Receptor Function Research Articles

Impact of Long-Term Cannabidiol (CBD) Treatment on Mouse Kidney Transcriptome.

Cannabidiol, which is one of the main cannabinoids present in Cannabis sativa plants, has been shown to have therapeutic properties, including anti-inflammatory and antioxidant effects that may be useful for treatment of various kidney conditions. This article investigates the effect of long-term cannabidiol (CBD) treatment on changes in the renal transcriptome in a mouse model. The main hypothesis was that systematic CBD treatment would affect gene expression associated with those processes in the kidney. The study was conducted on male C57BL/6J mice. Mice in the experimental groups received daily intraperitoneal injections of CBD at doses of 10 mg/kg or 20 mg/kg body weight (b.w.) for 28 days. After the experiment, kidney tissues were collected, RNA was isolated, and RNA-Seq sequencing was performed. The results show CBD's effects on changes in gene expression, including the regulation of genes related to circadian rhythm (e.g., Ciart, Nr1d1, Nr1d2, Per2, and Per3), glucocorticoid receptor function (e.g., Cyp1b1, Ddit4, Foxo3, Gjb2, and Pck1), lipid metabolism (e.g., Cyp2d22, Cyp2d9, Decr2 Hacl1, and Sphk1), and inflammatory response (e.g., Cxcr4 and Ccl28). The obtained results suggest that CBD may be beneficial for therapeutic purposes in treating kidney disease, and its effects should be further analyzed in clinical trials.

Harnessing nuclear receptors to modulate hepatic stellate cell activation for liver fibrosis resolution.

With the recent approval of Resmetirom as the first drug targeting nuclear receptors for metabolic dysfunction-associated steatohepatitis (MASH), there is promising way to treat MASH-associated liver fibrosis. However, liver fibrosis can arise from various pathogenic factors, and effective treatments for fibrosis due to other causes remain elusive. The activation of hepatic stellate cells (HSCs) represents a central link in the pathogenesis of hepatic fibrosis. Therefore, harnessing nuclear receptors to modulate HSC activation may be an effective approach to resolving the complex liver fibrosis caused by various factors. In this comprehensive review, we systematically explore the structure and physiological functions of nuclear receptors, shedding light on their multifaceted roles in HSC activation. Recent advancements in drug development targeting nuclear receptors are discussed, providing insights into their potential as rational and effective therapeutic targets for modulating HSC activation in the context of liver fibrosis. By elucidating the intricate interplay between nuclear receptors and HSC activation, this review contributes to the discovery of new nuclear receptor targets in HSCs for resolving hepatic fibrosis.

Regulation of presynaptic homeostatic plasticity by glial signalling in Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia among the elderly, affects numerous individuals worldwide. Despite advances in understanding the molecular underpinnings of AD pathology, effective treatments to prevent or cure the disease remain elusive. AD is characterized not only by pathological hallmarks such as amyloid plaques and neurofibrillary tangles but also by impairments in synaptic physiology, circuit activity and cognitive function. Synaptic homeostatic plasticity plays a vital role in maintaining the stability of synaptic and neural functions amid genetic and environmental disturbances. A key component of this regulation is presynaptic homeostatic potentiation, where increased presynaptic neurotransmitter release compensates for reduced postsynaptic glutamate receptor functionality, thereby stabilizing neuronal excitability. The role of presynaptic homeostatic plasticity in synapse stabilization in AD, however, remains unclear. Moreover, recent advances in transcriptomics have illuminated the complex roles of glial cells in regulating synaptic function in ageing brains and in the progression of neurodegenerative diseases. Yet, the impact of AD-related abnormalities in glial signalling on synaptic homeostatic plasticity has not been fully delineated. This review discusses recent findings on how glial dysregulation in AD affects presynaptic homeostatic plasticity. There is increasing evidence that disrupted glial signalling, particularly through aberrant histone acetylation and transcriptomic changes in glia, compromises this plasticity in AD. Notably, the sphingosine signalling pathway has been identified as being protective in stabilizing synaptic physiology through epigenetic and homeostatic mechanisms, presenting potential therapeutic targets for treating neurodegenerative disorders.

