British Journal of Pharmacology

G protein-gated inwardly rectifying potassium (GIRK2) channels regulate neuronal excitability and are implicated in neurodevelopmental disorders. A rare KCNJ6 variant, G154C (hGIRK2G154C), was identified in a patient with mild Keppen-Lubinsky syndrome features, contrasting with severe phenotypes linked to other selectivity filter mutations. Here we combined molecular dynamics simulations and patch-clamp electrophysiology to characterize the hGIRK2G154C mutant, revealing a widened selectivity filter that resulted in loss of potassium selectivity, aberrant sodium permeation, and loss of inward rectification, indicating a severe gain-of-function phenotype. An in silico and electrophysiological drug screen identified FDA-approved compounds, including nefazodone and eletriptan, that potently inhibited GIRK2 and GIRK2G154C through distinct blocking mechanisms. These findings elucidate the structural and functional impact of the G154C mutation and highlight potential pharmacological tools and therapeutic candidates for the treatment of GIRK2 channelopathies.

Cinnamaldehyde (CA) is extensively used as flavorant and in traditional medicine. CA is com-monly used in pain research as specific agonist of TRPA1, a polymodal cation channel expressed in nociceptive primary sensory neurons. However, we previously showed that CA inhibits the L-type Ca2+ channel in cardiac and smooth muscle cells, raising the possibility that other ion channels can be modulated by this compound as well. Here, we investigated whether CA exhibits affects the activity on the cardiac NaV1.5 channel. We used the whole-cell patch-clamp technique to record Na+ currents in HEK293T cells expressing the human NaV1.5 channel, as well as in cells expressing hNaV1.5 channels baring mutation in the binding pocket of local anesthetics (LA). Our results show that CA exhibit LA-like actions on hNaV1.5 channels: CA blocks hNaV1.5 currents in a tonic and voltage-dependent fashion. Residues F1760 and Y1767 are important for the blockade of NaV1.5 by CA and a double point mutation F1760A/Y1767A abolishes the blockade of INa by CA. We conclude that CA and LID share common structural determinants for the inhibition of Na+ channels and that CA has LA-like actions.

MK-801, a phencyclidine (PCP) derivative also known as dizocilpine, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). The NMDAr plays a critical role in mediating excitatory synaptic transmission in the central nervous system (CNS) and is important in regulating synaptic plasticity, learning, and memory. MK-801 is known to induce schizophrenia-like phenotypes in animal models by blocking the NMDAr. Several studies have reported that the cognitive impairment associated with schizophrenia is linked to functional defects of ion channels in parvalbumin-positive GABAergic interneurons. Kv3.1 is a voltage-gated K+ (Kv) channel involved in the rapid repolarization of the action potential in neurons; it is richly expressed in parvalbumin-positive GABAergic interneurons and is associated with fast, repetitive spike generation. A decrease of Kv3.1 in the CNS has been reported to be associated with schizophrenia. In the present study, the effect of MK-801 on Kv3.1 was investigated using the whole-cell patch-clamp technique in Chinese hamster ovary (CHO) cells stably expressing Kv3.1. MK-801 caused a concentration-dependent inhibition of Kv3.1, with an IC50 of 10.81 M and a Hill coefficient of 0.89. The blocking potency was stronger at depolarized potentials, indicating a voltage-dependent block. MK-801 also produced a use-dependent block, inducing progressive inhibition with repeated stimulation at increased frequencies (1 Hz and 2 Hz), consistent with a delay in recovery from inactivation of Kv3.1 in the presence of MK-801. In addition, MK-801 induced a hyperpolarizing shift in the voltage dependence of the steady-state inactivation curve of Kv3.1. Taken together, these results indicate that MK-801 blocks Kv3.1 expressed in CHO cells in a concentration-, voltage-, and state-dependent manner. Given the importance of Kv3.1 in parvalbumin-positive, fast-spiking GABAergic interneurons, these findings suggest that MK-801 may alter the firing patterns of these inhibitory neurons, contributing to the onset and symptoms of schizophrenia.

