British Journal of Pharmacology

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.

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.

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.

The genetic evidence for an essential role of sodium channel Nav1.7 in human pain conditions is compelling. Unfortunately, potent selective channel blockers of Nav1.7 are not useful analgesics because they have deleterious side effects, particularly on the autonomic nervous system. Here we have evaluated the downstream mechanisms induced by Nav1.7 loss in utero. Nav1.1 compensates for loss of Nav1.7 in sensory neurons and antisense Nav1.1 oligonucleotides diminish excitability in Nav1.7 embryonic null sensory neurons. Enhanced endogenous opioid signalling contributes substantially to analgesia. This is linked to altered gene expression that potentiates opioid signalling through lowered levels of proteins that terminate G-protein signalling (RGS4 and RGS8). There is also enhanced opioid drive through increased enkephalin levels linked to lowered sodium levels in Nav1.7 null neurons. Opioid signalling is the main driver of analgesia, but enhanced potassium channel expression (KCNC4 KCNK2, KCNMB2, KCNAB3) all implicated in pain control, may also contribute to the small level of naloxone-resistant analgesia. These findings suggest a potential new route to analgesia, through mimicking the events at Nav1.7 null sensory neuron terminals. Here we demonstrate potentiation of suzetrigine-evoked analgesia with RGS antagonists alone or in combination with normally inactive doses of morphine in formalin-evoked pain and CFA-induced inflammatory pain. Intriguingly, no analgesia is observed with acute pain. Cancer pain is under investigation. These data are of potential clinical interest.

Allosteric modulation of G protein-coupled receptors (GPCR) is a rapidly advancing area of drug discovery with considerable therapeutic potential. In this study, we used various bioluminescence resonance energy transfer (BRET) assay configurations to investigate the differential activity of LY3154207, a dopamine D1 receptor (D1R)- positive allosteric modulator (PAM), in potentiating D1R-mediated signaling pathways. Our data showed that LY3154207 demonstrated clear bias toward {beta}-arrestin recruitment over G-protein engagement. Among the G-protein subtypes, LY3154207 preferentially potentiated Gs signaling induced by DA. Interestingly, when tested for its effect on the Gs-selective D1R agonist DHX and the Golf-selective agonist TAV, LY3154207 further enhanced the selectivity of these compounds, suggesting a probe-dependent effect. We also studied the structural mechanism underlying this subtype selectivity and found that W123 and R130 in the intracellular loop 2 (ICL2) of D1R were crucial for the activity of LY3154207. Furthermore, the N domain of the G-protein appeared to make minor contributions to this G-protein subtype selectivity of LY3154207. Lastly, our data demonstrated that LY3154207 induced cAMP production in the absence of the orthosteric ligand, indicating that this compound is an ago-PAM.

The cytokine FL and its receptor, Fms-like tyrosine kinase 3 (FLT3), contribute to chronic pain and to the maladaptive effects of morphine but the intracellular mechanisms underlying these effects remain incompletely understood. Here, we investigated the contribution of the Raf-MEK-ERK pathway to FLT3dependent nociceptive signaling and to the modulation of morphine analgesia. Using HEK293T cells expressing human or mouse FLT3, primary dorsal root ganglion (DRG) neurons, and rodent models, we examined ERK activation, TRPV1 sensitization, and behavioral responses following FLT3 activation by its ligand, FL. FL induced concentration-dependent ERK phosphorylation through FLT3, while pharmacological inhibition of FLT3, Raf, or MEK reduced this response. In primary DRG neurons, MEK inhibition attenuated FLinduced potentiation of capsaicin-evoked calcium responses, supporting a role for MEK-ERK signaling in TRPV1 sensitization. In vivo, intrathecal FL increased ERK phosphorylation in DRGs and induced mechanical hypersensitivity, both of which were reduced by Raf or MEK inhibition. In FLT3/MOR-expressing cells, morphine potentiated FL-induced ERK activation, an effect reduced by naloxone and abolished by FLT3 inhibition. In rats, MEK inhibition prevented the FL-induced reduction of morphine analgesia and subsequent mechanical hypersensitivity. Together, these findings identify Raf-MEK-ERK signaling as an important downstream pathway linking neuronal FLT3 activation to nociceptor sensitization and impaired morphine analgesic efficacy.