The Emerging Role of PCSK9 in the Pathogenesis of Alzheimer's Disease: A Possible Target for the Disease Treatment.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease mainly caused by β-amyloid (Aβ) accumulation in the brain. Among the several factors that may concur to AD development, elevated cholesterol levels and brain cholesterol dyshomeostasis have been recognized to play a relevant role. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein primarily known to regulate plasma low-density lipoproteins (LDLs) rich in cholesterol and to be one of the main causes of familial hypercholesterolemia. In addition to that, PCSK9 is also recognized to carry out diverse important activities in the brain, including control of neuronal differentiation, apoptosis, and, importantly, LDL receptors functionality. Moreover, PCSK9 appeared to be directly involved in some of the principal processes responsible for AD development, such as inflammation, oxidative stress, and Aβ deposition. On these bases, PCSK9 management might represent a promising approach for AD treatment. The purpose of this review is to elucidate the role of PCSK9, whether or not cholesterol-related, in AD pathogenesis and to give an updated overview of the most innovative therapeutic strategies developed so far to counteract the pleiotropic activities of both humoral and brain PCSK9, focusing in particular on their potentiality for AD management.

Ligand Binding and Functional Effect of Novel Bicyclic α5 GABAA Receptor Negative Allosteric Modulators.

The significant importance of GABAA receptors in the treatment of central nervous system (CNS) disorders has been known for a long time. However, only in recent years have experimental protein structures been published that can open the door to understanding protein-ligand interactions and may effectively help the rational drug design for the future. In our previous work (Szabó, G. J. Med. Chem. 2022, 65(11), 7876), where a promising selective α5-GABAA negative allosteric modulator (NAM) was developed containing the 3-(4-fluorophenyl)-5-methyl-1,2-oxazole headgroup, we noticed a switch-like effect of a single nitrogen atom for the receptor function in some derivatives having a dihydro-naphthyridinone or dihydro-isoquinolinone moiety. Here, we focused on this chemotype, and a small set of compounds were designed to investigate ligand-receptor interactions experimentally and through computational methods. Elaborated compounds were tested against GABAA α1 and α5 subunit-containing receptors, and binding affinities and functional activities were measured. Starting from the published experimental structure of an engineered, homopentameric, basmisanil-binding GABAA receptor-like construct consisting of modified α5 subunits and an α1-containing GABAA structure, we created a new model of the ligand binding site at the α5/γ2 interface. Using this model, the measured ligand affinities were able to be reproduced well by free energy perturbation (FEP) calculations. In addition, calculations were able to explain the obtained structure-activity relationships, among others, the switch-like effect of the aromatic nitrogen position in the dihydro-naphthyridinone motif for the functional character, and suggest different binding poses for the ligands presenting silent versus negative allosteric effects in this set (SAMs vs. NAMs, respectively). We believe that our results can help design α5 selective GABAA negative allosteric modulators and better understand the GABAA receptor.

Pilot Screening of TREM1 Inhibitors in Cell-Based Reporter Assays Reflecting TREM1/DAP12 Receptor Assembly and Functionality.

Proinflammatory TREM1 receptors expressed on myeloid-derived cells have recently been recognized as a new oncogenic target in cancer, including gliomas. They are established chemotherapeutic targets in neurodegenerative Parkinson's and Alzheimer's diseases, and they also contribute to stroke and sepsis severities. TREM1 activation requires the TREM1/DAP12 interaction for receptor clustering and signal transduction coordinated by TREM1 ligands. Here, we established the quantitative cell-based high-throughput split luciferase assays of DAP12 dimerization, TREM1 dimerization, and TREM1/DAP12 interaction that allow screening of the inhibitory compounds with quantitative dose-responses, IC50 values, and specificity evaluation. The assays are based on the reconstitution of firefly luciferase activity during DAP12 dimerization, TREM1 dimerization, and TREM1/DAP12 interaction, leading to robust luminescence signals in the presence of luciferin. The ligand-dependent and -independent SCHOOL TREM1 inhibitory peptides were utilized for assay validation. Our pilot screen identified several compound scaffolds disrupting DAP12 dimerization, TREM1 dimerization, and the TREM1/DAP12 interaction. The compound potential mechanisms of action and binding sites in the TREM1 and DAP12 complexes were revealed using CB-Dock2 docking software. To our knowledge, this is the first report providing the first generation of pharmacological modulators for TREM1 receptors.

