Receptor Subunit Research Articles

Tyro3 promotes the maturation of glutamatergic synapses.

The receptor tyrosine kinase Tyro3 is abundantly expressed in neurons of the neocortex, hippocampus, and striatum, but its role in these cells is unknown. We found that neuronal expression of this receptor was markedly up-regulated in the postnatal mouse neocortex immediately prior to the final development of glutamatergic synapses. In the absence of Tyro3, cortical and hippocampal synapses never completed end-stage differentiation and remained electrophysiologically and ultrastructurally immature. Tyro3-/- cortical neurons also exhibited diminished plasma membrane expression of the GluA2 subunits of AMPA-type glutamate receptors, which are essential to mature synaptic function. Correspondingly, GluA2 membrane insertion in wild-type neurons was stimulated by Gas6, a Tyro3 ligand widely expressed in the postnatal brain. Behaviorally, Tyro3-/- mice displayed learning enhancements in spatial recognition and fear-conditioning assays. Together, these results demonstrate that Tyro3 promotes the functional maturation of glutamatergic synapses by driving plasma membrane translocation of GluA2 AMPA receptor subunits.

Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster.

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA.

Developmental loss of NMDA receptors results in supernumerary forebrain neurons through delayed maturation of transit-amplifying neuroblasts

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line (grin1−/−) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1−/− supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction.

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice

BackgroundGlutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. MethodsMale C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. ResultsCLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. ConclusionsOur results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

Differential functional consequences of GRIN2A mutations associated with schizophrenia and neurodevelopmental disorders

Human genetic studies have revealed rare missense and protein-truncating variants in GRIN2A, encoding for the GluN2A subunit of the NMDA receptors, that confer significant risk for schizophrenia (SCZ). Mutations in GRIN2A are also associated with epilepsy and developmental delay/intellectual disability (DD/ID). However, it remains enigmatic how alterations to the same protein can result in diverse clinical phenotypes. Here, we performed functional characterization of human GluN1/GluN2A heteromeric NMDA receptors that contain SCZ-linked GluN2A variants, and compared them to NMDA receptors with GluN2A variants associated with epilepsy or DD/ID. Our findings demonstrate that SCZ-associated GRIN2A variants were predominantly loss-of-function (LoF), whereas epilepsy and DD/ID-associated variants resulted in both gain- and loss-of-function phenotypes. We additionally show that M653I and S809R, LoF GRIN2A variants associated with DD/ID, exert a dominant-negative effect when co-expressed with a wild-type GluN2A, whereas E58Ter and Y698C, SCZ-linked LoF variants, and A727T, an epilepsy-linked LoF variant, do not. These data offer a potential mechanism by which SCZ/epilepsy and DD/ID-linked variants can cause different effects on receptor function and therefore result in divergent pathological outcomes.

The Influence of Perinatal Hypoxia (Asphyxia) on the Distribution of the α1 Subunit of the GABAA Receptor in the Neocortex of Newborn Rats

The Influence of Perinatal Hypoxia (Asphyxia) on the Distribution of the α1 Subunit of the GABAA Receptor in the Neocortex of Newborn Rats

Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

Causal relationship between inflammatory factors and cerebral small vessel disease: Univariate, multivariate, and summary-data-based mendelian randomization analysis.