Background: Milrinone, a phosphodiesterase-3 (PDE-3) inhibitor, is widely used to improve cardiac output in pediatric and adult patients. Yet, developmental differences in myocardial responsiveness to milrinone remain incompletely understood. In this study, we examined the impact of postnatal maturation on the acute cardiac effects of milrinone using an intact guinea pig heart model. Methods: Neonatal (0-2 days), juvenile (4-10 days), and adult (> 6 months) guinea pig hearts were excised, Langendorff-perfused, and cardiac metrics were evaluated under basal conditions and in response to acute milrinone treatment (15 minutes, 10 and 100 M sequential concentrations). Pseudo-electrocardiograms were recorded continuously and left ventricular pressure measurements were performed under sinus rhythm and in response to external pacing. Post-rest potentiation was used to assess contractile reserve, and optical action potentials and calcium transients were recorded. Results: Baseline left ventricular developed pressure (LVDP), action potential duration (APD), and calcium transient duration (CaD) increased with postnatal maturation, consistent with developmental cardiomyocyte remodeling and refinement of excitation-contraction coupling. Milrinone increased heart rate in all age groups, with the greatest chronotropic response at 100 M. Milrinone also increased ventricular contractility and relaxation across developmental stages, but the magnitude of the response varied with age and was attenuated during high frequency pacing. Adult hearts had the greatest increase in LVDP during sinus rhythm and robust post-rest potentiation, which were less pronounced in neonatal hearts. APD and CaD were shortened in neonatal and adult hearts, but were minimally affected in juveniles, indicating a non-linear developmental pattern. Conclusions: Milrinone exerts positive chronotropic, inotropic, and lusitropic effects throughout development, but the magnitude and frequency dependence of these responses vary with age. These findings suggest that postnatal maturation influences the cardiac response to PDE3 inhibition. These developmental differences highlight the importance of considering age as a biological factor in pediatric drug selection and dosing.

The full efficacy range of ligands at CB1 and CB2 (i.e., inverse agonists, neutral agonists, partial agonists, full agonists, and high-efficacy agonists) has been reported collectively across numerous laboratories and assay platforms. Canonical Gi/o and {beta}-arrestin signaling have each been suggested to shape physiological effects differently via different classes of cannabinoid ligands. These prior efficacy classifications, however, were obtained in multiple assay systems, often using indirect, amplified readouts and different reference agonists, making them difficult to compare across ligands. Here we profiled a structurally diverse panel of 22 cannabinoid ligands: synthetic cannabinoid receptor agonists (SCRAs), {Delta}-tetrahydrocannabinol ({Delta}-THC) analogues, endocannabinoid analogues, and antagonists/inverse agonists at CB1 and CB2 receptors using bioluminescence resonance energy transfer (BRET) assays, measuring Gi1 engagement and {beta}-arrestin 2 recruitment and normalizing every response to the reference full agonist CP55,940. At CB1, SCRAs and {Delta}-THC analogues acted as high-efficacy agonists. At CB1, these high-efficacy agonists were also highly efficacious in {beta}-arrestin 2 recruitment. Across the panel, the most consistent trend was a loss of agonist efficacy from CB1 to CB2. Among antagonists, inverse agonists suppressed constitutive CB1 activity more than neutral antagonists. Together, these CP55,940-normalized measurements provide a common efficacy scale and a framework for interpreting pharmacodynamics of CB1 and CB2 cannabinoid receptors.

Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.