To date, no pharmacotherapy has been approved for cocaine use disorder. Endocannabinoid signaling is closely related to dopamine-dependent reinforcement and may regulate cocaine-related behaviors. In this study, we used common marmosets (Callithrix jacchus) to investigate whether 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid lipid, reduces cocaine reinforcement in a non-restraint oral self-administration model. Marmosets performed oral cocaine self-administration under a fixed-ratio 1 schedule, and cocaine intake was confirmed using plasma benzoylecgonine detection. Dopamine transporter (DAT)-related positron emission tomography (PET) signal, DAT and G protein-coupled receptor 55 (GPR55)-associated fluorescent signals, GPR55/DAT immunofluorescence, and synaptosomal dopamine responses were assessed using 18F-N-(3-fluoropropyl)-2{beta}-carboxymethoxy-3{beta}-(4-iodophenyl) nortropane (18F-FP-CIT) PET imaging, ex vivo fluorescent ligand imaging, confocal microscopy, and dopamine quantification. Marmosets acquired oral cocaine self-administration with preferential active lever responding. Repeated cocaine self-administration reduced striatal DAT-related 18F-FP-CIT PET signal in vivo. In striatal slices, cocaine decreased fluorescent false neurotransmitter (FFN102)-associated DAT signal and increased T1117-associated GPR55 signal. Systemic 2-AG pretreatment reduced cocaine self-administration while restoring diminished circulating 2-AG levels. GPR55 and DAT immunofluorescent signals were colocalized in marmoset brain sections, and 2-AG enhanced cocaine-induced dopamine elevation in synaptosomal preparations. Taken together, these findings suggest that 2-AG attenuates cocaine-taking behavior and may be associated with GPR55/DAT-related dopaminergic responses in common marmosets.

We have examined how ibogaine may enhance synaptic plasticity and neural repair. Extracellular efflux analysis showed that ibogaine (3, 10 or 30 M) produced a rapid (<5 minutes) and concentration-dependent suppression of mitochondrial function and glycolysis in mouse cortical neurons. Cell-based imaging demonstrated this metabolic inhibition was preceded by an elevation of the mitochondrial membrane potential and followed by increased mitochondrial reactive oxygen species levels. Like ibogaine, which blocks potassium channels, mitochondrial Kv1.3 channel (mitoKv1.3) inhibitors produce concordant changes over a similar timeframe. Following treatment with ibogaine (3, 10 or 30 M) for 4 hours, a marked concentration-dependent rebound of energy production occurred 20 hours later suggestive of metabolic preconditioning. Indeed, proteomic profiling showed that oral ibogaine (100 mg/kg) administration to female mice produced changes in spinal cord protein abundance indicative of increasing metabolic stress from 0.25-24 hours that resolved by 72 hours. This was accompanied by activation, inhibition and reactivation of synaptic plasticity and neural repair signaling pathways at 0.25, 4-24 and 72 hours, respectively. Hence, by inducing transient metabolic stress, ibogaine may re-open a neurodevelopmental window characterized by enhanced synaptic plasticity and neural repair. Ibogaine binds with high affinity to the sigma-2 receptor. Like mitoKv1-3 inhibitors and sigma-2 receptor ligands, ibogaine transiently elevated AMP-activated protein kinase (0.25 hours) and temporally suppressed mechanistic target of rapamycin and lipid synthesis pathways (0.25-24 hours) implicated in enhanced synaptic plasticity and neural repair. These findings suggest ibogaine rewires and repairs neural circuits by a metabolic preconditioning mechanism, perhaps involving mitoKv1.3 and the sigma-2 receptor.

CNS4 is an agonist concentration-biased NMDAR modulator exhibiting GluN2 subtype-dependent activity. Here we evaluated the translational pharmacology of CNS4 across mice, rats, and client-owned surgical oncology dogs. In mice, CNS4 did not produce conditioned place preference, indicating lack of reward liability. CNS4 produced dose-dependent reductions in locomotor activity without motor impairment on accelerating rotarod testing. CNS4 also maintained body temperature 0.5 to 1 degree Celsius above the vehicle group in mice and dogs, consistent with NMDAR-dependent thermoregulatory activity and contrasting with hypothermic effects reported for NMDAR channel blockers such as ketamine. In fear-conditioning paradigms, CNS4 did not impair fear learning, memory consolidation, or fear expression. CNS4 reduced acute stress-induced sucrose preference, suggesting modulation of stress-responsive neural circuits. In a rat spinal nerve ligation model of neuropathic pain, both single-dose and three-day repeated CNS4 administration significantly reversed mechanical, pressure, and thermal hypersensitivity, supporting analgesic efficacy with no apparent evidence of rapid tolerance development. In client-owned surgical oncology dogs undergoing standard veterinary procedures, preliminary findings from 14 dogs (8 CNS4-treated, 6 vehicle-treated) demonstrated stable intraoperative mean arterial pressure and heart rate. Postoperatively, CNS4-treated dogs exhibited normal physiological recovery, with respiratory rate, heart rate, and body temperature returning toward the normal canine range, and required approximately 8% less propofol for anesthetic induction. Collectively, these findings support CNS4 as a novel NMDAR modulator with thermoregulatory, anti-agitative, analgesic, and autonomic-stabilizing properties without evidence of abuse liability, supporting continued translational development for neuropathic pain and possibly neuropsychiatric disorders.

