Biology subjects

Pattison, L. A.

Publications and source records attributed to Pattison, L. A..

17 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of action of a blood brain barrier shuttle antibody

The transferrin receptor has emerged as a prime target for transcytosis of antibody shuttles from the bloodstream into the brain parenchyma to deliver therapeutic payloads that treat neurological disorders. However, how the transferrin receptor-antibody binding mode impacts avidity, degradation, pH sensitivity and delivery remains underexplored. To address this, we determined the cryo-EM structure of mouse transferrin receptor 1 bound to the model brain shuttle antibody 8D3. In combination with cell binding and localisation studies we show that 8D3 can structurally support small-scale inter-receptor cross-linking, such as in self-contained pairs, that cause avidity but do not induce receptor redistribution or degradation. The structure can also explain pH-dependent binding modes, and we show how some of these antibody variants with graded sensitivities regulate brain penetration in vivo. Overall, our study illuminates how distinct molecular features of antibody binding impact transferrin receptor behaviour and brain delivery to inform on future shuttle design.

neuroscience↗

bioRxiv2026

Endosomal GPR65 signaling in fibroblast-like synoviocytes promotes inflammatory cytokine release and nociceptive neuron sensitization.

GPR65 is a proton-sensing G protein-coupled receptor implicated in inflammatory pain. In fibroblast-like synoviocytes (FLS), GPR65 activation promotes the release of proinflammatory cytokines capable of sensitizing sensory neurons. Following stimulation by protons, the synthetic agonist BTB09089, and the glycosphingolipid psychosine GPR65 undergoes internalization; however, the contribution of this trafficking to downstream signaling remains unclear. Using heterologous cell systems, the molecular mechanisms governing GPR65 internalization were first defined. Pharmacological and genetic inhibition of internalization revealed that intracellular trafficking is required for activation of extracellular-signal-related kinase (ERK) in the nucleus and transcriptional responses, indicating a spatially restricted signaling program originating from endosomes. The physiological relevance of this pathway was then examined in primary mouse FLS. Inhibition of endogenous GPR65 internalization reduced the ability of the conditioned media from BTB09089 stimulated FLS to sensitize dorsal root ganglia sensory neurons, thus linking receptor trafficking to pro-nociceptive function. Together these findings identify receptor internalization as a key determinant of nuclear ERK signaling and transcription downstream of GPR65 and demonstrate that endosomal signaling is required for pro-nociceptive activity of GPR65 in FLS. One-sentence summaryEndosomal internalization of GPR65 is required to coordinate gene transcription and proinflammatory cytokine production that drive neuronal sensitization.

pharmacology and toxicology↗

bioRxiv2025

AAV-mediated overexpression of Prdm12 in knee-innervating afferents reduces inflammatory joint pain and neuronal hyperexcitability in mice

Inflammatory joint pain features in numerous musculoskeletal disorders that affect millions globally. The Prdm12 gene encodes a conserved zinc finger transcriptional regulator expressed selectively in the nervous system. In humans, PRDM12 mutations can cause congenital insensitivity to pain (CIP) or midface toddler excoriation syndrome (MiTES). Prdm12 is prominently expressed in developing somatosensory ganglia, where it plays a crucial role in nociceptive neuron development, its expression being maintained in mature C-LTMRs (C-low threshold mechanoreceptors) and nociceptive neurons. Despite enhanced understanding of Prdm12s role in neuronal excitability and pain behavior, the impact of Prdm12 overexpression in mature nociceptive neurons has not been explored. Here, we conducted intravenous injection of AAV-PHP.S viral vectors encoding Prdm12-GFP (Prdm12-AAV) or GFP alone (Control-AAV), observing no overt changes in mouse behavior. When examining the properties of Prdm12 overexpressing sensory neurons in vitro, we observed an increase in rheobase alongside decreased neuronal responses to capsaicin and ATP, indicating a downregulation of TRPV1 and P2X ion channels activity, respectively. We next conducted intraarticular administration of viral constructs in female mice to determine how Prdm12 overexpression in knee-innervating sensory neurons alters their excitability and influences inflammatory joint pain induced by intraarticular administration of complete Freunds adjuvant (CFA). Prdm12 overexpression in knee-innervating neurons decreased inflammation-induced changes in digging and weight bearing, prevented inflammation-induced neuronal hyperexcitability, and decreased macroscopic voltage-gated ion channel conductance. Our findings illustrate that Prdm12 overexpression strongly modulates neuronal excitability in adult animals, highlighting its importance in pain perception and its potential as an analgesic target. SummaryOverexpression of the transcriptional regulator Prdm12 in knee-innervating neurons of mice reduces inflammatory joint pain and counteracts inflammation-induced neuronal hyperexcitability.