To explore the impact of inflammatory factors on the incidence of cerebral small vessel disease (CSVD), we performed a mendelian randomization (MR) study to analyze the causal relationship between multiple inflammatory factors and CSVD imaging markers and utilized summary-data-based mendelian randomization (SMR) analysis to infer whether the impact of instrumental variables (IVs) on disease is mediated by gene expression or DNA methylation. Using public databases such as UKB and IEU, and original genome-wide association studies, we obtained IVs related to exposure (inflammatory factors) and outcome (CSVD imaging markers). We performed the inverse variance weighted, weighted median, and MR-Egger methods to assess causal effects between exposure and outcome in univariate MR analysis. To evaluate their heterogeneity, a series of sensitivity analyses were conducted, including the Cochrane Q test, MR-Egger intercept test, MR-Presso, and leave-one-out analysis. We also applied mediation and multivariate MR analysis to explore the interactions between positive exposures on the same outcome. Additionally, we conducted the SMR, which utilizes instruments within or near relevant genes in blood or brain tissues, to elucidate the causal associations with CSVD markers. ABO Univariate MR of multiple cohorts revealed that the risk of small vessel stroke (SVS) increases with elevated levels of TNF-related apoptosis-inducing ligand (TRAIL, OR, 1.23, 95% CI, 1.08-1.39) and interleukin-1 receptor-like 2, (IL-1RL2, OR, 1.29, 95% CI, 1.04-1.61). IL-18 was a potential risk factor for extensive basal ganglia perivascular space burden (BGPVS, OR, 1.02, 95% CI, 1.00-1.05). Moreover, the risk of extensive white matter perivascular space burden (WMPVS) decreased with rising levels of E-selectin (OR, .98, 95% CI, .97-1.00), IL-1RL2 (OR, .97, 95% CI, .95-1.00), IL-3 receptor subunit alpha (IL-3Ra, OR, .98, 95% CI, .97-1.00), and IL-5 receptor subunit alpha (IL-5Ra, OR, .98, 95% CI, .97-1.00). Mediation and multivariate MR analysis indicated that E-selectin and IL-3Ra might interact during the pathogenesis of WMPVS. SMR estimates showed that TRAIL-related IVs rs5030044 and rs2304456 increased the risk of SVS by increasing the expression of gene Kininogen-1 (KNG1) in the cerebral cortex, particularly in the frontal cortex (βsmr=.10, Psmr=.003, FDR=.04). Instruments (rs507666 and rs2519093) related to E-selectin and IL-3Ra could increase the risk of WMPVS by enhancing DNA methylation of the gene ABO in blood tissue (βsmr=.01-.02, Psmr=.001, FDR=.01-.03). According to MR and SMR analysis, higher levels of TRAIL increased the risk of SVS by upregulating gene expression of KNG1 in brain cortex tissues. In addition, protective effects of E-selectin and IL-3a levels on WMPVS were regulated by increased DNA methylation of gene ABO in blood tissue.

Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis

ABSTRACT: Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. “Basal-like” breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10–15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.

Abstract TP245: Identifying Ischemic Stroke Etiology Through Cytokine Biomarkers

Background: Ischemic stroke can arise from various etiologies, such as cardioembolism, atherosclerosis, small vessel disease or cryptogenic. Accurate identification of stroke etiology is vital for secondary stroke prevention. Accumulating evidence suggests that an imbalance of neuroprotective and neuroinflammatory cytokines may contribute to stroke outcomes. Interleukin-2 receptor subunit α (IL-2Rα), a cell surface protein that regulates lymphocyte activation, has been shown to have either pro-atherosclerotic or atheroprotective roles through activation of lymphocytes or regulatory T cells, respectively. We hypothesized that distinct cytokine profiles, and in particular IL-2Rα, may correlate with specific stroke etiology. Methods: Plasma samples were collected from patients with ischemic stroke that were admitted to Memorial Hermann Hospital, Houston by UTHealth biobank. ELISA was used to analyze the plasma samples for levels of IL-2Rα. The relationship between stroke etiology (TOAST criteria) and IL-2Rα was assessed using ANOVA and then adjusted for co-morbidities using multilinear regression models. Results: The mean age of patients was 63.4 years, 35.2% were women, 87% had hypertension, 48.1% had diabetes mellitus, and 53.7% had hyperlipidemia. IL-2Rα levels were significantly elevated in patients with ischemic stroke due to large-artery atherosclerosis (53.5±4.7 pg/ml) as compared to cardioembolic (35.6±6.5 pg/ml), small vessel disease (38.2±7.4pg/ml) or cryptogenic etiology 32±7.8; p<0.01). Conclusions: Our study suggests that IL-2Rα is significantly elevated in ischemic strokes caused by atherosclerosis compared to other ischemic etiologies. This suggests the role of lymphocytic activation in strokes from intracranial and extracranial atherosclerotic disease, which may be used as a therapeutic target. It also identifies a novel biomarker for ischemic strokes from atherosclerosis. Future studies identifying the upstream and downstream pathways that lead to IL-2Rα activation and stroke in atherosclerotic lesions, will further add to our understanding of the characteristics of atherosclerotic lesions that lead to ischemic strokes.

