti-rabbit Cy3 for MUOC1 and donkey anti-goat Cy2 for TRIBV1, and the slides were mounted with DAPI to be visualized under the microscope. Figure 2 is showing that fluorescence can label mitochondria to reveal the prolific mitochondrial distribution in mouse cell bodies on the left and central axons on mouse DRG sensory neurons in superficial dorsal horns on the right, and figure 3 shows that human DRG-derived cells can be labeled with fluorescence for visualization, and these are images pre-treatment. In the HD10.6 human cells, the BAM-15-stimulated N-control cells demonstrated positive labeling with equal intensity for MUOC1, with projections seen branching off the axons indicating potential subcellular structures. We realized after staining that the TRIBV1 antibody we selected only recognizes mouse cells, so the HD10.6 cells did not show positive labeling for TRIBV1. In the mouse DRG cells, there was positive labeling for MUOC1 seen in the TRIBV1-labeled neurons. So in this figure 4, the mouse DRG cells showing positive labeling for MUOC1 in A, and the mouse showing positive labeling for TRIBV1 in B, and MUOC1 and TRIBV1 overlaid together in the mouse DRGs, showing that MUOC1 labels and correlates with the same TRIBV1-labeled neurons in C. And slide D shows the HD10.6 human DRG-derived cells showing positive labeling for MUOC1 as well. MUOC1 is a novel kinase channel that has not been previously studied in sensory neurons, channel that has not been previously studied in sensory neurons. We have found that MUOC1 is enriched in nociceptive sensory neurons in both mouse and human cell lines, and likely they're nociceptors. Because MUOC1 is part of the AMPK master regulator family, along with metformin, a drug that has been used as an AMPK activator for years, and is one of the most prescribed drugs in the world for diabetes, we think that MUOC1 may have closely related functions that have not yet been investigated. One limitation of our study was that we could not determine if the staining for MUOC1 in the HD10.6 cells correlated 100% with the TRIBV1-labeled neurons. This can be corrected and addressed by using guinea pig anti-TRIBV1 antibodies in subsequent studies. The importance of using human cell lines in pain signaling pathway modeling was a critical step towards developing a highly novel analgesic agent for humans that is non-addictive. Thank you very much.

cell signaling

mitochondrial uncoupling

non-addictive analgesics

opioid epidemic

sensory neurons