Opioid use disorder (OUD) remains a major public health concern, with potent synthetic opioids driving overdose deaths. Understanding how synthetic opioids like fentanyl alter brain circuits to promote persistent use is critical for advancing OUD treatment. The ventral tegmental area (VTA), which projects to several other brain regions including prefrontal cortex (PFC), nucleus accumbens (NAc) and amygdala (Amy), contributes to both the euphoric and dysphoric phases of OUD. Projection target influences the baseline properties and opioid-induced neuroadaptations of VTA neurons, yet less is known about the molecular adaptations that occur across distinct VTA projections during spontaneous opioid withdrawal. Here, we investigated transcriptional responses to acute spontaneous fentanyl withdrawal in projection-defined VTA neurons. Following chronic fentanyl exposure and 24h of spontaneous withdrawal in male and female mice, we sequenced ribosome-associated RNA from VTA neurons projecting to PFC, NAc, or Amy. We found that fentanyl withdrawal produced divergent transcriptional adaptations in VTA neurons projecting to PFC compared with VTA-NAc and VTA-Amy neurons, whereas adaptations in VTA-NAc and VTA-Amy neurons were more concordant. Weighted gene co-expression network analysis (WGCNA) identified fentanyl-responsive modules across VTA projections, with gene ontology analysis linking these modules to distinct biological processes. We further used regulatory enrichment analysis to identify candidate transcriptional regulators associated with fentanyl-responsive co-expression modules. Together, our data demonstrate that VTA neurons undergo distinct molecular adaptations during fentanyl withdrawal based on their projection target, providing new insight into molecular mechanisms relevant to OUD.

Background: More than 90% of rare diseases lack an approved therapy, partly reflecting economic barriers in developing treatments for small patient populations. A common bottleneck is costly and resource-intensive hit-to-lead prioritization. Congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) is an ultra-rare neurodevelopmental disease often caused by de novo gain-of-function variants in the sodium leak channel NALCN, with no approved treatments. Methods: We developed a cost and resource-efficient, AI-enabled translational drug discovery framework integrating phenotypic high-throughput screening (HTS), structural biology foundation models for hit-to-lead prioritization, and orthogonal experimental validation. We applied this framework for drug repurposing to identify NALCN inhibitors as therapeutic candidates for CLIFAHDD. We screened ~1,200 approved drugs using a fluorescence-based membrane potential assay in doxycycline-inducible HEK293 cells stably expressing the NALCN channelosome. Boltz-2 modeling predicted binding affinity and mode to prioritize hits. Lead repurposing candidates were selected by a multidisciplinary expert review panel. Lead candidates were evaluated using patch-clamp electrophysiology in HEK293 cells expressing wild-type or CLIFAHDD-associated NALCN variants, primary mouse hippocampal neurons, and a C. elegans NALCN gain-of-function model. Results: HTS identified 11 candidate NALCN inhibitors. Boltz-2 prioritized candidates based on predicted binding affinity and predicted binding within the NALCN pore, consistent with a direct pore-blocking mechanism. Aprepitant emerged as the lead candidate after multidisciplinary evaluation based on its predicted mechanism, pharmacologic properties, blood-brain barrier penetration, established safety profile, and regulatory approval in children and adults. Patch-clamp electrophysiology confirmed partial inhibition of NALCN-mediated sodium leak currents in HEK293 cells (IC50;=2.22 M), with comparable potency against Cav3.1 (IC50=1.63 M). In primary mouse hippocampal neurons, aprepitant reduced sodium leak currents, hyperpolarized resting membrane potential, and decreased spontaneous firing. Aprepitant rescued hyperlocomotor and dystonic phenotypes in a C. elegans gain-of-function NALCN model. There was notable concordance between AI-predicted hits, phenotypic screening hits, and experimental validation. Conclusion: These findings identify aprepitant as a lead therapeutic candidate for CLIFAHDD, and establish an AI-enabled translational drug discovery framework integrating phenotypic screening, structural modeling, and experimental validation for NALCN channelopathies. This scalable and resource-conscious strategy may accelerate hit-to-lead prioritization and drug discovery for rare and ultra-rare diseases lacking effective therapies.