Mutation of novel ethanol-responsive lncRNA Gm41261 impacts ethanol-related behavioral responses in mice.

Chronic alcohol exposure results in widespread dysregulation of gene expression that contributes to the pathogenesis of Alcohol Use Disorder (AUD). Long noncoding RNAs are key regulators of the transcriptome that we hypothesize coordinate alcohol-induced transcriptome dysregulation and contribute to AUD. Based on RNA-Sequencing data of human prefrontal cortex, basolateral amygdala and nucleus accumbens of AUD versus non-AUD brain, the human LINC01265 and its predicted murine homolog Gm41261 (i.e., TX2) were selected for functional interrogation. We tested the hypothesis that TX2 contributes to ethanol drinking and behavioral responses to ethanol. CRISPR/Cas9 mutagenesis was used to create a TX2 mutant mouse line in which 306 base-pairs were deleted from the locus. RNA analysis revealed that an abnormal TX2 transcript was produced at an unchanged level in mutant animals. Behaviorally, mutant mice had reduced ethanol, gaboxadol and zolpidem-induced loss of the righting response and reduced tolerance to ethanol in both sexes. In addition, a male-specific reduction in two-bottle choice every-other-day ethanol drinking was observed. Male TX2 mutants exhibited evidence of enhanced GABA release and altered GABAA receptor subunit composition in neurons of the nucleus accumbens shell. In C57BL6/J mice, TX2 within the cortex was cytoplasmic and largely present in Rbfox3+ neurons and IBA1+ microglia, but not in Olig2+ oligodendrocytes or in the majority of GFAP+ astrocytes. These data support the hypothesis that TX2 mutagenesis and dysregulation impacts ethanol drinking behavior and ethanol-induced behavioral responses in mice, likely through alterations in the GABAergic system.

Histone Methyltransferase G9a Plays an Essential Role on Nicotine Preference in Zebrafish.

Psychostimulants regulate behavioral responses in zebrafish via epigenetic mechanisms. We have previously shown that DNA methylation and histone deacetylase (HDAC) inhibition abolish nicotine-induced conditioned place preference (CPP) but little is known about the role of histone methylation in addictive-like behaviors. To assess the influence of histone methylation on nicotine-CPP, zebrafish were treated with a histone (H3) lysine-9 (K9) dimethyltransferase G9a/GLP inhibitor, BIX-01294 (BIX), which was administered before conditioning sessions. We observed a dual effect of the inhibitor BIX: at high doses inhibited while at low doses potentiated nicotine reward. Transcriptional expression of α6 and α7 subunits of the nicotinic acetylcholine receptor and of G9a, DNA methyl transferase-3, and HDAC-1 was upregulated in zebrafish with positive scores for nicotine-CPP. Changes in relative levels of these mRNA molecules reflected the effects of BIX on nicotine reward. BIX treatment per sé did not affect transcriptional levels of epigenetic enzymes that regulate trimethylation or demethylation of H3. BIX reduced H3K9me2 protein levels in a dose-dependent manner in key structures of the reward pathway. Thus, our findings indicated that different doses of BIX differentially affect nicotine CPP via strong or weak inhibition of G9a/GLP activity. Additionally, we found that the lysine demethylase inhibitor daminozide abolished nicotine-CPP and drug seeking. Our data demonstrate that H3 methylation catalyzed by G9a/GLP is involved in nicotine-CPP induction. Dimethylation of K9 at H3 is an important epigenetic modification that should be considered as a potential therapeutic target to treat nicotine reward and perhaps other drug addictions.

Combining a noble gas with radiotherapy: glutamate receptor antagonist xenon may act as a radiosensitizer in glioblastoma

BackgroundIonotropic glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as a radiosensitizer in GB.MethodsWe designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine.ResultsThe gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Indications for the presence of glutamate receptor subunits were present in glioblastoma-derived and HeLa cells. Significantly reduced clonogenic survival by xenon was shown in U87 and U251 at irradiation doses of 4–8 Gy and 2, 6 and 8 Gy, respectively (p < 0.05). Clonogenic survival was further reduced by the addition of memantine, showing a significant effect at 2–8 Gy for both glioblastoma cell lines (p < 0.05). Xenon did not significantly reduce the surviving fraction of HeLa cells until a radiation dose of 8 Gy.ConclusionThe developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled indications of radiosensitizing properties of xenon gas in glioblastoma.

