Pharmacological Research

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3{beta} sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 {micro}M and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at 4/{delta} subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure- activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Repeated exposure to hypoxia (oxygen levels below sea-level atmospheric conditions, [~]21%) alternated with regular voluntary exercise, known colloquially as Living High, Training Low, or simply High-Low, is used by elite athletes to boost exercise benefits and athletic performance. While paradigms of High-Low training have been utilized by Olympic athletes for decades, the therapeutic potential of a High-Low regimen in the context of neurotrauma has yet to be investigated. This long-term experiment evaluated the independent and combined effects of repeated hypoxic exposure and voluntary exercise on functional outcomes within the context of preclinical spinal cord injury (SCI). We hypothesized that combinatorial High-Low training enhances functional recovery, beyond either exercise or repeated exposures to hypoxia alone, to improve outcomes after SCI. Adult female rats (n=62) underwent a high-cervical hemisection (LC2H) to model spinal cord injury. At 6 weeks post-SCI, treatment (access to exercise wheel, repeated exposure to normobaric hypoxia at rest, or alternation of both) began in the surviving subjects (n=49). Despite initiation of treatment beyond the acute post-injury phase, High-Low therapy significantly improved respiratory function and prevented the development of SCI-associated anxiety-like behaviors. Notably, repeated in vivo exposure to normobaric hypoxia induced a shift in peripheral T cell profiles, characterized by increased CD4+ and reduced CD8+ expression. These findings indicate that combining repeated exposure to hypoxia with voluntary exercise as a therapy could promote recovery in the existing spinal cord-injured population. Collectively, this work provides a foundational first step for further investigation of High-Low training as a rehabilitation therapy for individuals living with SCI.

SGLT2 inhibitor (SGLT2i)-induced diabetic hyperketonemia is a life-threatening acute complication of diabetes. While Celastrol has been reported to exert beneficial effects on obesity; its potential role in ketogenesis remains unclear. In this study, Celastrol administration significantly attenuates the fasting-induced elevation of blood {beta}-hydroxybutyrate. Moreover, a 7-day course of Celastrol (1 mg/kg/day) leads to reductions in body weight and fat mass. Mechanistically, Celastrol specifically downregulates HMGCS2 expression and suppressess hepatic ketogenesis through inhibiting PPAR expression in the short term ([≤] 2 days). However, after prolonged treatment for 7 days, Celastrol modulates both PPARand serum free fatty acids (FFAs) levels. Furthermore, anti-ketogenic effect of Celastrol is abolished in Ppar{square} /{square} mice. Importantly, Celastrol effectively ameliorates SGLT2i-induced hyperketonemia. In summary, Celastrol curbs hepatic ketone overproduction in a PPAR-dependent manner, indicating its protective potential against SGLT2i-induced hyperketonemia.

Background: Potassium is involved in multiple physiological processes in the body, and hyperkalemia is a common, potentially life-threatening condition. Objective: The aim of our study was to examine the association between plasma potassium levels, and 30-day mortality in patients presenting to an emergency department with normo- or hyperkalemia. Design: Retrospective Cohort study. Setting: Emergency Departments in the Capital region of Denmark Participants: Persons attending Emergency Departments in the Capital Region of Denmark from 2017--2021 with a plasma potassium level of at least 3.5 mM measured within 4 hours after arrival. Measurements: The study was based on data from Danish National Registries and electronic patient records. We performed Kaplan-Meier survival analyses and unadjusted and adjusted cox regression analyses utilizing plasma [K+] 3.5--4.4 mM as the reference group for 30-day mortality hazard ratios (HRs). Results: A total of 248,453 patients were included with a median age of 60 years (Q1;Q3 42;75), and 6,959 (2.8%) died within 30 days. Mortality was 2.2% for potassium level 3.5--4.4 mM, 6.9% for 4.5--4.9 mM, 17.1% for 5.0--5.9 mM, and 26.9% for [≥] 6.0 mM. Unadjusted 30-day HRs were 3.2 (95%CI: 3.0--3.4) for [K+] 4.5--4.9 mM, 8.6 (95%CI: 7.9--9.3) for [K+] 5.0--5.9 mM, and 14.7 (95%CI: 12.5--17.0) for [K+] [≥]6.0 mM. Adjusted HRs were 1.4 (1.3--1.5), 2.10 (1.9--2.3), and 2.4 (2.0--2.8), respectively. Limitations: Risk of residual confounding. Missing data. No access to data regarding in-hospital treatment. Conclusion: Plasma potassium levels above 4.4 mM were associated with increased 30-day mortality among patients presenting to emergency departments. Primary funding source: Department of Emergency Medicine, Copenhagen University hospital, Bispebjerg and Frederiksberg Hospital.

