Biology subjects

Jensen, D. D.

Publications and source records attributed to Jensen, D. D..

7 recordsLinked to original sources

bioRxiv2026

Endocytosis and Compartmentalized Intracellular Signaling of the Prostaglandin Receptor EP4 Mediate Pain

Prostaglandin E2 (PGE2) is recognized as a major mediator of inflammatory pain. However, the intracellular signaling mechanisms by which PGE2 mediates pain remain unclear. Here, we show that pain-like responses evoked by PGE2 in dorsal root ganglion (DRG) nociceptors are mediated by internalization and compartmentalized signaling of the EP4 receptor. The EP4-selective agonist L-902,688 induced immediate nocifensive behavior and prolonged mechanical allodynia in mice and sensitized isolated DRG nociceptors. Pharmacological inhibition of clathrin- and dynamin-mediated endocytosis (using pitstop2 and dyngo4a) and siRNA knockdown of Dnm1 prevented EP4-induced nociception and sensitization, implicating receptor trafficking in pain signaling. Using genetically-encoded biosensors, we monitored EP4 trafficking and downstream signaling within subcellular compartments of HEK293 cells. PGE2 stimulated dynamin- and {beta}-arrestin-dependent EP4 trafficking from the plasma membrane to early, late and recycling endosomes and Golgi apparatus, and mobilized intracellular EP4 pools from the endoplasmic reticulum. PGE2 induced the assembly of EP4, G proteins and {beta}-arrestin signaling complexes in endosomes and the Golgi apparatus. Inhibition of EP4 endocytosis suppressed intracellular cAMP production and ERK activation. Our findings reveal that EP4-mediated nociceptive signaling originates from intracellular compartments and suggest that modulating EP4 internalization and subcellular signaling dynamics could offer a novel strategy for inflammatory pain management.

cell biology↗

bioRxiv2026

Label-free real-time imaging of mitochondrial matrix volume changes and permeability transition in living cells

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and cristae dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the sub-organellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K+ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K+ influx leads to increase of mitochondrial matrix volume while stimulation of K+ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.

cell biology↗

bioRxiv2026

The contribution of endocytic mediators to itch transmission

Chronic itch is a major burden, impacting the quality of life for one in four adults, and is closely associated with increased levels of anxiety, depression, and suicide. Despite its widespread burden, the precise mechanisms driving and sustaining chronic itch remain poorly understood. Non-histaminergic itch transmission in the spinal cord relies on the synaptic vesicle (SV) release of gastrin-releasing peptide (GRP) and the subsequent binding to the Gastrin-Releasing Peptide Receptor (GRPR). While SV exocytosis facilitates neurotransmitter release, such as GRP, SV endocytosis, mediated by key proteins including clathrin, adaptor associated kinase 1 (AAK1), and Dynamin (Dnm) are essential for retrieving and recycling SV from the presynaptic membrane to maintain signaling. Building upon evidence that AAK1- and Dnm-mediated endocytosis are viable targets to reverse pain, we characterized the role of these endocytic mediators in the regulation of synaptic transmission in itch pathways. We localized mRNA encoding AAK1, Dnm1 and Dnm3 within GRP positive neurons of the mouse dorsal root ganglia (DRG). Genetic and pharmacological disruption of AAK1 significantly reduced scratching behavior compared to control groups. This anti-pruritic effect correlated with confirmed Aak1 mRNA knockdown in both the DRG and spinal cord. Similarly, siRNA mediated knockdown of Dnm1 and Dnm3 in the DRGs also reduced scratching behavior. Crucially, these treatments decreased GRP release without altering locomotor activity or anxiety-like behaviors. Together, these findings suggest that the disruption of SV recycling reduces itch related signaling and behavior without affecting normal motor functions, providing a new approach for chronic pruritus.

neuroscience↗

bioRxiv2025

Endosomal MrGPRX1 signaling sensitizes TRPV1 to enhance itch

G protein-coupled receptors (GPCRs) and TRPV (transient receptor potential vanilloid) channels are crucial for signal transduction in physiological processes, including neurotransmission, pain, and itch. Downstream effectors of GPCR signaling can directly stimulate TRPV channels or enhance their sensitivity to stimuli, a process known as TRPV sensitization. Traditionally, GPCRs are activated at the cell surface by extracellular agonists, triggering signaling cascades. Recent evidence suggests GPCRs continue to signal from intracellular organelles. The human Mas-related G-protein coupled receptor X1 (MrGPRX1) is a GPCR expressed in primary sensory neurons involved in nociception and pruritus. Recent studies demonstrated how intracellular GPCR signaling regulates neuronal activity. However, there is no evidence characterizing MrGPRX1 trafficking or intracellular signaling. Herein, we characterized MrGPRX1 signaling within the endosomal network and its role in sensitizing TRPV1 channels to enhance itch signaling. Utilizing subcellular targeted biosensors, we demonstrated MrGPRX1 can traffic and signal from endosomes. Immunofluorescence analysis showed that MrGPRX1 internalizes following BAM8-22 stimulation. BRET assays revealed that MrGPRX1 activation induces Gq and {beta}-arrestin-1 recruitment to the plasma membrane and early endosomes. Inhibition of dynamin or clathrin blocked BAM8-22-induced MrGPRX1 endocytosis and decreased nuclear extracellular signal-regulated kinase (ERK) signaling. Calcium signaling confirmed that MrGPRX1-mediated TRPV1 sensitization is mediated by protein kinase C and ERK activation. Our findings reveal a novel role for MrGPRX1 endosomal signaling in TRPV1 sensitization. Understanding the mechanisms of MrGPRX1 signaling offers valuable insights into differentiating between pain and itch pathways, aiding in the development of targeted therapies for chronic pain and persistent itch.

neuroscience↗

bioRxiv2025

Characterization and targeting of the endosomal signaling of the gastrin releasing peptide receptor in pruritus.