Gabapentin (GBP) and pregabalin (PGB) are widely used gabapentinoids. Previously, we have demonstrated, for the first time, that GBP and PGB modulate the nociceptive response to noxious heat in C. elegans at an optimal concentration. In the current study, we use C. elegans and paired thermal nociception assays with direct internal drug concentration measurements to characterize the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of both compounds. Neither drug altered baseline mobility or quadrant preference, confirming that behavioral effects reflected genuine antinociceptive action. Both GBP and PGB produced dose- and time-dependent reductions in thermal avoidance, with 500 uM exposures generating a biphasic, V-shaped time course in which suppression of thermal sensitivity deepened before partially reversing. This partial reversal occurred later with PGB than with GBP. Internal concentrations confirmed dose-dependent absorption and retention for both drugs, yet at 500 uM, internal drug levels remained elevated through 360 min even as behavioral avoidance recovered, indicating that the recovery limb reflects active counter-regulation rather than passive clearance, consistent with previously reported transcriptional and proteomic signatures. Exposure-response profiles were notably flat, suggesting a saturable pharmacodynamic ceiling. Molecular modeling revealed conserved electronic pharmacophores supporting shared alpha-2-delta engagement, alongside shape-descriptor differences that may contribute to divergent absorption kinetics. These findings position C. elegans as a valuable model for dissecting gabapentinoid PK/PD relationships. Beyond mechanistic insight, these findings support the continued investigation of C. elegans as a screening platform whose validation could help address the 3R (Replacement, Reduction, Refinement) principles guiding animal research.

We published recently that one of the main constituents of cannabis products, cannabidiol (CBD), is an efficacious negative allosteric modulator (NAM) of the mu opioid receptor (MOR1) (Bosquez-Berger et al., 2023). Here, we investigated how the presence of cannabidiol (CBD) is associated with fentanyl (FEN) binding across MOR1 conformations. We performed molecular dynamics simulations of systems containing FEN alone or FEN+CBD in three mouse MOR1 conformational backgrounds: active-like 5C1M, inactive-like 4DKL, and a modeled Morph50 intermediate between the 5C1M and 4DKL conformations. Three independently seeded 200 ns trajectories were analyzed per model and condition (18 trajectories total), with the trajectory treated as the independent unit. Across the matched 0-200 ns window, consensus CBD contacts and CBD-associated changes in FEN contacts were strongly state dependent. Corrected intracellular TM3 to TM6 analyses separated the expected active-like, intermediate, and inactive-like backgrounds but did not identify a CBD-associated shift that was consistent across both geometric definitions and all three replicates. Equal-weight replicate-composite density maps preserved both the shared ligand distributions and this between-trajectory variability. These descriptive results support receptor-state-dependent CBD, FEN, MOR1 interactions while emphasizing the limited inferential power of three trajectories per condition.

The neutrophil NADPH oxidase is activated by signals generated by formyl peptide receptor (FPR) agonists recognized by FPR1 (fMLF) and FPR2 (WKYMVM), respectively. Also, the antagonists cyclosporin H (specific for FPR1) and PBP10 (specific for FPR2) inhibit the NADPH oxidase activity when induced by the two peptide agonists. When bound to its receptor, the non-activating positive allosteric modulator Cmp58, specific for the free fatty acid 2 receptor (FFA2R), affects not only the response induced by agonists specific for FFA2R, but also the activating potency but not the efficacy of the two FPR activating peptides. Even though Cmp58 is without effect on the efficacy of the response induced by the FPR agonists, the inhibitory effect of the respective FPR specific antagonists is reduced. This sensitivity shift was reversed by an FFA2R specific antagonist suggesting that two different signals generated by the FPRs activate the NADPH oxidase. According to a receptor trans-regulation model, the activated FPRs generate signals that directly activate the NADPH oxidase and signals that activate the allosterically modulated FFA2Rs to elicit activation of the NADPH.