neuroscience↗

bioRxiv2024

The osteoarthritis associated sphingolipid sphingomyelin 34:1 causes inflammatory pain in mice

ObjectiveOsteoarthritis (OA) has a multifactorial pathogenesis, pain being the main symptom driving clinical decision making. Dysregulation of multiple mediators occurs in OA, the roles of many remaining to be identified. In dogs and humans with OA, synovial fluid lipidome dysregulation occurs, some findings being replicated in the plasma lipidome in a mouse OA model. One upregulated lipid is the sphingomyelin N-palmitoyl-D-erythro-sphingosylphosphorylcholine (d18:1/16:0), referred to here as SM. This study aimed to determine if SM causes joint pain and neuronal hyperexcitability in mice. DesignThe effects of SM or a structurally related ceramide (CM) on mouse sensory neuron excitability were measured using patch-clamp electrophysiology, as well as the ability of intraarticular SM and CM to induce inflammatory pain in mice. ResultsIncubation of sensory neurons with 1 {micro}M SM decreased rheobase, compared to incubation with vehicle (p-adj = 0.0000146, 95% confidence interval (CI): 50.20, 76.73) or CM (p-adj = 0.138, CI: 103.45, 171.55). Similarly, SM induced mechanical hypersensitivity in mice compared to mice receiving vehicle (p-adj = 0.000003, 95% confidence interval (CI): 166.82, 251.63) or CM (p-adj = 0.055, 95% CI: 218.28, 268.12), which was coupled with a significant decrease in rheobase of knee-innervating neurons isolated from SM-injected mice compared to those receiving vehicle (p-adj = 0.0138, CI: 50.19, 76.73) or CM (p-adj = 1.0, CI: 103.45, 171.55). ConclusionsThe results generated demonstrate that a dysregulated lipidome can contribute to inflammatory OA pain, further work being necessary to determine the mechanism by which SM exerts its activity.

neuroscience↗

bioRxiv2024

Pro-inflammatory mediators sensitise Transient Receptor Potential Melastatin 3 cation channel (TRPM3) signalling in mouse sensory neurons

Pro-inflammatory mediators can directly activate pain-sensing neurons, known as nociceptors. Additionally, these mediators can potentiate or sensitise ion channels and receptors expressed by these cells through transcriptional and post-translational modulation, leading to nociceptor hypersensitivity. A well-characterised group of ion channels that subserve nociceptor sensitisation is the transient receptor potential (TRP) superfamily of cation channels. For example, the roles of TRP channels vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in nociceptor sensitisation and inflammatory pain have been extensively documented. In the case of TRP melastatin 3 (TRPM3), however, despite the increasing recognition of this channels role in inflammatory pain, the mechanisms driving its sensitisation during inflammation remain poorly understood. Here, we found that an inflammatory soup of bradykinin, interleukin 1{beta} (IL-1{beta}) and tumour necrosis factor (TNF) sensitised TRPM3 function in isolated mouse sensory neurons; IL-1{beta} and TNF, but not bradykinin, independently potentiated TRPM3 function. TRPM3 expression and translocation to the membrane remained unchanged upon individual or combined exposure to these inflammatory mediators, which suggests post-translational modification occurs. Finally, using the model of complete Freunds adjuvant-induced knee inflammation, we found that pharmacological blockade of TRPM3 does not alleviate inflammatory pain, which contrasts with previous reports using different pain models. We propose that the nuances of the immune response may determine the relative contribution of TRPM3 to nociceptive signalling in different neuro-immune contexts. Collectively, our findings improve insight into the role of TRPM3 sensitisation in inflammatory pain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/612393v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@11e90acorg.highwire.dtl.DTLVardef@1ac1c2aorg.highwire.dtl.DTLVardef@6d116forg.highwire.dtl.DTLVardef@93c605_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

neuroscience↗

bioRxiv2024

Activation of the proton-sensing GPCR, GPR65 on fibroblast-like synoviocytes contributes to inflammatory joint pain.