Reference Ranges and Determinants of Thyroid Function and TSH Receptor Antibodies During Early Pregnancy in Nepal.

Different definitions of thyroid dysfunction during pregnancy may lead to under or overtreatment. The aims of this study were to (1) define population-based pregnancy-specific reference ranges for thyroid dysfunction during early pregnancy in Nepal and assess the impact of antibody positivity, (2) quantify the diagnostic impact of population-based reference ranges compared with current practice and (3) assess the determinants of thyroid function and antibody positivity. A total of 800 healthy pregnant women aged 20-40 years in the Bhaktapur municipality were included. Population-based reference ranges for thyroid hormones levels were defined as 2.5th and 97.5th percentiles using competitive immunoluminometric assay design. Thyroid disease cases and those with recommended treatment indications were calculated using current non-pregnancy new reference ranges. Multivariate regression analyses were performed to identify the determinants of thyroid hormones and antibody levels. Median gestational age was 11 weeks. The reference interval was 0.05-3.69 µLU/mL for thyroid stimulating hormone (TSH) and 8.89-15.28 pg/mL for free tetraiodothyronine (fT4) after excluding thyroid peroxidase antibody-positive women. Compared with the current non-pregnancy reference ranges, the new calculations increased the number of women who required treatment from 5 to 12 (0.9% increase). We identified 19 women (2.4%) who were positive for TSH receptor antibody (TRAb). We could not identify the determinants of TRAb positivity, and TRAb positivity was not associated with TSH or fT4 levels. We found meaningful changes using population-based pregnancy-specific TSH and fT4 reference intervals and encourage further studies in other low- and middle-income settings. Our findings suggest that population screening for TRAb is not clinically meaningful. Clinical Trial Registration: U1111-1183-4093.

Development of a RIPK1 degrader to enhance antitumor immunity.

The scaffolding function of receptor interacting protein kinase 1 (RIPK1) confers intrinsic and extrinsic resistance to immune checkpoint blockades (ICBs) and emerges as a promising target for improving cancer immunotherapies. To address the challenge posed by a poorly defined binding pocket within the intermediate domainof RIPK1, here we harness proteolysis targeting chimera (PROTAC) technologyto develop a RIPK1 degrader, LD4172. LD4172 exhibits potent and selective RIPK1 degradation both in vitro and in vivo. Degradation of RIPK1 by LD4172 triggers immunogenic cell death, enhances tumor-infiltrating lymphocyte responses, and sensitizes tumors to anti-PD1 therapy in female C57BL/6J mice. This work reports a RIPK1 degrader that serves as a chemical probe for investigating the scaffolding functions of RIPK1 and as a potential therapeutic agent to enhance tumor responses to ICBs therapy.

Therapeutic potential of dopamine and serotonin in inflammation and cancer: Insights into immune regulation.