Background and Purpose: Among new psychoactive substances, synthetic cannabinoid receptor agonists (SCRAs) encompass considerable structural diversity and show wide variation in cannabinoid receptor 1 (CB1) potency and efficacy, but the molecular and receptor-interaction characteristics underlying these differences remain unclear. We assessed the pharmacological profiles of 16 SCRAs and examined structural features associated with differences in CB1 potency and efficacy. Experimental Approach: CB1 agonist activity was measured in a CB1-G15-based Ca2+ assay, and cataleptic effects were assessed in mice. Ligand-receptor interactions were characterized by molecular docking to an active-state CB1 structure. Principal component analysis (PCA) was applied to residue-level interaction profiles, and the resulting scores were tested for associations with CB1 potency and efficacy. Key Results: Potency and efficacy varied widely across cellular and animal assays, with several SCRAs exhibiting different pharmacological responses across the two experimental systems. PCA identified an interaction pattern whose PC1 scores were significantly associated with Emax but not EC50, with contributions from activation-related CB1 residues including the PHE200-TRP356 toggle switch. Molecular descriptor analysis likewise identified structural features significantly associated with in vitro and in vivo Emax. Conclusion and Implications: Our findings suggest that CB1 efficacy is associated with distinct receptor-interaction patterns among structurally diverse SCRAs. Combining functional pharmacology with receptor-level interaction analysis provides structural insight into SCRA efficacy and may help prioritize emerging compounds for further pharmacological and behavioral evaluation.

Panic disorder (PD) is a chronic and highly disabling psychiatric disorder characterised by recurrent and unexpected panic attacks, with underlying neurobiological mechanisms poorly understood. Emerging evidence suggests that inflammatory processes may contribute to the triggering of panic attacks, with a subset of PD patients exhibiting alterations in circulating cytokine levels, while animal studies indicate that an immunoresponsive microglial phenotype may contribute to the disorders pathophysiology. Minocycline, a tetracycline-class antibiotic that crosses the blood-brain barrier, exerts anti-inflammatory effects and has demonstrated therapeutic potential in attenuating panic attacks, supporting its potential as an alternative strategy for reducing PD-related symptoms by modulating microglial activity. In this study, we investigated whether exposure of male Sprague-Dawley rats to a panicogenic stimulus, hypoxia (7% O2), is followed by microglial morphological remodelling in the midbrain periaqueductal grey (PAG), a well-known panic-associated structure, at baseline and after minocycline treatment. The effects of hypoxia on the expression of panic-like jumping behaviour were measured during the respiratory challenge, whereas microglial morphology was assessed at 1, 6, or 24h following the aversive stimulus. In a second experiment, the effects of minocycline (30 mg/kg, i.p., administered once daily for 5 days) on the immediate behavioural responses to hypoxia were compared with those produced by an acute administration of alprazolam (2 mg/kg, i.p.), a benzodiazepine widely used in the clinical management of PD. Minocycline effects on microglial morphology in the PAG were also assessed. Our findings show that hypoxia elicited robust jumping behaviour, without affecting overall locomotion. This panicogenic effect was accompanied by marked and time-dependent microglial morphological changes in the dorsomedial (dmPAG) and ventrolateral (vlPAG) columns of the PAG, consistent with a shift towards an immunoresponsive phenotype. Notably, minocycline, similarly to alprazolam, reduced the number of jumps, indicating a panicolytic effect, while also preventing hypoxia-induced microglial remodelling in the dmPAG. Altogether, these findings suggest a role for microglia in regulating hypoxia-induced panic- like behaviour, indicating that microglial inhibition, as achieved here with minocycline, represents a promising therapeutic strategy for preventing panic attacks.