Chronic pruritus is a major unmet clinical problem affecting one in four adults. G protein-coupled receptors (GPCRs) are key receptors driving itch signaling and are a therapeutic target for itch relief. The endosomal signaling of GPCRs provides new challenges for understanding how GPCR signaling is regulated, how endosomal signaling of GPCRs contributes to disease states like chronic pruritus and opens new targets for therapeutic development. The Gastrin releasing peptide receptor (GRPR) is a key mediator of pruritus in the spinal cord. Yet, little is known about the molecular mechanisms regulating GRPR signaling in pruritus, if GRPR can signal from endosomes, or the role of endosomal GRPR in the development of pruritus. Here we show the importance of internalization and endosomal signaling of GRPR in pruritus. Agonist induced GRPR internalization and trafficking was quantified using BRET or microscopy while endosomal-mediated ERK signaling was measured using compartmentalized FRET biosensors. Recruitment of G proteins to endosomes was measured with NanoBit BRET. pH sensitive mesoporous silica nanoparticles (MSN) which accumulated in endosomes were used to deliver RC-3095, a GRPR specific antagonist, intracellularly to block endosomal signaling of GRPR. MSN-RC proved more effective than free RC-3095 at inhibiting chloroquine scratching in mice. Our results demonstrate a critical role for GRPR endosomal signaling in itch sensation. These results highlight the ability of endosomally targeted antagonist to inhibit GRPR signaling and provide a new target for developing therapeutics that block GRPR mediated pruritus. Significance StatementGPCRs are dynamic signaling receptors that can continue to signal following internalization and trafficking to endosomes. Using subcellular targeted BRET and FRET based biosensors we can quantify the recruitment of signaling partners like G proteins and arrestins to GRPR from the endosomal compartment. Inhibition of clathrin and dynamin mediated endocytosis allowed to differentiate plasma membrane vs endosomal signaling of GRPR. pH sensitive nanoparticles loaded with the GRPR antagonist RC-3095 are endocytosed to the endosomal network where they specifically target and block endosomal GRPR signaling. Intrathecal injection of RC-3095 loaded nanoparticles blocked chloroquine induced scratching behavior in mice. Thus, intracellular GRPR drives itch sensation and targeted inhibition of intracellular GRPR signaling is a more effective strategy to treat pruritus.

neuroscience↗

bioRxiv2024

Nanomedicines targeting signaling of protease-activated receptor 2 in organelles provide sustained analgesia

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of GPCR signaling in organelles are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically-encoded biosensors targeted to subcellular compartments were used to analyze organelle-specific signaling of protease-activated receptor 2 (PAR2); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to selectively antagonize intracellular PAR2 signaling of pain. PAR2 agonists evoked sustained activation of PAR2, Gq and {beta}-arrestin-1 in early, late and recycling endosomes and the cis- and trans-Golgi apparatus, and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with organelle-targeted biosensors. Dendrimer and core-shell polymeric NPs accumulated in early and late endosomes of HEK293 cells, colonic epithelial cells and nociceptors, detected by confocal imaging of fluorescent NPs. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR2 antagonist. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR2, Gq and {beta}-arrestin-1 activation in endosomes and the Golgi apparatus and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs targeted neurons, sites of PAR2 expression. Both NP-AZ3451 formulations, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. Thus, organelle-specific PAR2 signals in colonocytes and nociceptors mediate pain. Antagonism of PAR2 in organelles, rather than at the plasma membrane, provides effective pain relief. Significance StatementOnce activated at the cell surface, many GPCRs internalize and continue to signal. The mechanisms and physiological relevance of intracellular GPCR signaling are uncertain. By using organelle-targeted biosensors, we detected sustained activation of the GPCR, PAR2, and its effectors in early, late and recycling endosomes, the cis- and trans-Golgi apparatus, and the cytosol and nucleus. NPs that delivered AZ3451, a PAR2 antagonist, to endosomes disrupted these intracellular signals, whereas unencapsulated AZ3451 was minimally effective. After intracolonic administration to mice, NPs accumulated in colonocytes and neurons. NP-encapsulated AZ3451, but not unencapsulated AZ3451, reversed pain in preclinical models of inflammatory bowel disease. Thus, intracellular PAR2 signaling mediates pain and antagonism of intracellular rather than plasma membrane PAR2 provides effective therapy.

physiology↗

bioRxiv2024

Targeting the Schwann Cell EP2/cAMP Nanodomain to Block Pain but not Inflammation

Analgesia by non-steroidal anti-inflammatory drugs (NSAIDs) is ascribed to inhibition of prostaglandin (PG) biosynthesis and ensuing inflammation. However, NSAIDs have life-threatening side effects, and inhibition of inflammation delays pain resolution. Decoupling the mechanisms underlying PG-evoked pain vs. protective inflammation would facilitate pain treatment. Herein, we reveal that selective silencing of the PGE2 EP2 receptor in Schwann cells via an adeno-associated viral vector abrogates the indomethacin-sensitive component of pain-like responses in mice elicited by inflammatory stimuli without affecting inflammation. In human Schwann cells and in mice, EP2 activation and optogenetic stimulation of adenylyl cyclase evokes a plasma membrane-compartmentalized cyclic adenosine monophosphate (cAMP) signal that, via A-kinase anchor protein-associated protein kinase A, sustains inflammatory pain-like responses, but does not delay their resolution. Thus, an unforeseen and druggable EP2 receptor in Schwann cells, via specific cAMP nanodomains, encodes PG-mediated persistent inflammatory pain but not protective inflammation.

pharmacology and toxicology↗