Background-Synuclein (-Syn) plays a central role in Parkinsons disease (PD). Under pathological conditions, -Syn aggregates into toxic oligomers and fibrils that act as damage-associated molecular patterns (DAMPs), stimulating microglial reactivity. This -Syn-microglia axis creates a self-perpetuating cycle of neuroinflammation and neurodegeneration, accelerating dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) and contributing to motor deficits. Moreover, -Syn pathology spreads through the brain, disrupting synaptic plasticity in cognitive regions like the cortex and hippocampus, leading to early cognitive decline. Thus, targeting -Syn aggregation and its inflammatory consequences presents a promising dual-hit therapeutic strategy for PD. MethodsThis study investigates the therapeutic potential of 3-monothiopomalidomide (3MP), a novel thalidomide derivative designed to reduce neuroinflammation with a potentially better safety profile than Pomalidomide (POM). The neuroprotective and anti-inflammatory effects of 3MP were evaluated in rat primary mesencephalic mixed neuron-microglia cultures exposed to human -Syn oligomers (H-SynOs). Anti-aggregation activity was assessed via Thioflavin T (ThT) assays and Thioflavin S (ThS) staining in SH-SY5Y cells. Finally, the anti-aggregation, anti-inflammatory, and neuroprotective effects of 3MP were evaluated in vivo in a rat model of PD induced by intracerebral infusion of H-SynOs. ResultsIn primary cell cultures, 3MP dose-dependently reduced -Syn-induced neuronal death and microglial inflammatory responses. It also significantly inhibited -Syn aggregation in vitro in the ThT assay and in SH-SY5Y cells exposed to -Syn protofibrils, outperforming POM. When chronically administered in vivo, 3MP preserved dopaminergic neurons within the SNpc and yielded functional benefits on motor and cognitive readouts. Notably, 3MP markedly attenuated -Syn aggregates induced by the H-SynOs infusion in the SNpc more efficiently than POM, as shown by reduced intraneuronal staining for pSer129--Syn+ and reduced pSer129-Syn in both cytoplasmic and phagolysosomal compartments of microglia. In addition, mesencephalic and cortical inflammatory microgliosis that followed to intranigral H-SynOs-infusion, were significantly dampened by 3MP. ConclusionsOverall, 3MP emerges as a dual-action drug candidate capable of modulating neuroinflammation and -Syn aggregation and thereby disrupting the -Syn-driven inflammatory cycle. Its neuroprotective effects and favourable safety profile support its potential as a disease-modifying therapy for PD, with promising implications for clinical translation.