In pre-clinal studies, VC108, a novel GPR39 inhibitor, increased myocardial O2 tension (mpO2) during different phases of acute myocardial infarction (AMI). The purpose of this work was to investigate the temporal effects of VC108 on mpO2 using a novel population PK/PD model developed from preclinical data using healthy animals and animals subjected to AMI protocols. PK/PD simulations were then performed to explore mpO2 dynamic in untested experimental scenarios. VC108 PK data fitted well to a two-compartment model with linear elimination with body weight, sex, and nominal dose identified as significant covariates influencing clearance and/or central compartment volume. Applying indirect effect model principles for representing mpO2 flux, a structural population PK/PD model was developed to capture mpO2 dynamics during different AMI phases: before and during coronary occlusion and after reperfusion. The model successfully captured basal mpO2 homeostasis, ischemic decline, and reperfusion overshoot, while quantifying the contribution of VC108 to collateral blood flow and functional recovery. Dose response relationships were simulated to measure the effect of different VC108 dose levels on permanent damage, i.e. a reduction in mpO2 at steady state after AMI. The probability of remaining below predefined damage thresholds (mpO2 reduction of 5% to 20%) increased with dose in both sexes, with females consistently achieving greater protection that plateaued around 1 mg/kg of VC108 for higher permanent damage. This model represents a valuable tool to describe, for the first time, VC108 efficacy data and pharmacokinetics with ischemia/reperfusion dynamics and quantify its effect in untested dose levels.

The psychiatric utility of ketamine is limited by its dissociative and systemic side effects. Recently, to enable precision ketamine pharmacotherapy, we introduced SonoKet, ketamine-loaded acoustically activatable liposomes that enable focused ultrasound (FUS)-targeted ketamine delivery to millimeter-sized brain regions. In initial studies, we observed that SonoKet uncaging targeted ketamine to the ultrasound-treated brain region, while inducing greater electrophysiologic and behavioral functional effects than dose-matched free ketamine. To further define these uncaging-potentiated neuromodulatory effects, we used solid-phase microextraction (SPME) coupled to LC-MS/MS to investigate the effect of ultrasound and SonoKet uncaging on key neurotransmitters in real-time. SPME probes were used to sample ketamine, its metabolites, and glutamate, GABA, serotonin (5-HT), and dopamine in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and retrosplenial cortex (RsC) of awake rats. Sampling occurred before and after intravenous administration of either SonoKet, free ketamine, or saline, with FUS targeted to either a frontolimbic or caudal brain region. FUS alone did not yield significant changes in neurotransmitter concentration, nor did it affect the pharmacodistribution of free ketamine. In contrast, FUS generally increased the neurotransmitter response to free ketamine, suggesting an ultrasonic potentiation of ketamine neuromodulation. SonoKet (0.75 mg/kg) uncaging with FUS elicited further elevations in glutamate, GABA, and 5-HT within the FUS-targeted region, along with an increase in dopamine in the NAc when the frontolimbic region was sonicated. These increases were similar to or higher than those induced by 10 mg/kg free ketamine alone or 0.75 mg/kg free ketamine combined with FUS, especially with frontolimbic SonoKet uncaging. Altogether, FUS potentiates ketamine-induced neuromodulation, with spatially specific and synergistically greater effects when ketamine is spatially localized via ultrasonic uncaging. This strategy could augment ketamine pharmacotherapy for psychiatric diseases, while limiting its dissociative and abuse liabilities. HighlightsO_LIFocused ultrasound potentiated ketamine-driven glutamate, serotonin, and dopamine release C_LIO_LILocalized ketamine delivery with SonoKet uncaging drove synergistically greater region-specific neurochemical responses C_LIO_LIUncaging boosts ketamine effects at a fraction of the ketamine dose C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/741494v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1745214org.highwire.dtl.DTLVardef@1b8e06borg.highwire.dtl.DTLVardef@95a840org.highwire.dtl.DTLVardef@15924ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

How water intake is initiated and maintained following V2 vasopressin receptor antagonism remains poorly understood. To elucidate the role of the V1b receptor in managing dehydration stress induced by V2 antagonism, we used deep learning-based computer vision to analyze drinking behavior in V1b knockout (V1bKO) and wild-type (WT) mice. While total water access and intake volume were comparable between genotypes, V1bKO mice exhibited distinct temporal dynamics. Modeling cumulative intake with the Hill equation revealed that the time required to reach 50\% of maximal water access was significantly shorter in V1bKO mice than in WT mice. This accelerated drinking effectively mitigated increases in serum osmolality and body weight loss. A reduced Hill's coefficient in V1bKO mice indicates a reduction of the rapid, cooperative-like water accumulation seen in WT mice. Furthermore, elevated basal hemoglobin levels in V1bKO mice were independent of dehydration, as confirmed via bone marrow transplant. Analysis of movement trajectories revealed that V1bKO mice exhibit a lower proportion of vertical movement (required for nozzle access) despite similar total distances traveled. Collectively, our results demonstrate that the V1b receptor critically regulates water-seeking behavior and osmotic homeostasis.