Inflammation is associated with localised acidosis, however, attributing physiological and pathological roles to proton-sensitive receptors is challenging due to their diversity and widespread expression. Here, agonists of the proton-sensing GPCR, GPR65, were systematically characterised. The synthetic agonist BTB09089 (BTB) recapitulated many proton-induced signalling events and demonstrated selectivity for GPR65. BTB was used to show that GPR65 activation on fibroblast-like synoviocytes (FLS), cells that line synovial joints, results in the secretion of pro-inflammatory mediators capable of recruiting immune cells and sensitising sensory neurons. Intra-articular injection of BTB resulted in GPR65-dependent sensitisation of knee-innervating neurons and nocifensive behaviours in mice. Stimulation of GPR65 on human FLS also triggered the release of inflammatory mediators and synovial fluid samples from human osteoarthritis patients were shown to activate GPR65. These results suggest a role of GPR65 in mediating cell-cell interactions that drive inflammatory joint pain in both mice and humans.

neuroscience↗

bioRxiv2023

Digging deeper into pain - an ethological behavior assay correlating well-being in mice with human pain experience.

The pressing need for safer, more efficacious analgesics is felt worldwide. Pre-clinical tests in animal models of painful conditions represent one of the earliest checkpoints novel therapeutics must negotiate before consideration for human use. Traditionally, the pain status of laboratory animals has been inferred from evoked nociceptive assays which measure their responses to noxious stimuli. The disconnect between how pain is tested in laboratory animals and how it is experienced by humans may in part explain the shortcomings of current pain medications and highlights a need for refinement. Here, we survey human chronic pain patients who assert that everyday aspects of life, such as cleaning and leaving the house, are affected by their on-going level of pain. Accordingly, we test the impact of painful conditions on an ethological behavior of mice, digging. Stable digging behavior was observed over time in naive mice of both sexes. By contrast, deficits in digging were seen following acute knee inflammation. The analgesia conferred by meloxicam and gabapentin was compared in the monosodium iodoacetate knee osteoarthritis model, meloxicam more effectively ameliorating digging deficits, in line with human patients finding meloxicam more effective. Lastly, in a visceral pain model, the decrease in digging behavior correlated with the extent of disease. Ultimately, we make a case for adopting ethological assays, such as digging, in studies of pain in laboratory animals, which we believe to be more representative of the human experience of pain and thus valuable in assessing clinical potential of novel analgesics in animals.

neuroscience↗

bioRxiv2023

Inhibition of sensory neuron driven acute, inflammatory, and neuropathic pain using a humanised chemogenetic system

Hyperexcitability in sensory neurons is known to underlie many of the maladaptive changes associated with persistent pain. Chemogenetics has shown promise as a means to suppress such excitability, yet chemogenetic approaches suitable for human applications are needed. PSAM4-GlyR is a modular system based on the human 7 nicotinic acetylcholine and glycine receptors, which responds to inert chemical ligands and the clinically-approved drug, varenicline. Here, we demonstrated the efficacy of this channel in silencing both mouse and human sensory neurons by the activation of large shunting conductances after agonist administration. Virally-mediated expression of PSAM4-GlyR in mouse sensory neurons produced behavioural hyposensitivity upon agonist administration, which was recovered upon agonist washout. Importantly, stable expression of the channel led to similar reversible behavioural effects even after 10 months of viral delivery. Mechanical and spontaneous pain readouts were also ameliorated by PSAM4-GlyR activation in acute and joint pain inflammation models. Furthermore, suppression of mechanical hypersensitivity generated by a spared nerve injury model of neuropathic pain was also observed upon activation of the channel. Effective silencing of behavioural hypersensitivity was reproduced in a human model of hyperexcitability and clinical pain: PSAM4-GlyR activation decreased the excitability of human induced pluripotent stem-cell-derived sensory neurons and spontaneous activity due to a gain of function NaV1.7 mutation causing inherited erythromelalgia. Our results demonstrate the contribution of sensory neuron hyperexcitability to neuropathic pain and the translational potential of an effective, stable and reversible human-based chemogenetic system for the treatment of pain.