Targeting GluR3 in Depression and Alzheimer's Disease: Novel Compounds and Therapeutic Prospects.

The present study investigates the interrelated pathophysiology of depression and Alzheimer's disease (AD), with the objective of elucidating common underlying mechanisms. Our objective is to identify previously undiscovered biogenic compounds from the NuBBE database that specifically interact with GluR3. This study examines the bidirectional association between depression and AD, specifically focusing on the role of depression as a risk factor in the onset and progression of the disease. In this study, we utilize pharmacokinetics, homology modeling, and molecular docking-based virtual screening techniques to examine the GluR3 AMPA receptor subunit. The compounds, namely ZINC000002558953, ZINC000001228056, ZINC000000187911, ZINC000003954487, and ZINC000002040988, exhibited favorable pharmacokinetic profiles and drug-like characteristics, displaying high binding affinities to the GluR3 binding pocket. These findings suggest that targeting GluR3 could hold promise for the development of therapies for depression and AD. Further validation through in vitro, in vivo, and clinical studies is necessary to explore the potential of these compounds as lead candidates for potent and selective GluR3 inhibitors. The shared molecular mechanisms between depression and AD provide an opportunity for novel treatment approaches that address both conditions simultaneously.

Anti-N-methyl-D-aspartate receptor-associated encephalitis: A review of clinicopathologic hallmarks and multimodal imaging manifestations.

Anti-N-methyl-D-aspartate receptor-associated encephalitis (NMDARE) is a rare immune-mediated neuroinflammatory condition characterized by the rapid onset of neuropsychiatric symptoms and autonomic dysfunction. The mechanism of pathogenesis remains incompletely understood, but is thought to be related to antibodies targeting the GluN1 subunit of the NMDA receptor with resultant downstream dysregulation of dopaminergic pathways. Young adults are most frequently affected; the median age at diagnosis is 21 years. There is a strong female predilection with a female sex predominance of 4:1. NMDARE often develops as a paraneoplastic process and is most commonly associated with ovarian teratoma. However, NMDARE has also been described in patients with small cell lung cancer, clear cell renal carcinoma, and other benign and malignant neoplasms. Diagnosis is based on correlation of the clinical presentation, electroencephalography, laboratory studies, and imaging. Computed tomography, positron emission tomography, and magnetic resonance imaging are essential to identify an underlying tumor, exclude clinicopathologic mimics, and predict the likelihood of long-term functional impairment. Nuclear imaging may be of value for prognostication and to assess the response to therapy. Treatment may involve high-dose corticosteroids, intravenous immunoglobulin, and plasma exchange. Herein, we review the hallmark clinicopathologic features and imaging findings of this rare but potentially devastating condition and summarize diagnostic criteria, treatment regimens, and proposed pathogenetic mechanisms.

Pathogenicity Prediction of GABAA Receptor Missense Variants

AbstractVariants in the genes encoding gamma‐aminobutyric acid type A (GABAA) receptor subunits are associated with epilepsy. To date, over 1000 clinical variants have been identified in these genes. However, the majority of these variants lack functional studies and their clinical significance is uncertain although accumulating evidence indicates that proteostasis deficiency is the major disease‐causing mechanism. Here, we apply two state‐of‐the‐art modeling tools, namely AlphaMissense and Rhapsody to predict the pathogenicity of saturating missense variants in genes that encode the major subunits of GABAA receptors in the central nervous system, including GABRA1, GABRB2, GABRB3, and GABRG2. We demonstrate that the predicted pathogenicity correlates well between AlphaMissense and Rhapsody. In addition, AlphaMissense pathogenicity score correlates modestly with plasma membrane expression, peak current amplitude, and GABA potency of the variants that have available experimental data. Furthermore, almost all annotated pathogenic variants in the ClinVar database are successfully identified from the prediction, whereas uncertain variants from ClinVar partially due to the lack of experimental data are differentiated into different pathogenicity groups. The pathogenicity prediction of GABAA receptor missense variants provides a resource to the community as well as guidance for future experimental and clinical investigations.