Introduction: Osteoarthritis (OA) is a degenerative joint condition characterized by chronic pain and the need for pain management. Locally targeting the endocytotic AP2 complex in nociceptors presents a potential strategy for providing sustained pain relief in individuals with OA. Objective: We investigated whether pain behavior associated with OA can be mitigated by genetically silencing the AP2alpha2 subunit of the AP2 complex in nociceptors and by pharmacologically inhibiting the AP2 complex through the intraarticular administration of a small lipidated decoy peptide. Method: Monoiodoacetate (MIA) was employed to induce knee joint OA in mice and rats. Pain behavior was assessed using dynamic weight-bearing and von Frey filaments. Upon confirmation of established OA pain behavior, in vivo AP2alpha2 genetic knockdown in mice was achieved through sciatic nerve transfection of a targeting AP2alpha2 short hairpin RNA (shRNA). To pharmacologically target endocytosis, a single intraarticular injection of peptide was administered into the arthritic knee of rats. The injection contained either the AP2 inhibitor peptide or a scrambled peptide control. Results: Pain behavior was significantly reduced after both genetic and pharmacological disruption of AP2-driven endocytosis. Animals treated with the Ap2 inhibitor peptide exhibited reduced pain behavior throughout the 28-day assay period. Following the completion of behavioral testing, arthritic knee joints and contralateral healthy knee joints were subsequently collected to assess the impact of the treatment on disease progression. Micro-computed tomography analysis revealed a preservation of bone volume in the arthritic joints that received the AP2 inhibitor peptide treatment, in contrast to the scrambled peptide group. Conclusion: These findings demonstrate that the inhibition of nociceptor endocytosis by a small lipidated peptide presents a promising approach to provide sustained relief from joint pain in individuals with arthritis.

Neuropathic pain involves spinal microglial activation and metabolic changes.Forsythoside B (FB) has anti-inflammatory effects, but its role in neuropathic pain and microglial glycolysis remains unclear. We investigated the effects and mechanisms of FB in mice with chronic constriction injury (CCI) and in LPS-stimulated BV2 microglia.FB reduced mechanical allodynia and thermal hyperalgesia in CCI mice. FB also reduced the spinal levels of HIF-1, HK2, PKM2, LDHA, and CD86. It decreased the proportion of IBA1+/CD86+ cells and the levels of IL-1{beta}, IL-6, and TNF-. In LPS20 stimulated BV2 microglia, FB reduced glycolysis-related proteins, proinflammatory markers, and cytokine release. FB also reduced glycolytic activity and partly restored basal mitochondrial respiration. FB did not change Hif1a mRNA levels. However, it Manuscript Click here to access/download;Manuscript;Manuscript.docx increased HIF-1 ubiquitination and promoted its proteasome-dependent degradation. HIF-1 overexpression partly reversed the effects of FB on glycolytic enzymes, CD86 expression, and proinflammatory cytokine production. Immunoprecipitation followed by mass spectrometry and UbiBrowser analysis identified USP7 as a candidate HIF27 1-associated deubiquitinase. Molecular docking and cellular thermal shift assays supported a possible interaction between FB and USP7. FB reduced the association between USP7 and HIF-1. USP7 knockdown produced effects similar to those of FB on HIF-1 ubiquitination and downstream glycolytic and inflammatory responses.Overall, FB alleviated neuropathic pain and reduced proinflammatory microglial responses. These effects may involve the promotion of HIF-1 degradation through modulation of the USP7-HIF-1 regulatory axis.

In human dorsal root ganglia (DRG), and trigeminal (TG) neurons, the various voltage-gated sodium channel (Nav) isoforms play critical roles in the firing of action potentials, which drive electrical impulses that encode somatosensations including, itch, and pain. The SCN11A gene encodes the tetrodotoxin (TTX)-resistant voltage-gated sodium channel Nav1.9, characterized by unique gating properties. Unlike other isoforms, the Nav1.9 channel activates and inactivates slowly and has a hyperpolarized voltage-dependence of activation and depolarized voltage-dependence of inactivation. This leads to a large window current that has been suggested to function as a regulator of the resting membrane potential of neurons. Mutations in Nav1.9 channels lead to congenital insensitivity to pain (gain-of-function) or familial episodic pain syndrome (loss-of-function) suggesting the channel is a critical mediator of pain. Despite its relevance in pain pathophysiology, most existing data relies on rodent models or heterologous expression systems, leaving the specific pharmacology and biophysical behavior of these channels in human primary neurons largely unknown. In this study, we pharmacologically isolated and characterized native Nav1.9 channel currents in human DRG and TG neurons to compare their biophysical profiles. Our findings reveal significant kinetic and voltage-dependent differences between the two populations. Specifically, Nav1.9 channels in TG neurons exhibit a right-shifted steady-state inactivation curve, a larger window current, and faster activation kinetics compared to those in DRG neurons. In addition, conditions that simulate inflammatory states in-vivo greatly potentiates the Nav1.9 currents consistent with similar observations in rodent models. By detailing these distinct biophysical properties, this research offers crucial insights into Nav1.9 channel function relevant for drug discovery efforts aimed at developing analgesics for both acute and chronic pain.