This review explores the significant roles of neurotransmitters, focusing on dopamine and serotonin, in inflammation and cancer. These neurotransmitters are vital for neural signaling and play crucial roles in various physiological and pathological processes. We conducted an extensive literature review, focusing on studies published between 2013 and 2024, using databases such as PubMed, Google Scholar, and Scopus. Studies were selected based on relevance to neurotransmitter synthesis, receptor function, and their involvement in diseases like cancer and neurodegenerative disorders. The key search terms included "dopamine", "serotonin", "inflammation", and "cancer". Dopamine and serotonin are synthesized intracellularly and function as pivotal signaling molecules within the central nervous system (CNS) and peripheral systems. Through their respective receptors, dopamine and serotonin influence immune responses and the functionality of various bodily systems. Dysregulation in their signaling pathways is associated with a range of cancers and imbalances within the immune system. The interplay between dopamine and serotonin systems extends beyond neural communication, significantly affecting immune responses and inflammation. Dopamine's role in modulating immune cell activity highlights its potential in treating inflammatory conditions and cancer. Similarly, serotonin's extensive physiological impact underscores the importance of targeting 5-HT pathways in various disorders. Future research should focus on developing therapeutic strategies that leverage these neurotransmitters' regulatory functions in both the CNS and peripheral systems.

The C5a/C5aR1 axis promotes migration of tolerogenic dendritic cells to lymph nodes, impairing the anticancer immune response.

The precise mechanisms by which the complement system contributes to the establishment of an immunosuppressive tumor microenvironment (TME) and promotes tumor progression remain unclear. In this study, we investigated the expression and function of complement C5a receptor 1 (C5aR1) in human and mouse cancer-associated dendritic cells (DCs). First, we observed an overexpression of C5aR1 in tumor-infiltrating DCs, compared to DCs from blood or spleen. C5aR1 expression was restricted to type 2 conventional DCs (cDC2) and monocyte-derived DCs (moDCs), which displayed a tolerogenic phenotype capable of inhibiting T-cell activation and promoting tumor growth. C5aR1 engagement in DCs drove their migration from tumors to tumor-draining lymph nodes, where C5a levels were higher. We used this knowledge to optimize an anticancer therapy aimed at enhancing DC activity. In three syngeneic tumor models, C5aR1 inhibition significantly enhanced the efficacy of poly I:C, a Toll-like receptor 3 (TLR3) agonist, in combination with PD-1/PD-L1 blockade. The contribution of C5aR1 inhibition to the antitumor activity of the combination treatment relied on type 1 conventional DCs (cDC1s) and antigen-specific CD8+ T cells, required lymphocyte egress from secondary lymphoid organs, and was associated with an increase in interferon gamma (IFNγ) signaling. In conclusion, our study highlights the importance of the C5a/C5aR1 axis in the biology of cancer-associated DCs and provides compelling evidence for the therapeutic potential of modulating the complement system to enhance DC-mediated immune responses against tumors.

Dual Activation-Induced Marker Combinations Efficiently Identify and Discern Antigen-Specific and Bystander-Activated Human CD4+ T Cells.

Identifying activated T lymphocytes and differentiating antigen-specific from bystander T cells is crucial for understanding adaptive immune responses. This study investigates the efficacy of activation-induced markers (AIMs) in distinguishing these cell populations. We measured the expression of commonly used AIMs (CD25, CD38, CD40L, CD69, CD137, HLA-DR, ICOS, and OX40) in an in vitro T-cell activation system and evaluated their sensitivity, specificity, and positive predictive value. We demonstrated that individual AIMs, while specific in detecting activated CD4+ T cells, poorly discriminate between antigen-specific and bystander activation, as assessed by a discriminative capacity (DC) score we developed. Our analysis revealed that dual AIM combinations significantly enhanced the ability to distinguish antigen-specific from bystander-activated T cells, achieving DC scores above 90%. These combinations also improved positive predictive value and specificity with a modest reduction in sensitivity. The CD25hi/ICOShi combination emerged as the most efficient, with an average sensitivity of 84.35%, specificity of 99.7%, and DC score of 90.12%. Validation through T-cell cloning and antigen re-stimulation confirmed the robustness of our predictions. This study provides a practical framework for researchers to optimize strategies for identifying and isolating antigen-specific human CD4+ T lymphocytes and studying their phenotype, function, and T-cell receptor repertoire.