Heterologous investigation of metabotropic and ionotropic odorant receptors in ab3A neurons of Drosophila melanogaster.

In insects, antennal ionotropic receptors (IRs) and odorant receptors (ORs) are among the main sensors of olfactory cues. To functionally characterize the subunits from these receptors, the use of ab3A neurons from transgenic Drosophila melanogaster represented one of the most powerful tools, allowing the identification of ligands (deorphanization) and decrypting their pharmacological properties. However, further investigation is needed to shed light on possible metabotropic functionalities behind insect olfactory receptors and test potentials from the up-to-now-used empty neuronal systems to express subunits belonging to variegate receptor classes. In this project, we adopted the most updated system of Drosophila ab3A empty neurons to test various olfactory receptors, ranging from human ORs working as metabotropic G-protein coupled receptors to insect ionotropic IRs and ORs. Testing transgenic Drosophila expressing human ORs into ab3A neurons by single sensillum recording did not result in an OR response to ligands, but it rather re-established neuronal spiking from the empty neurons. When transgenic D. melanogaster expressed ionotropic IRs and ORs, both heterologous and cis-expressed IRs were non-functional, but the Drosophila suzukii OR19A1 subunit responded to a wide asset of ligands, distinguishing phasic or tonic compound-dependent effects. Despite the use of Drosophila ab3A neurons to test the activation of some metabotropic and ionotropic receptor subunits resulted non-functional, this study deorphanized a key OR of D. suzukii demonstrating its binding to alcohols, ketones, terpenes, and esters.

P470 Crohn’s disease patients with Ustekinumab-induced remission are characterized by high baseline p35 cytokines subunits in LPMC and high IL-23 receptor expression in the lamina propria Th1 cells

Abstract Background Crohn’s disease (CD) is a chronic relapsing disorder of the gastrointestinal tract characterized by excessive inflammation sustained by the induction and expansion of Th1 and Th17 cells. Ustekinumab (UST), a fully humanized monoclonal antibody, is currently used for the therapy of CD patients. It acts by neutralizing both IL12 and IL23, essential for the maturation and expansion of Th1 and Th17 cells, by binding the common subunit p40. To be effective UST needs, in addition to the expression of target cytokines, the presence of cells responsive to IL12 and IL23, thus cells with IL12 and IL23 receptors. Methods This is a prospective, observational, single-center open-label study. All CD patients with clinical indications to receive UST for active luminal disease were enrolled. Patients had baseline endoscopy to confirm disease activity by SES-CD and to collect biopsies to isolate LPMC. Real-time pcr was performed to evaluate IL12 (p40 and p35) and IL23 (p40 and p19) cytokines subunits and IL12 (IL12Rb1 and IL12Rb2) and IL23 (IL12Rb1 and IL23R) receptors subunits expression in remitters (R) and not remitters (NR). Moreover, LPMCs were analyzed by flow cytometry, and IL12Rb2 and IL23R expression in Th1, Th17, and type 1 Innate lymphoid cells (iLC) cells were evaluated. Results were analyzed for clinical remission status (HBI&amp;lt;5) at week 16. Differences between groups were analyzed by Mann-Whitney test and statistical significance was considered for p&amp;lt;0.05. Results From April 2019 to November 2022, 13 CD patients were enrolled, whose clinical and demographic characteristics are enclosed in Table 1. At baseline there was no difference in IL12Rb2, ILRb1, and IL23R expression between R and NR (Fig.1). Regarding cytokines subunits, there was no difference in p19 but there was a significantly higher expression of p35 subunits in R than in NR (mean 1,52 vs 0,34; p=0,001) (Fig.1). Flow cytometry showed no difference in Th and ILCs subgroups frequency between R and NR. IL12Rb2 and IL23R were also equally expressed by Th17, Th1/Th17 and ILC1 cells in the two groups. In contrast, Th1 cells, IL23R expression was higher in R group as compared to NR (mean 20,6% vs 6,8; p=0.009) (Fig.2). Conclusion In CD patients treated with UST, higher expression p35 in LPMC and higher expression of IL23R in the lamina propria Th1 cells was associated with remission at week 16.