Background and PurposeSerotonergic psychedelics affect behavior and physiology, but the relationships among these effects remain poorly understood. In rodents, the head-twitch response is used as a measure of psychedelic-like activity, yet it does not capture changes in physiological state or the performance of learned behaviors. Here, we investigated the acute effects of the 5-HT2A receptor agonist TCB-2 across several behavioral and physiological measures and examined how these effects were modified by pretreatment with the 5-HT2A receptor antagonist volinanserin. Experimental ApproachMice were tested in head-fixed and freely moving conditions. During a learned auditory trace-conditioning task, we measured licking, pupil area, eye position, and blinking. We measured locomotor activity in an open field and quantified head-twitch responses using a DeepLabCut-based method. To examine the contribution of 5-HT2A receptors, mice were pretreated with the 5-HT2A receptor antagonist volinanserin. Key ResultsTCB-2 caused pupil constriction without detectable changes in eye position or blinking when administered alone. TCB-2 also reduced licking at the highest dose, but the cue-locked temporal pattern of licking remained evident. In freely moving mice, TCB-2 reduced locomotor activity and produced a dose-dependent increase in head-twitch responses. Volinanserin partially attenuated TCB-2-induced pupil constriction and reduced head-twitch responses under some conditions, but it did not consistently prevent the other effects of TCB-2. Conclusions and ImplicationsTCB-2 produced distinct effects across physiological and behavioral measures rather than a uniform disruption of behavioral function. Pronounced pupil constriction and head-twitch responses occurred without detectable changes in eye position or blinking, while the temporal organization of conditioned licking was retained despite a reduction in its magnitude. The incomplete and variable effects of volinanserin preclude definitive conclusions about the receptor mechanisms underlying each response. Combining automated head-twitch detection with physiological and task-related measurements provides a broader framework for comparing the pharmacological profiles of serotonergic compounds. What is already knownO_LIClassical psychedelics produce characteristic effects primarily through serotonin 5-HT2A receptor activation. C_LIO_LIHead-twitch responses capture only one dimension of psychedelic-like drug action. C_LI What this study addsO_LITCB-2 reduced locomotion and licking while preserving the cue-locked pattern of conditioned licking. C_LIO_LIPupil constriction occurred without detectable changes in eye position or blinking. C_LI Clinical significanceO_LIMultidimensional phenotyping can distinguish the physiological and behavioral profiles of serotonergic compounds. C_LIO_LIComplementary measures may improve preclinical evaluation of emerging serotonergic therapeutics. C_LI

Lysosomal storage disorders (LSDs), a group of inherited genetic diseases, are often associated with early-onset neurodegeneration and refractory epileptic seizures. In CLN2 disease, an LSD caused by recessively inherited dysfunction of lysosomal serine protease Tripeptidyl Peptidase 1 (TPP1), lysosomes are functionally impaired through a characteristic accumulation of subcellular materials. Here, we develop and apply a whole-organism screening workflow in tpp1-/- zebrafish to identify small molecules that suppress epileptic seizures - a hallmark of the human disease - in this model. Among 640 US Food and Drug Administration-approved drugs, pregnenolone, an endogenous precursor for steroid biosynthesis, efficiently suppresses seizures and cell death in tpp1-/- zebrafish. Using a semi-automated high-content workflow, we further show that pregnenolone normalizes lysosomal architecture in tpp1-/- zebrafish. Pregnenolone stimulates steroid hormone biosynthesis and related gene expression, which is dysregulated in tpp1-/- zebrafish. Taken together, tpp1-/- zebrafish are a suitable model to study CLN2 disease, in which we have identified pregnenolone as a candidate with therapeutic properties.

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.