A growing body of evidence demonstrates that signaling pathways of endogenous lipids (endolipids) modulate the reinforcing properties of opioids including reward and withdrawal. Many of these studies are focused on the endocannabinoid (eCB) system, and its primary eCB ligands, Anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The central hypothesis is that modulation of the eCB system through eCB receptors and enzymes may improve therapeutic outcomes for opioid use disorder (OUD). However, outcomes in preclinical and clinical studies using the low efficacy, CB1/CB2 orthosteric agonist, THC, found limited to no effectiveness, suggesting that targeting this aspect of CB1/CB2 is not a useful therapeutic tool for OUD. Previous studies finding that genetic deletion or pharmacological inhibition of endolipid-regulating enzymes (including eCBs) can alter behavioral sensitivity to opioids provide insight into an alternative approach. If opioid use dysregulates a wide range of endolipid biosynthesis and metabolism, then a clearer understanding of these changes, especially in signaling ligands, will provide a novel avenue to both understand the underlying physiological changes with opioid use as well as providing novel targets for therapeutic interventions. In this study, we tested the hypothesis that if morphine dysregulates multiple endolipid signaling systems in the brain and body, and this dysregulation evolves over time, then these differential effects will be measurable by changes in endolipid levels. Using an Acute (30 minutes post injection 20mg/kg) and a Chronic paradigm (5 days, twice daily, 20mg to 100mg/kg escalating dose) we measured 100 targeted endolipids in 8 brain regions, plasma, liver, and feces in male mice. In the Acute condition, we found that the most screened endolipids were changed in the striatum (42%), while the fewest were changed in the thalamus (19%) and 38% in the plasma. In the Chronic condition, 79% of plasma endolipids were changed. The highest level of change in the CNS was in the cortex (39%). Levels of AEA and 2-AG were largely unchanged; however, levels of the N-acyl GABAs, N-acyl valines, N-acyl taurines, and specific bile acids (e.g. DCA, TCA) showed the most dynamic changes by treatment group. These results provide information on novel endolipid signaling pathways that may contribute to the unwanted side effects of opioids, such as dependence and withdrawal, and provide novel avenues for the development of therapeutic strategies.

Cervical vagus nerve stimulation (VNS) suppresses conditioned fear in rodents and may serve as an adjunct to exposure-based therapy for anxiety disorders. Non-invasive auricular VNS reduces conditioned fear also in humans, although the underlying mechanisms remain unclear. Using a contextual fear conditioning paradigm, in which brief re-exposure induces reconsolidation and prolonged re-exposure promotes extinction learning in mice, we examined auricular VNS-induced attenuation of fear. Auricular VNS during prolonged but not during brief re-exposures reduced freezing, indicating selective facilitation of extinction learning. Stimulation during the latter half of prolonged re-exposure, while extinction learning was progressing, reduced fear responses. As a neural circuit mechanism, we focused on projections from the posterior insular cortex (pIC), which receives interoceptive signals, including vagal input, to the medial prefrontal cortex (mPFC), which regulates fear. Auricular VNS increased c-Fos expression in pIC neurons projecting to the mPFC subdivisions: infralimbic and dorsal peduncular cortex (IL/DP). Optogenetic activation of the pIC-IL/DP pathway mimicked the extinction-facilitating effects of auricular VNS, whereas inhibition blocked them. These results demonstrate that auricular VNS promotes extinction learning of contextual fear memory by activating the pIC-IL/DP circuit, providing insight into how vagal afferent stimulation engages insular-prefrontal networks to modulate fear.

Background and Purpose High novelty-seeking is an endophenotype associated with greater vulnerability to drug addiction, marked by a hyperdopaminergic state and reduced dopamine D2 receptor (D2R) levels in the dorsal striatum (DS). G protein-coupled receptor kinases (GRKs) regulate desensitization, internalization, and recycling of these receptors, including the D2R. This study investigated the relevance of GRK2 and 3 (GRK2/3) in novelty-seeking behavior and striatal dopamine dynamics. Experimental Approach Individual novelty-seeking trait was assessed in Sprague Dawley rats using an open-field test. D2R, GRK2, and GRK5 protein levels were measured in synaptosomal DS samples using Western Blot. Pharmacological inhibition of GRK2/3 was assessed using Cmpd101. We tested the effect of systemic Cmpd101 administration on contextual novelty discrimination and local Cmpd101 perfusion on baseline and stimulus-evoked extracellular dopamine (DA) levels in the DS. Key Results Locomotor activity in a novel environment inversely correlated with the synaptosomal GRK2 and D2R protein levels in the DS. Systemic Cmpd101 treatment modulated the locomotor discrimination index in a novelty phenotype-dependent manner. Moreover, local Cmpd101 perfusion in the DS elicited a sustained elevation in basal extracellular dopamine levels and significantly blunted subsequent stimulus-evoked DA release, both effects independent of novelty-seeking phenotype. Conclusion and Implications Together, our findings link GRK2/3 signaling to individual differences in novelty-seeking and striatal dopaminergic function. These results suggest a potential relevance to drug addiction vulnerability.