BackgroundTriple-negative breast cancer (TNBC) presents significant therapeutic limitations due to its aggressive heterogeneity and the rapid emergence of adaptive resistance to apoptosis-based regimens. Addressing these challenges requires polypharmacological strategies capable of modulating multiple signalling networks simultaneously. While the Cannabis sativa phytocomplex offers a vast chemical space for multi-target intervention, the quantitative pharmacological basis of its synergistic interactions remains largely uncharacterised. PurposeThis study aimed to deconstruct the synergistic landscape of high-purity phytocannabinoids (CBD, CBG, CBD-A) in combination with the sesquiterpene {beta}-caryophyllene (BCP) against TNBC, using MDA-MB-231 as a primary model and Hs578T as a validation line. MethodsGrowth Rate (GR) inhibition metrics and the SynergyFinder+ framework were used to map pharmacological interactions across four reference models. Subcellular dynamics and phenotypic transitions were characterised by high-resolution label-free holotomographic microscopy combined with live-cell kinetic imaging and single-cell fate mapping. ResultsTwo highly potent synergistic clusters were identified for CBD-CBG-BCP combinations, with ZIP, HSA, and Bliss synergy scores exceeding 65. CBD-A exhibited minimal interaction potential and was excluded from ternary studies. GR-based quantification further revealed that these combinations produced net cytotoxicity (GR < 0) at sub-IC concentrations of each component. Single-cell fate mapping by holotomographic microscopy identified a temporally ordered death programme: an initial phase of extensive cytoplasmic vacuolisation associated with focal perinuclear space swelling and progressive nuclear compression, morphological hallmarks of autosis, which is followed by a transition to apoptotic execution. The autotic nature of the primary death phase was confirmed by pharmacological rescue with digoxin, a selective inhibitor of the Na,K-ATPase. To the best of our knowledge, this sequential engagement of autosis followed by apoptotic execution represents the first documented instance of such a two-stage death programme in any cellular model. ConclusionThese findings provide robust evidence that specific phytocannabinoid-terpene ratios engage a Na,K-ATPase-regulated autotic programme as an upstream commitment step, followed by apoptotic execution, effectively circumventing the caspase-independent resistance mechanisms characteristic of TNBC. This study establishes a rational, quantitatively validated framework for transitioning from empirical botanical use to evidence-based, multi-target cannabinoid polypharmacology in aggressive breast cancer.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

Major depressive disorders (MDD) are predicted to become the first cause of burden of disease worldwide in 2030, but 30% of patients still do not respond to antidepressants. Current rodent models of MDD mainly result either from one genetic or one environmental risk factor exposure, not recapitulating the multifactorial and polygenic nature of MDD. We recently generated a polygenic mouse model of MDD from selective breeding after mild stress in the Tail Suspension Test (TST), named H-TST. Here, we selected animals exhibiting high immobility during the Forced Swim Test (FST) to generate a new stable polygenic model of MDD, called H-FST. Unlike our previous H-TST model, H-FST mice did not exhibit any anxiety-or anhedonia-like behaviors, nor did they display any sleep disturbances. Moreover, H-TST and H-FST mice showed opposite response after administration of various antidepressant treatments. The gene expression level in the prefrontal cortex of H-TST and H-FST mice revealed little overlap in genes and biological pathways associated with depressive-like behaviors and opposite dysregulation of excitatory/inhibitory synaptic imbalance. Finally, these two models allowed in humans the identification biomarkers of treatment response specific of clinical subgroup of patients.

Chemotherapy-induced neuropathic pain (CINP) is a frequent and debilitating adverse effect of anti-tumor therapies, for which current treatments are largely non-specific and offer limited efficacy. Identifying molecular mechanisms that drive CINP may enable the development of targeted therapeutic strategies. Here, we demonstrate that paclitaxel-induced mechanical pain hypersensitivity in mice occurs independently of classical Nav1.8+ nociceptors but critically depends on TrkB+ sensory neurons. Transcriptomic analysis of TrkB+ sensory neurons revealed selective expression of Scn5a, which encodes the voltage-gated sodium channel Nav1.5, a channel classically associated with cardiac excitability. Importantly, SCN5A expression was also detected in human primary sensory neurons, indicating potential translational relevance. Functional studies further showed that Scn5a knockdown, using small interfering RNA, significantly attenuates paclitaxel-induced mechanical pain hypersensitivity. Together, these findings identify TrkB+ sensory neurons as key drivers of CINP and reveal Nav1.5 as a previously unrecognized contributor to chemotherapy-induced neuropathic pain. Targeting Nav1.5 in TrkB+ sensory neurons may therefore represent a novel therapeutic strategy for the treatment of CINP.