Metabolomic profiling of germinated and non-germinated Lablab purpureus seeds: antioxidant properties and α-amylase inhibitory activities for diabetes management

Diabetes is characterized by prolonged hyperglycemia and disruptions in carbohydrate, lipid, and protein metabolism, stemming from inadequate insulin production, impaired insulin receptor functioning, or a combination of both. Conventional diabetes medications like biguanides and sulphonylureas, are widely used and raise concerns about potential side effects with prolonged usage. In this context, legumes emerge as promising candidates due to their significance in traditional diets globally and associated health benefits. Despite being challenging to digest due to anti-nutritive factors, germination, a simple bioprocessing technique, significantly enhances nutritional aspect of the seeds. This study focuses on Lablab purpureus, an underutilized legume, employing a metabolomic approach to explore compounds in germinated and non-germinated seeds. Metabolomic profiling identified 125 compounds in non-germinated and 80 compounds in germinated seeds, revealing unique compounds in each type with potential health benefits. The study identified therapeutically important metabolites such as alkaloids, flavonoids, terpenoids, and saponin in both the germinated and non-germinated seeds. A notable change in the phytochemical composition (total phenol, flavonoid, and total ascorbic acid content) of germinated seeds was observed compared to the non-germinated seeds flour. An increased fold change (1.15, 1.5 and 1.65) was observed in the total phenol, flavonoid, and total ascorbic acid content in germinated seeds compared to non-germinated seeds, alongside higher antioxidant levels in terms of DPPH, ABTS, and FRAP. The IC50 value for α-amylase inhibitory activity was noted to be 2.05 ± 0.05 mg/ml in germinated samples while 0.79 ± 0.00 mg/ml was observed in the non-germinated Lablab purpureus seeds. Therefore, displaying greater α-amylase inhibitory activity in the non-germinated seeds, possibly due to their unique biochemical composition. Nevertheless, even germinated seeds demonstrated appreciable α-amylase inhibitory activity. Therefore, these findings suggest that germination process significantly influences seed biochemistry and helps to raise the phytochemical composition, while the unique composition of the metabolites in the non-germinated seeds could have impact on the α-amylase inhibitory activity. Thus, study suggests Lablab purpureus as a promising functional food source with diverse health-promoting attributes, particularly in diabetes management.Graphical

Understanding paralogous epilepsy–associated GABAA receptor variants: Clinical implications, mechanisms, and potential pitfalls

Recent discoveries have revealed that genetic variants in γ-aminobutyric acid type A (GABAA) receptor subunits can lead to both gain-of-function (GOF) and loss-of-function (LOF) receptors. GABAA receptors, however, have a pseudosymmetrical pentameric assembly, and curiously diverse functional outcomes have been reported for certain homologous variants in paralogous genes (paralogous variants). To investigate this, we assembled a cohort of 11 individuals harboring paralogous M1 proline missense variants in GABRA1, GABRB2, GABRB3, and GABRG2. Seven mutations (α1P260L, α1P260S, β2P252L, β3P253L, β3P253S, γ2P282A, and γ2P282S) in α1β2/3γ2 receptors were analyzed using electrophysiological examinations and molecular dynamics simulations. All individuals in the cohort were diagnosed with developmental and epileptic encephalopathy, with a median seizure onset age of 3.5 mo, and all exhibited global developmental delay. The clinical data for this cohort aligned with established GABAA receptor GOF but not LOF cohorts. Electrophysiological assessments revealed that all variants caused GOF by increasing GABA sensitivity by 3- to 23-fold. In some cases, this was accompanied by LOF traits such as reduced maximal current amplitude and enhanced receptor desensitization. The specific subunit mutated and whether the mutation occurred in one or two subunits within the pentamer influenced the overall effects. Molecular dynamics simulations confirmed similar structural changes from all mutations, but with position-dependent asymmetry. These findings establish that paralogous variants affecting the 100% conserved proline residue in the M1 transmembrane helix of GABAAR subunits all lead to overall GOF traits. The unexpected asymmetric and mixed effects on receptor function have broader implications for interpreting functional analyses for multimeric ion-channel proteins.