GABAergic inhibitory neurons play a central role in shaping network dynamics, and accordingly, their dysfunction is implicated in the pathophysiology of various psychiatric, neurodevelopmental, and neurological disorders. In the past decade, studies on vasoactive intestinal polypeptide-expressing inhibitory neurons (VIP+ neurons) have positioned them as critical components of this circuitry. In particular, VIP+ neurons have been proposed to function within a canonical disinhibitory circuit motif, preferentially inhibiting other interneuron subtypes, to ultimately modulate the gain and activity of local pyramidal neurons. However, their contribution to psychiatrically relevant behavioral endophenotypes is still not fully understood, limiting our ability to target these neurons for therapeutic purposes. In the present study, we systematically assessed the functional contribution of VIP+ neurons to affective and social behaviors. To this end, we used a chemogenetic approach to manipulate the activity of VIP+ neurons in core corticolimbic regions, including the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC). The consequences of either activating or inhibiting VIP+ neurons in these regions were observed while mice underwent a battery of behavioral tasks assessing anxiety-related avoidance behaviors, social interest and recognition, and associative fear learning. While sustained modulation of VIP+ neurons had no significant effect on anxiety-related or social behaviors in any of the brain regions assessed, it produced a robust reduction in associative fear learning specifically in the mPFC. These findings demonstrate that the consequences of sustained VIP+ neuron modulation depend on both anatomical location and behavioural context. Moreover, they identify VIP+ neurons in the mPFC as potentially clinically relevant targets for interventions aimed at modulating fear-related circuits.

Background: Sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular mortality and chronic kidney disease (CKD) progression in cardiovascular-kidney-metabolic (CKM) syndrome, but this benefit derives primarily from reductions in heart failure and cardiovascular mortality rather than in atherothrombotic events. Sotagliflozin, which inhibits both intestinal SGLT1 and renal SGLT2, is the only drug in this class shown to lower rates of myocardial infarction and stroke in CKD. However, its mechanism remains unclear, and its effectiveness in non-diabetic CKD has not been studied. We hypothesized that dual SGLT1/2 inhibition reprograms systemic substrate utilization toward fatty acid oxidation, which may partially account for this protective effect. Methods: CKD was induced in male low-density lipoprotein receptor-deficient mice by 5/6 nephrectomy. Sham and CKD mice received a high-fat diet and drinking water containing sotagliflozin, empagliflozin, or vehicle for 16 weeks, at doses that produced equivalent fractional excretion of glucose. We quantified atherosclerosis en face and in the aortic sinus. Indirect calorimetry, echocardiography, hepatic RNA sequencing, plasma acylcarnitine profiling, and in vivo deuterium labeling of palmitate were performed. Results: At matched levels of glucosuria and plasma total cholesterol, sotagliflozin decreased body mass, atherosclerosis burden, triglycerides, and TNF- more than empagliflozin, and raised postprandial total GLP-1 approximately 3-fold, while both drugs similarly improved albuminuria and diastolic function. Sotagliflozin increased energy expenditure independent of body mass and lowered respiratory exchange ratio despite compensatory hyperphagia. Sotagliflozin reduced hepatic lipogenic genes and de novo lipogenic flux, while increasing {beta}-oxidation and ketogenesis; empagliflozin only activated the oxidative pathway. Conclusions: In experimental CKM syndrome, dual SGLT1/2 inhibition reduces atherosclerosis and metabolic syndrome more effectively than selective SGLT2 inhibition and is associated with reprogramming of hepatic lipid metabolism. These results suggest that sotagliflozin could be an attractive candidate for mitigating atherosclerotic risk in non-diabetic CKD.