Globally, about 264 million individuals across all age groups are impacted by depression, a prevalent central nervous system (CNS) condition. Chronic and enduring depression might result in significant health consequences. Numerous pharmaceutical antidepressants exist for the management of mild to severe depression, largely functioning by modifying neurotransmitter levels in the brain. Nevertheless, these drugs frequently induce a variety of side effects, such as insomnia, constipation, exhaustion, drowsiness, and anxiety. Saffron (Crocus sativus L.) is widely acknowledged as a natural antidepressant with little adverse effects. This study investigated the potential antidepressant mechanisms of saffrons principal bioactive compounds safranal, crocin, and picrocrocin via molecular docking against critical target proteins associated with depression, namely the dopamine transporter (DAT), serotonin transporter (SERT), and monoamine oxidase B (MAO-B). Molecular docking was conducted with AutoDock 4.2 to assess the binding affinity and interaction energy of these drugs with the target proteins. Furthermore, Discovery Studio facilitated the viewing and study of both interacting and non-interacting residues at the docking sites, juxtaposing these interactions with those of established inhibitors in crystal structures. The permeability of the blood-brain barrier (BBB), pharmacokinetic characteristics, and toxicity profiles of saffron components were evaluated using SWISS ADME, DataWarrior, and Osiris Molecular Property Explorer. Among the evaluated elements, safranal had the greatest potential as a competitive inhibitor of the dopamine transporter, according to its notable blood-brain barrier permeability, robust binding affinity, and analogous interaction residues in comparison to nortriptyline, a recognized inhibitor. Our findings indicate that safranal may be a viable natural alternative to traditional antidepressants, with minimized adverse effects.

Prognostic stratification in multiple myeloma (MM) relies on staging systems that assign patients to fixed categories at diagnosis and discard the temporal information that accumulates during treatment. We developed a dynamic multimodal framework that predicts residual overall survival using observation windows ranging from 1 to 18 months post-diagnosis. The model integrates DeepInsight-transformed gene expression representation, longitudinal laboratory measurement trajectories across 10 analytes, and treatment history for three drug classes through an adaptive fusion mechanism that accounts for missing clinical observations. On the MMRF CoMMpass cohort (n = 752), five-fold cross-validation yielded a concordance index (C-index) of 0.773 {+/-} 0.024 and a time-dependent AUC at a 1-year prediction horizon (tdAUC1yr) of 0.789 {+/-} 0.021, outperforming all evaluated baseline methods including DeepSurv (0.633 {+/-} 0.095) and random survival forests (0.636 {+/-} 0.024) on matched cross-validation splits. Modality ablation identified longitudinal laboratory measurements as the strongest individual contributor (C-index 0.693); the DeepInsight spatial encoding of gene expression yielded higher discrimination than a multilayer perceptron (MLP) baseline operating on the same features (0.624 vs. 0.596). Kaplan-Meier analysis showed significant prognostic group separation at all primary landmarks (log-rank p < 0.001; hazard ratios 3.46-3.93). A distilled student model retaining only the DeepInsight representation and five baseline clinical features achieved C-index 0.672 and tdAUC1yr 0.740 on an independent microarray cohort (GSE24080, n = 507) without retraining. Interpretability analysis identified prognostic associations consistent with established myeloma biology, including ubiquitin-proteasome pathway genes, endoplasmic reticulum stress markers, and Interferon Alpha Response pathway enrichment.