neuroscience↗

bioRxiv2022

Sensitisation of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Whats more, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we employed Ca2+ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca2+ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca2+ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitise the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca2+ imaging experiments, IL-13-mediated sensitisation of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signalling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pro-nociceptive signalling downstream of IL-13. Key Points SummaryThe pro-inflammatory cytokine IL-13 is elevated in gastrointestinal (GI) diseases and known to sensitise sensory neurons. This study confirms a role for IL-13 in colonic afferent sensitisation and defines a role for downstream JAK and p38 MAPK signalling in colonic mechanosensitisation. IL-13-mediated increase in [Ca2+]i in capsaicin-sensitive sensory neurons is dependent on p38 MAPK and JAK signalling. IL-13-induced sensitisation of colonic afferents to noxious mechanical distension is abolished by inhibition of p38 MAPK and JAK. We have built on the current understanding of IL-13 and its neuronal interactions, highlighting the therapeutic potential of targeting p38 MAPK and JAK signalling pathways to treat visceral pain in GI disease.

physiology↗

bioRxiv2022

Dysregulation of ADAM10 shedding activity as a mechanism of cancer resistance in the naked mole-rat

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts (MPSF) ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+-dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted wound closure in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

cancer biology↗

bioRxiv2022

Human mesenchymal stem cells and derived extracellular vesicles reduce sensory neuron hyperexcitability and pain-related behaviors in a mouse model of osteoarthritis

Osteoarthritis (OA) is a common degenerative joint disease characterized by joint pain and stiffness. In humans, mesenchymal stem cells (MSCs) and derived extracellular vesicles (MSC-EVs) have been reported to alleviate pain in knee OA. Here, we used the destabilization of the medial meniscus (DMM) mouse model of OA to investigate mechanisms by which MSCs and MSC-EVs influence pain-related behavior. We found that MSC and MSC-EV treated DMM mice displayed improved OA pain-related behavior (i.e. locomotion, digging and sleep) compared to untreated DMM mice. Improved behavior was not the result of reduced joint damage, but rather knee-innervating sensory neurons from MSC and MSC-EV treated mice did not display the hyperexcitability observed in untreated DMM mice. Furthermore, we found that MSC-EVs normalize sensory neuron hyperexcitability induced by nerve growth factor in vitro. Our study suggests that MSCs and MSC-EVs may reduce pain in OA by direct action on peripheral sensory neurons. TeaserMesenchymal stem cells and secreted extracellular vesicles normalize sensory neuron excitability to reduce pain.

neuroscience↗

bioRxiv2022

Sensitisation of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity

Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNF is linked to pain in both patients with inflammatory bowel disease and irritable bowel syndrome, and has been shown to sensitise colonic sensory neurons. Somatic, TNF- triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesised that TNFR1-evoked p38 MAPK activity may also be responsible for TNF sensitisation of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglion sensory neurons, we observed TNF-mediated increases in intracellular [Ca2+] and sensitisation of capsaicin responses. The sensitising effects of TNF were dependent on TNFR1 expression and attenuated by p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNF pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNF-induced sensitisation of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in GI disease. Abstract figure legendTNF sensitised Ca2+ responses to the TRPV1 agonist capsaicin in dorsal root ganglion sensory neurons. Sensitisation was TNFR1-dependent and attenuated by inhibition of p38 MAPK. Direct Ca2+ responses to TNF were TRPV1-and TRPA1-dependent. In ex vivo colonic afferent recordings, TNF increased sensitivity to noxious ramp distension and capsaicin, both of which were absent in TNFR1-/- tissue or blocked by inhibition of p38 MAPK. These findings establish a role for TNFR1, p38 MAPK and TRPV1 in TNF-mediated sensitisation of colonic afferents. Key Points SummaryO_LITNF sensitises sensory neurons and colonic afferents to the TRPV1 agonist capsaicin. C_LIO_LITNF-mediated sensitisation of sensory neurons and colonic nociceptors is dependent on TNFR1 expression. C_LIO_LITNF sensitisation of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK. C_LI

neuroscience↗

bioRxiv2021

Behavioral and nociceptor states of inflammatory painacross timescales in 2D and 3D