Trigeminal pain produces persistent facial hypersensitivity that is difficult to treat. Ion channels contribute to the pathology of trigeminal pain, but the consequences of their modulation remain poorly understood. We previously demonstrated that voltage-gated sodium channel Nav1.7 is a therapeutic target that can be indirectly modulated via interaction with collapsin mediated response protein 2 (CRMP2) to reduce membrane expression of Nav1.7 and trigeminal ganglion neuronal excitability. We used the Foramen Rotundum Inflammatory Constriction of the Trigeminal InfraOrbital Nerve (FRICT-ION) model of trigeminal neuropathic pain to characterize behavioral and supraspinal effects of indirectly modulating the peripheral CRMP2-Nav1.7 interaction via our novel Compound 194 (C194). FRICT-ION produced mechanical and cold allodynia, which were reduced by C194, but had no effect on non-evoked affective behaviors. Despite behavioral improvement, C194 did not normalize heightened glutamatergic activity in the parabrachial nucleus (PBN), revealing a dissociation between behavioral analgesia and activity of a central pain circuit. These findings extend mechanistic studies of C194 in the trigeminal system and demonstrate efficacy of CRMP2-Nav1.7 disruption in modulating facial pain-like behavior. These results support the CRMP2-Nav1.7 axis as a promising therapeutic target while highlighting the need to define how peripheral and central adaptations interact to shape persistent facial 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.

Ogerin, a positive regulator of GPR68, reduces infarct volume and attenuates blood-brain barrier (BBB) leakage post transient middle cerebral artery occlusion (tMCAO). These data support the potential use of Ogerin for ischemic protection. For therapeutic interventions, tissue distribution, kinetics, and brain penetration are important characteristics for a compound. For these reasons, we performed liquid chromatography-mass spectrometry (LC-MS) analysis of Ogerin in mice. At 3 hours following intraperitoneal (IP) injection, liver exhibited highest level of Ogerin among all major organs assessed. We further analyzed time-dependent changes in Ogerin concentration in serum following intravenous delivery. Brain Ogerin level was ~140 ng/g at 2 hour and ~0.06ng/g at 24 hours. The brain-to-serum ratio of Ogerin was ~0.6 at 5 hr. To determine the effect of Ogerin on signaling, we collected samples 24 hours after tMCAO with or without Ogerin delivered and performed Western blotting. Consistent with the diminished level of Ogerin in brain at 24 hour, tMCAO increased similar changes in phosphorylation of PKA substrates and PKC substrates in vehicle- and Ogerin-treated groups. The pharmacokinetic data suggests that Ogerin is effective mainly during acute hours after injection. This finding generates important insights on the design of effective protocol for Ogerin delivery to potentiate GPR68 function in vivo.

Alcohol use disorder is associated with persistent neuroadaptations and cognitive impairments, particularly during withdrawal, yet the temporal dynamics and interactions between these processes remain poorly understood. In the present study, we investigated time-dependent alterations in the oxytocin receptor (OTR), -opioid receptor (MOPr), metabotropic glutamate receptor 5 (mGlu5R), and translocator protein (TSPO) following chronic ethanol exposure and withdrawal, and their relationship to behavioral impairments. Male C57BL/6J mice were exposed to a 10-day escalating-dose ethanol liquid diet paradigm followed by 1, 4, or 7 days of withdrawal. Motor coordination and recognition memory were assessed using rotarod and novel object recognition tests, and receptor binding was quantified using autoradiography across multiple brain regions. Chronic ethanol exposure induced motor deficits that persisted during early withdrawal, whereas recognition memory impairments emerged after 7 days of withdrawal. Ethanol exposure and withdrawal produced region- and time-dependent alterations across all systems examined, with increased OTR binding in septal regions and transient reductions in basomedial amygdala OTR binding during intermediate withdrawal that normalized following prolonged withdrawal, transient increases in MOPr binding in striatal regions at 4 days of withdrawal, increased mGlu5R binding in the nucleus accumbens core, basolateral amygdala, and hippocampus, and early transient increases in TSPO binding indicative of neuroimmune activation. Notably, mGlu5R binding in the caudate-putamen and amygdala was associated with recognition memory deficits in a withdrawal stage-dependent manner. Bioinformatic analysis of human postmortem hippocampal transcriptomic datasets from individuals with alcohol use disorder revealed no consistent changes in GRM5 expression, suggesting that mGlu5R dysregulation may be driven by post-transcriptional mechanisms. These findings demonstrate that ethanol withdrawal is characterized by temporally distinct neuroadaptations across multiple systems and identify mGlu5R as a key correlate of withdrawal-associated memory impairment, highlighting its potential as a translational therapeutic target.