BackgroundSpinal cord injury (SCI) triggers persistent neuroinflammation, gliosis, neuronal loss, and demyelination, leading to motor deficits and neuropathic pain. Botulinum neurotoxin type A (BoNT/A) has shown anti-inflammatory and neuroprotective effects in acute SCI, but its potential in the chronic phase remains unclear. This study investigates whether combining BoNT/A with electrical muscle stimulation (EMS) enhances recovery in chronic SCI. MethodsAdult mice with severe thoracic SCI (paraplegic) underwent EMS (30 min/day for 10 non-consecutive days starting 3 days post-injury) or no stimulation. Fifteen days after SCI, animals received a single intrathecal injection of BoNT/A (15 pg/5 L) or saline. Functional recovery was assessed up to 60 days as well as in moderate and mild SCI mice, neuropathic pain onset and maintenance were evaluated. Spinal cord tissue was analysed for astrocytic and microglial morphology, neuronal and oligodendroglia survival, myelin protein expression, and in vitro effects on oligodendrocyte precursor cells (OPCs). The phenotype of hindlimb muscles was evaluated through morphological and gene expression analyses. ResultsEMS was able to counteract muscle atrophy and fibrosis, and when combined with BoNT/A, also denervation. Moreover, the combination restored hindlimb motor function in chronic SCI, whereas BoNT/A or EMS alone were ineffective. Neuropathic pain, a common comorbidity associated with SCI, was mitigated by BoNT/A treatment even when administered in the chronic phase. BoNT/A reduced astrocytic hypertrophy and excitatory synapse association and was associated with a morphology-based redistribution of microglial profiles toward a resting-like classification, decreased apoptosis, and increased neuronal and oligodendroglia survival. Myelin basic protein expression was significantly elevated in vivo. In vitro, BoNT/A promoted OPC differentiation into myelinating oligodendrocytes, increased process complexity, and upregulated Myelin basic protein, galactocerebroside C, proteolipid protein, and myelin oligodendrocyte glycoprotein under both proliferative and differentiating conditions. Cleaved SNAP25 colocalization with OPC confirmed direct BoNT/A internalization and activity. ConclusionsBoNT/A exerts multi-cellular neuroprotective actions in chronic SCI, supporting neuronal and oligodendroglia survival, reducing neuroinflammation, enhancing remyelination and the combination with EMS promotes substantial recovery of muscle homeostasis within a permissive microenvironment shaped by early stimulation. Its efficacy depends on a permissive microenvironment achieved through EMS. These results provide strong rationale for the clinical evaluation of BoNT/A as a therapeutic strategy for chronic SCI.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

BACKGROUNDMono-ADP ribosylation is a post-translational modification that regulates various cellular physiological processes, including cell cycle progression, genomic stability, transcription, and cellular protein turnover. PARP16 is an endoplasmic reticulum (ER)-localized mono-ADP-ribosyltransferase that has been shown to regulate the unfolded protein response and maintain ER homeostasis under stress conditions. Despite its established role in ER stress signaling, the functional significance of PARP16 in cardiac pathophysiology, particularly in cardiac hypertrophy and heart failure, remains poorly understood. In this study, we aim to investigate the role of PARP16 in cardiac hypertrophy and heart failure using in vitro and mouse model systems. METHODSWe analysed PARP16 expression in human heart failure samples as well as in heart failure-based mouse models. We evaluated gene expression by RT-PCR, immunoblotting, and confocal microscopy to understand the role of PARP16 in heart failure under phenylephrine- or isoproterenol-treated conditions. We also investigated the role of PARP16 in regulating cardiac function in genetically engineered mouse models, including whole-body PARP16 knockout, cardiac-specific PARP16 knockout, inducible cardiac-specific PARP16 knockout, and cardiac-specific PARP16 Transgenic mice. We performed echocardiography to assess cardiac function. We also used an in vitro primary cardiomyocyte system to knock down and overexpress PARP16. We performed RNA sequencing and mass spectrometry, followed by molecular docking, molecular dynamics simulation, immunoprecipitation, and luciferase assay to characterise the molecular mechanism by which PARP16 regulates cardiac function. RESULTSHuman heart failure samples showed reduced PARP16 expression. PARP16 expression was also significantly reduced in models of heart failure, including the hearts of isoproterenol-treated C57B/L6 mice and phenylephrine-treated primary cardiomyocytes. PARP16-deficient NRCMs showed signs of pathological remodelling. Whole-body, cardiac-specific, and inducible cardiac-specific PARP16 KO mice exhibited cardiac remodelling and dysfunction. In contrast, cardiac-specific PARP16-overexpressing mice were protected from iso-induced cardiac hypertrophy. Mechanistically, several hypertrophic signalling pathway genes are dysregulated in PARP16 knockout mouse hearts concomitant with upregulated NFAT1 transcriptional activity and nuclear translocation. PARP16 binds to and catalytically downregulates NFAT activity, thereby maintaining cardiac function. Mass spectrometry analysis showed that PARP16 is involved in ADP-ribosylation of NFAT1 at E398 and T533. Pharmacological inhibition of NFAT activation attenuates structural and functional abnormalities associated with PARP16 deficiency. CONCLUSIONSPARP16 binds to and inhibits NFAT1 activity to regulate cardiac function in mice, and its downregulation may activate NFAT1 signalling, leading to hypertrophy. In this manner, PARP16 plays a critical role in cardiac hypertrophy and failure and may serve as a potential therapeutic target for the treatment of heart failure.