Ongoing pain is often driven by direct activation of pain-sensing neurons and neuroimmune mediated sensitization. These heightened states of pain alter physiology, reduce motor function, and alter motivation to engage in normal behaviors. The complexity of the pain state has evaded a comprehensive definition, especially in nonverbal animals. Here in mice, we capture the physiological state of sensitized pain neurons at different time points post-inflammation and used computational tools to automatically map behavioral signatures of evoked and spontaneous displays of pain. First, retrograde labeling coupled with electrophysiology of neurons innervating the site of localized inflammation defined critical time points of pain sensitization. Next, we used high-speed videography combined with supervised and unsupervised machine learning tools and uncovered sensory-evoked defensive coping postures to pain. Using 3D pose analytics inspired by natural language processing, we identify movement sequences that correspond to robust representations of ongoing pain states. Surprisingly, with this analytical framework, we find that a commonly used anti-inflammatory painkiller does not return an animals behavior back to a pre-injury state. Together, these findings reveal the previously unidentified signatures of pain and analgesia at timescales when inflammation induces heightened pain states.

neuroscience↗

bioRxiv2020

Intra-articular AAV-PHP.S mediated chemogenetic targeting of knee-innervating DRG neurons alleviates inflammatory pain in mice

ObjectiveJoint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee-innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. Therefore, our objective was to evaluate whether the newly engineered adeno-associated virus (AAV) serotype, AAV-PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intra-articular knee injection. MethodsAAV-PHP.S virus packaged with dTomato fluorescent protein and either excitatory (Gq) or inhibitory (Gi) designer receptors activated by designer drugs (DREADDs) was injected into the knee joint of adult mice. Labelling of DRG neurons by AAV-PHP.S from the knee was evaluated using immunohistochemistry. Functionality of Gq- and Gi-DREADDs was evaluated using whole-cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing and rotarod, before and after intra-peritoneal administration of the DREADD activator, Compound 21. ResultsWe show that AAV-PHP.S can deliver functional genes into the DRG neurons when injected into the knee joint in a similar manner to the well-established retrograde tracer, fast blue. Short-term activation of AAV-PHP.S delivered Gq-DREADD increases excitability of knee neurons in vitro, without inducing overt pain in mice when activated in vivo. By contrast, in vivo Gi-DREADD activation alleviated complete Freunds adjuvant mediated knee inflammation-induced deficits in digging behavior, with a concomitant decrease in knee neuron excitability observed in vitro. ConclusionsWe describe an AAV-mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.

neuroscience↗

bioRxiv2019

Sensitization of knee-innervating sensory neurons by tumor necrosis factor-α activated fibroblast-like synoviocytes: an in vitro, co-culture model of inflammatory pain

Pain is a principal contributor to the global burden of arthritis with peripheral sensitization being a major cause of arthritis-related pain. Within the knee joint, distal endings of dorsal root ganglion neurons (knee neurons) interact with fibroblast-like synoviocytes (FLS) and the inflammatory mediators they secrete, which are thought to promote peripheral sensitization. Correspondingly, RNA-sequencing has demonstrated detectable levels of pro-inflammatory genes in FLS derived from arthritic patients. This study confirms that stimulation with tumor necrosis factor (TNF-), results in expression of pro-inflammatory genes in mouse and human FLS (derived from OA and RA patients), as well as increased secretion of cytokines from mouse TNF- stimulated FLS (TNF-FLS). Electrophysiological recordings from retrograde labelled knee neurons co-cultured with TNF-FLS, or supernatant derived from TNF-FLS, revealed a depolarized resting membrane potential, increased spontaneous action potential firing and enhanced TRPV1 function, all consistent with a role for FLS in mediating the sensitization of pain-sensing nerves in arthritis. Therefore, data from this study demonstrate the ability of FLS activated by TNF- to promote neuronal sensitization, results that highlight the importance of both non-neuronal and neuronal cells to the development of pain in arthritis.

neuroscience↗

bioRxiv2019

In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3

Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is instrumental in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an 2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks 2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.

pharmacology and toxicology↗

bioRxiv2018

Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner.

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individuals quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freunds adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labelled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-hours after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-hours in culture, but was not observed after 48-hours in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist A-425619 reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

neuroscience↗