Ion Channels as Potential Drug Targets in Dry Eye Disease and Their Clinical Relevance: A Review.

Dry eye disease (DED) is a common multifactorial disorder characterized by a deficiency in the quality and/or quantity of tear fluid. Tear hyperosmolarity, the dysfunction of ion channel proteins, and eye inflammation are primarily responsible for the development and progression of DED. Alterations in the structure and/or function of ion channel receptors (transient receptor potential ankyrin 1 (TRPA1), transient receptor potential melastatin 8 (TRPM8), transient receptor potential vanilloid 1 and 4 (TRPV1 and TRPV4)), and consequent hyperosmolarity of the tears represent the initial step in the development and progression of DED. Hyperosmolarity triggers the activation of ion channel-dependent signaling pathways in corneal epithelial cells and eye-infiltrated immune cells, leading to the activation of transcriptional factors that enhance the expression of genes regulating inflammatory cytokine production, resulting in a potent inflammatory response in the eyes of DED patients. A persistent and untreated detrimental immune response further modifies the structure and function of ion channel proteins, perpetuating tear hyperosmolarity and exacerbating DED symptoms. Accordingly, suppressing immune cell-driven eye inflammation and alleviating tear hyperosmolarity through the modulation of ion channels in DED patients holds promise for developing new therapeutic strategies. Here, we summarize current knowledge about the molecular mechanisms responsible for the inflammation-induced modification of ion channels leading to tear hyperosmolarity and immune cell dysfunction in DED patients. We also emphasize the therapeutic potential of the newly designed immunomodulatory and hypo-osmotic solution d-MAPPS™ Hypo-Osmotic Ophthalmic Solution, which can activate TRPV4 in corneal epithelial cells, stabilize the tear film, enhance natural cytokine communication, and suppress detrimental immune responses, an important novel approach for DED treatment.

Haloperidol-Induced Catalepsy and Its Correlations with Acetylcholinesterase Activity in Different Brain Structures of Mice.

Antipsychotic medicines are used to treat several psychological disorders and some symptoms caused by dementia and schizophrenia. Haloperidol (Hal) is a typical antipsychotic usually used to treat psychosis; however, its use causes motor or extrapyramidal symptoms (EPS) such as catalepsy. Hal blocks the function of presynaptic D2 receptors on cholinergic interneurons, leading to the release of acetylcholine (ACh), which is hydrolyzed by the enzyme acetylcholinesterase (AChE). This study was designed to investigate the Hal-inhibitory effects on AChE activity in regions representative of the cholinergic system of mice and potential associations between cataleptic effects generated by Hal using therapeutic doses and their inhibitory effects on AChE. The distribution of the AChE activity in the different regions of the brain followed the order striatum > hippocampus > (prefrontal cortex/hypothalamus/ cerebellum) > brainstem > septo-hippocampal system. In ex vivo assays, Hal inhibited AChE activity obtained from homogenate tissue of the striatum, hippocampus, and septo-hippocampal system in a concentration-dependent manner. The inhibitory concentration of 50% of enzyme activity (IC50) indicated that the septo-hippocampal system required a higher concentration of Hal (IC50 = 202.5 µmol·L-1) to inhibit AChE activity compared to the striatum (IC50 = 162.5 µmol·L-1) and hippocampus (IC50 = 145 µmol·L-1). In in vivo assays, male Swiss mice treated with concentrations of Hal higher than 0.1 mg·kg-1 induced cataleptic effects. Positive correlations with Spearman's correlation were observed only between the lack of cataleptic effect and the decreased AChE activity of the hippocampus in the mice treated with 0.01 mg·kg-1 of Hal but not in the striatum and septo-hippocampal system. Our results suggest that Hal could increase cholinergic effects via AChE inhibition, in addition to its dopamine antagonist effect, as an alternative approach to the treatment of behavioral disturbances associated with dementia.