BackgroundNeonatal global hypoxic-ischemic cerebral injury is a leading cause of infant mortality and lifelong disability. Current rodent models do not replicate neonatal global cerebral ischemia (nGCI) and reperfusion injury. Here, we developed and characterized a rodent model of cardiac arrest and cardiopulmonary reperfusion (CA/CPR) to induce nGCI, producing acute systemic ischemia, mild neuronal injury, white matter alterations, and motor and memory deficits. MethodsRat pups underwent CA/CPR or sham procedure on postnatal day 9-11. CA/CPR in rat pups was performed under anesthesia while intubated. Asystole was induced with intravenous (IV) KCl and maintained for 10-14 minutes. Resuscitation included oxygen ventilation, chest compressions, and IV epinephrine. ResultsTwelve minutes of asystole provided an optimal balance between survival and systemic injury. Behavioral testing on postoperative day (POD) 7 revealed memory impairments. Despite the absence of overt neuronal death in the hippocampus or cerebellum, we observed evidence of glial activation and white matter alterations. ConclusionThis novel rodent model of nGCI addresses limitations in existing models while offering clinically relevant features to support future mechanistic and translational research. ImpactO_LIThis study validates cardiac arrest and cardiopulmonary resuscitation (CA/CPR) as a novel model for neonatal global cerebral ischemia (nGCI), complementing existing rodent models of unilateral and permanent injury by enabling investigation of both global ischemia and reperfusion injury. C_LIO_LInGCI results in memory impairment in the absence of overt neuronal cell death. Functional deficits are associated with neuroinflammatory responses in the hippocampus, white matter, and cerebellum. C_LIO_LINeonatal CA/CPR induces global cerebral ischemia which uniquely allows investigation of hindbrain structures, such as cerebellum, which are typically spared in existing rodent models of neonatal hypoxia-ischemia. C_LI

Serotonergic psychedelics have attracted considerable interest as promising therapeutic agents. However, the molecular mechanisms linking their acute hallucinogenic-like effects to longer-lasting neuroplastic responses remain incompletely understood, partly because of the scarcity of native neural models suitable for mechanistic studies. Here, we developed a neural stem cell-derived in vitro model capable of differentiating into neuronal and glial lineages and, after characterization, used it to investigate the molecular pharmacology of serotonergic psychedelics. A panel comprising tryptamines, phenethylamines and ergolines, including psychedelic compounds and selected non-psychedelic analogues, was evaluated alongside ketamine and TrkB agonists. Endpoints included dendritogenesis, synaptogenesis, immediate-early gene induction, BDNF expression and lactate production. TrkB silencing abolished dendritogenic responses to serotonergic psychedelics, ketamine and TrkB agonists, whereas 5-HT2A receptor silencing selectively impaired serotonergic psychedelic-induced plasticity and altered TrkB-dependent responses. Most serotonergic compounds also increased synaptogenesis and induced c-Fos and Egr-2 expression, although ligand-specific differences were evident, particularly for psilocin and the phenethylamines DOI and Ariadne. Uncoupling of Gq/11 or Gi/o protein-dependent signaling differentially modified neuroplastic and transcriptional responses, indicating a ligand and endpoint dependent contribution of both pathways. Serotonergic psychedelics further induced a 5-HT2A receptor dependent lactate response that was generally sensitive to disruption of either Gq/11 or Gi/o protein coupling. Taken together, these findings support a model in which serotonergic psychedelics recruit an integrated 5-HT2A-TrkB signaling network with distinct structural, transcriptional and metabolic outputs, and establish this neural stem cell-derived system as a valuable platform for screening and dissecting the signaling basis of psychedelic action.