P656 Plasma Inflammation-related Proteins Predict the Therapeutic Effect of 5-ASA in Patients with Ulcerative Colitis

Abstract Background 5-aminosalicylate acid (5-ASA) is the first line therapy for mild to moderate ulcerative colitis (UC). However, up to 56% of patients on 5-ASA treatment could not achieve mucosal healing, which is the therapeutic target of UC. At present, predictors for patients’ response to 5-ASA are lacking. Our aim was to identify circulating markers to predict the therapeutic effect of 5-ASA in patients with UC. Methods Plasma samples were collected from patients with active UC at baseline and analyzed using the OLINK panel (Target 96 Inflammation). Effective therapy was defined as achieving clinical remission within 8 weeks or mucosal healing within 1 year of 5-ASA treatment. Orthogonal Projections to Latent Structures Discriminant Analyses (OPLS-DA) were implemented to correlate patient groups to plasma proteins in linear multivariate models. Machine learning models (ML) were constructed to predict ineffective therapy in UC patients. Results Among the 36 UC patients involved (media age 38.5 years old, men 61.6%), 18 patients belonged to the effective group and the ineffective group, respectively. In multivariate analysis, circulating inflammatory protein profiles could distinguish the patients who did not respond well to 5-ASA treatment (Figure 1A). Elevated levels of Axin 1 (p=0.012), sulfotransferase 1A1 (ST1A1) (p=0.032) and interleukin 15 receptor subunit alpha (IL-15RA) (p=0.022), and decreased level of interleukin 2 (IL-2) (p=0.036) were observed in UC patients refractory to 5-ASA therapy (Figure 1B). Receiver operating characteristic curves (ROC) for predicting the therapeutic efficacy of 5-ASA were constructed. The areas under ROC (AUC) of Axin 1, ST1A1, IL-15RA and IL-2 for the prediction of ineffective 5-ASA therapy were 0.744 (p=0.012, sensitivity 83.3%, specificity 72.2%), 0.710 (p=0.031, sensitivity 94.4%, specificity 55.6%), 0.704 (p=0.037, sensitivity 88.9%, specificity 50%) and 0.704 (p=0.037, sensitivity 77.8%, specificity 61.1%), respectively (Figure 1C). Combination of the four protein markers improved the performance of prediction (AUC 0.860 to 1.000, sensitivity 72.2%-100%, specificity 83.3%-100% across all machine learning classifiers) (Figure 1D). Conclusion We identified four plasma inflammation-related proteins associated with the therapeutic effect of 5-ASA in UC. The ML models demonstrated excellent performance in predicting the therapeutic effect of 5-ASA, which could help clinicians identify UC patients who are refractory to 5-ASA in the early course of disease and timely initiation of treatment escalation.

P176 TYK2 inhibition attenuates the stimulation of sensory neurons with hyper-IL-6

Abstract Background Abdominal pain and diarrhoea are a cause of significant morbidity for people with colitis. Recent single cell RNA sequencing of sensory neurons innervating the gut highlights a likely role for sensory nerves in the pathophysiology of these symptoms based on the marked expression of cytokine receptors from the IL-6 receptor family in gut projecting sensory neurons. In keeping with the reported downstream signalling pathway for these receptors marked co-expression of TYK2 transcripts were also found along with IL-6 signal transducer (GP130) in colonic nociceptors characterised by expression of the capsaicin receptor TRPV1. These observations suggest that TYK2 inhibitors may ameliorate symptoms of pain and diarrhoea in people with colitis by preventing neurogenically mediated gut contractility and nociceptor activation. Methods TYK2 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to the hyper-IL-6 fusion protein of the soluble IL-6 receptor and IL-6, which transactivates the GP130 receptor subunit to mimic IL-6 family receptor signalling following pre-treatment with vehicle, the TYK2 inhibitor Deucravacitinib (100nM) or the pan-JAK inhibitor Pyridone-6 (2 μM). Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of hyper-IL-6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons which was significantly attenuated by pre-treatment with the TYK2 specific inhibitor Deucravacitinib (p&amp;lt;0.01) or the pan JAK inhibitor Pyridone-6 (p = &amp;lt;0.05). Conclusion Findings demonstrate that inhibition of TYK2 inhibitor prevents sensory neuron activation in response to IL-6 trans-signalling. This data suggests that TYK2 inhibitors may have utility for the treatment of abdominal pain in people with colitis.