Behavioral decline in Shank3Δex4–22 mice during early adulthood parallels cerebellar granule cell glutamatergic synaptic changes

BackgroundSHANK3, a gene encoding a synaptic scaffolding protein, is implicated in autism spectrum disorder (ASD) and is disrupted in Phelan-McDermid syndrome (PMS). Despite evidence of regression or worsening of ASD-like symptoms in individuals with PMS, the underlying mechanisms remain unclear. Although Shank3 is highly expressed in the cerebellar cortical granule cells, its role in cerebellar function and contribution to behavioral deficits in ASD models are unknown. This study investigates behavioral changes and cerebellar synaptic alterations in Shank3Δex4–22 mice at two developmental stages.MethodsShank3Δex4–22 wildtype, heterozygous, and homozygous knockout mice lacking exons 4–22 (all functional isoforms) were subjected to a behavioral battery in both juvenile (5–7 weeks old) and adult (3–5 months old) mouse cohorts of both sexes. Immunostaining was used to show the expression of Shank3 in the cerebellar cortex. Spontaneous excitatory postsynaptic currents (sEPSCs) from cerebellar granule cells (CGCs) were recorded by whole-cell patch-clamp electrophysiology.ResultsDeletion of Shank3 caused deficits in motor function, heightened anxiety, and repetitive behaviors. These genotype-dependent behavioral alterations were more prominent in adult mice than in juveniles. Reduced social preference was only identified in adult Shank3Δex4–22 knockout male mice, while self-grooming was uniquely elevated in males across both age groups. Heterozygous mice showed little to no changes in behavioral phenotypes in most behavioral tests. Immunofluorescence staining indicated the presence of Shank3 predominantly in the dendrite-containing rosette-like structures in CGCs, colocalizing with presynaptic markers of glutamatergic mossy fiber. Electrophysiological findings identified a parallel relationship between the age-related exacerbation of behavioral impairments and the enhancement of sEPSC amplitude in CGCs.LimitationsOther behavioral tests of muscle strength (grip strength test), memory (Barnes/water maze), and communication (ultrasonic vocalization), were not performed. Further study is necessary to elucidate how Shank3 modulates synaptic function at the mossy fiber-granule cell synapse in the cerebellum and whether these changes shape the behavioral phenotype.ConclusionsOur findings reveal an age-related exacerbation of behavioral impairments in Shank3Δex4–22 mutant mice. These results suggest that Shank3 may alter the function of glutamatergic receptors at the mossy fiber-cerebellar granule cell synapse as a potential mechanism causing cerebellar disruption in ASD.

Revisiting the follicle-stimulation hormone receptor expression and function in human myometrium and adipose tissue

BackgroundExtragonadal follicle-stimulating hormone receptor (FSHR) expression at low levels has been shown in several normal and tumor tissues, including myometrium and adipose tissue. FSH-FSHR signaling in the myometrium has been suggested to regulate uterine contractile activity and the timing of labor. In contrast, FSH-FSHR has been linked to the activation of brown/beige fat thermogenesis in adipose tissue. The issue of extragonadal FSHR expression and its functionality remains contentious within the scientific community, as contradictory findings necessitate further independent and critical analyses. Hereby, we re-investigated the FSHR expression and its functionality in normal non-pregnant (M-NP) and pregnant (N-P) human myometrium, as well as in human visceral (VAT) and subcutaneous (SCAT) adipose tissue (AT).MethodsFSHR expression at mRNA (real-time qPCR, RNAscope in situ hybridization) and protein (immunohistochemical staining) levels in adipose tissue, myometrium, and adipocytes were evaluated. Myometrium and adipocytes were treated with recombinant (rh)FSH to study its effects on functional pathways. Myometrium contractile activity was measured using a force transducer with digital output and the DASYLab software unit. Cyclic adenosine monophosphate (cAMP) production by myometrium explants and adipocytes was measured using a cAMP ELISA Kit. The activation of the AKT pathway in myometrium and adipocytes was analyzed by Western blot analysis.ResultsContrary to previous observations, we found no expression of FSHR at either mRNA or protein levels in M-NP, N-P, VAT, and SCAT. Treatment with recombinant human FSH (rhFSH) showed no effect on cAMP production or phosphorylation of AKT in M-NP, N-P, as well as in VAT and SCAT. rhFSH treatment did not influence contractile activity in M-NP, N-P.ConclusionsThese findings suggest that the FSHR signaling pathway does not regulate myometrial contractility during pregnancy. Additionally, the absence of FSHR expression in both VAT and SCAT implied that FSHR does not play a role in the functional signaling pathways in adipose tissues. In conclusion, our findings contradict earlier data on the involvement of FSH-FSHR signaling in regulating myometrial contractility near term, as well as in adipose tissue function.