Background Emergency departments (EDs) in high-income countries face rising demand, workforce shortages and crowding. We investigated whether prehospital point-of-care blood gas analysis (BGA), used by emergency physicians, is associated with higher ambulatory treatment rates and improved patient selection for hospital admission. Methods We retrospectively analysed routinely collected data from a pilot implementation of a mobile blood gas analyser in physician-staffed emergency medical services (EMS) in Jena, Germany (July 2023 to May 2024). Adult emergency patients receiving prehospital BGA were compared with propensity score-matched EMS controls without BGA. Primary outcomes were the proportion treated on scene and, among transported patients, the hospital admission rate. Secondary outcomes were 30-day safety among ambulatory patients and associations between BGA parameters and disposition. We used standardised mean differences to assess balance and receiver operating characteristic analysis for lactate thresholds. Results Of 109 patients receiving prehospital BGA, 98 met inclusion criteria after excluding 9 patients with missing NACA scores, 1 on-scene death and 1 invalid age record; these were matched to 390 controls (total n = 488). Baseline demographics, severity and vital signs were well balanced. Ambulatory treatment was markedly higher in the BGA cohort compared with matched controls (27.6% vs 8.7%; OR 3.98, 95% CI 2.26 to 7.01; p<0.001). No ambulatory BGA patient required ED re-attendance or repeat EMS contact within 30 days. Among transported patients, 58% in the BGA cohort were admitted to hospital, compared with an overall regional ED conversion rate of approximately 30%. Lactate [&ge;]2.6 mmol/L was the most influential parameter for disposition decisions, with elevated lactate and acid-base disturbances strongly associated with transport and admission. Conclusion Prehospital BGA was associated with fourfold higher ambulatory treatment rates (27.6%) and a twofold higher ED conversion rate among the patients who were transported (58%), indicating improved risk stratification and resource allocation. These findings suggest that integrating objective biochemical data into prehospital assessment may enhance treat-and-refer decision-making and support more efficient use of limited emergency care capacity.

Glioma is the most common primary malignant tumor of the brain, and accumulating evidence indicates that neuronal activity plays a pivotal role in tumor progression. In this study, neuronal activity is modulated in vitro using potassium chloride (KCl)-induced depolarization and midazolam (MDZ)-mediated suppression. MDZ is a neuronal activity modulation medication, commonly used for sedation, anxiolysis, and amnesia in clinics. After treatment, conditioned media derived from these neuronal cultures are subsequently co-cultured with glioma cells. EdU incorporation assays demonstrate that MDZ significantly inhibits glioma cell proliferation in vitro. Furthermore, an orthotopic xenograft glioma model is established to assess the anti-tumor efficacy of MDZ in vivo, as evaluated by tumor volume and Ki-67 immunostaining. Mechanistically, insulin-like growth factor 1 (IGF1) is identified as the neuronal-activity-regulated factor that promotes glioma growth through activation of the PI3K/AKT signaling pathway. Moreover, transcriptomic profiling of brain tissues reveals that MDZ attenuates neuronal activity and downregulates neuron-derived growth factors in both glioma and non-tumor regions, thereby exerting anti-tumor effects in vivo. Collectively, these findings demonstrate that MDZ suppresses glioma progression by suppressing neuronal activity and inhibiting neuron-derived trophic factors, providing new insights into the development of therapeutic strategies for glioma.

The muscarinic acetylcholine receptor 1 (mAChR1, M1) has been identified as a primary target for Alzheimers disease (AD) and better understanding of the receptor biology, especially in regard to biased signalling of the receptor, will allow for the development of improved drugs targeting cholinergic dysfunction in AD. The aim of this study was to determine the contribution of phosphorylation of M1 to the learning and memory (LM) effects of M1 agonism. The contribution of M1 phosphorylation dependent signalling in LM was assessed using the mAChR1 positive allosteric modulator, VU0486846, in a scopolamine (1.5 mg/kg) induced LM deficit model in mice expressing HA-tagged M1 (M1-WT), phosphorylation deficient HA-tagged M1 (M1-PD), or mice deficient in M1 (M1-KO). LM was assessed using a fear conditioning (FC) testing paradigm where context and cued memory retrieval was measured 24 hrs after training and a higher level of freezing indicated intact memory. Results demonstrated that scopolamine induced a significant LM deficit in both context and cued retrieval in M1-WT mice which was partially rescued by VU0486846 confirming a contribution of M1 signalling in LM. The scopolamine induced deficit in contextual retrieval in M1-KO mice was not rescued by VU0486846, which is an M1 selective ligand, while scopolamine did not induce a deficit in cued retrieval in M1-KO mice. In M1-PD mice, scopolamine induced a LM deficit in contextual retrieval, however this was also not rescued by VU0486846 treatment. Similarly to M1-KO animals, M1-PD mice did not display a scopolamine induced deficit in cued retrieval. When freezing responses were compared across strains, M1-PD mice displayed a deficit compared to M1-WT and M1-KO mice in contextual retrieval, while both M1-PD and M1-KO mice displayed a deficit compared to M1-WT mice in cued retrieval. These results demonstrate that although M1 agonism can restore a LM deficit in both contextual and cued testing paradigms, only the cued retrieval response is dependent on the M1. Additionally, biased Gq M1 signalling is not sufficient to restore contextual memory and requires phosphorylation of the receptor. Furthermore, biased M1 signalling results in LM deficits not seen with KO of the receptor. Overall, these results reiterate the importance of considering the bias of ligands when developing M1 agonists for dementia in the future.

Antipsychotic drugs are the first-line treatment for psychosis yet their mechanism of action remains poorly understood, largely due to the challenge to faithfully model psychosis preclinically. Here, we focus on the emerging concept that psychosis can be caused by brain autoimmunity and present a novel mouse model of anti-N-methyl-D-aspartate-receptor (anti-NMDAR) encephalitis, a condition that manifests with psychosis and autoanti-bodies against the NMDAR. We devised a new mRNA-based approach to immunize mice against the NMDAR. Immunized mice developed psychosis-like behaviors that were caused by anti-NMDAR autoantibodies leading to phagocytosis of NMDARs by brain microglia. The antipsychotic drug clozapine rescued psychosis-like behaviors and, remarkably, reduced anti-NMDAR autoantibody levels and antibody-mediated phagocytosis of NMDARs. The immunomodulatory effects of clozapine were confirmed in a mouse model of systemic lupus erythematosus. Our results demonstrate that clozapine suppresses autoimmunity driving psychosis-like behaviors, raising the possibility that immunomodulation contributes to antipsychotic drug action. HIGHLIGHTSO_LImRNA immunization against the NMDAR induces psychosis-like behavior in mice C_LIO_LIAnti-NMDAR autoantibodies are sufficient for psychosis-like behavior C_LIO_LIMicroglial phagocytosis of NMDARs mediates psychosis-like behavior induced by anti-NMDAR autoanti-bodies. C_LIO_LIClozapine reduces anti-NMDAR autoantibodies, microglial phagocytosis and psychosis-like behavior, consistent with immunomodulation as a potential mechanism of antipsychotic drug action. C_LI