The modulation of cholecystokinin receptor 1 in the NAc core input from VTA on METH-induced CPP acquisition

BackgroundMethamphetamine (METH) is a potent psychostimulant that interferes the functionality of various brain regions and nervous connections, leading to addiction. The nucleus accumbens core (NAcC), primarily composed of gamma-aminobutyric acid (GABAergic) neurons, serves as a critical nucleus intimately related to addictive behavior. Previous research has indicated the involvement of cholecystokinin (CCK) receptors in drug addiction, yet the precise function of CCK receptors within the neural circuitry mediating METH-induced addiction remains elusive. MethodsMETH-induced conditioned place preference (CPP) model was established in mice. In CCK receptor 1 conditional knockout (CCK1Rflox/flox) or CCK receptor 2 conditional knockout (CCK2Rflox/flox) mice, we then utilized the adeno-associated virus (AAV) transfection system to knock out the specific CCK receptor subtype and explore the function of the CCK receptors in the ventral tegmental area (VTA) to NAcC circuit during METH-induced CPP acquisition. ResultsDuring the acquisition of METH-induced CPP, the expression of CCK1R, but not CCK2R, was upregulated specifically in NAcC. Genetic disruption of either CCK1R in the NAcC effectively hindered METH-induced CPP acquisition and prevented the hyper-excitability of neurons triggered by METH. Furthermore, CCK is released by dopaminergic neurons in the VTA, projecting to the NAcC. Notably, specifically knocking out CCK1R in the VTADA → NAcCGABA circuit blocked the presynaptic release and synaptic plasticity enhancement induced by METH. ConclusionsThese discoveries highlight the critical effect of CCK1R in the VTADA → NAcCGABA circuit on METH-induced CPP acquisition and provide a more comprehensive understanding of the mechanisms underlying CCK receptors contributing to the METH-induced addictive behavior.

Genetic variation of HPA axis activity in farm animals and beyond.

Many experimental data in several species clearly demonstrate the important genetic contribution to variations in HPA axis activity. The influence of corticosteroid hormones on adaptive processes and on production traits such as growth rate, feed efficiency, carcass composition and meat quality is a strong impetus to the search for the molecular bases of these differences for efficient genetic selection. Three main sources of genetic variability have been documented so far in farm animal species, the adrenal cortex sensitivity to ACTH regulating corticosteroid hormone production, the bioavailability of corticosteroid hormones and especially corticosteroid-binding globulin capacity, and glucocorticoid receptor function. The effect of single mutations may be dependent on the genetic background and genetic variation of cortisol levels may have different functional consequences depending on the molecular mechanisms responsible for this change. Understanding the genetic basis of HPA axis activity allows the development of genomic tools and breeding technologies aimed at improving adaptive capacity and stress tolerance in farm animals and their use as valuable models for the genetic study of the HPA axis and the correlation with adaptation, metabolism and other functions regulated by adrenal hormones, and associated pathologies (obesity, cardiovascular, etc.). The next step will be to explore HPA axis variability from a system genetics perspective including the multiple sources of variation and their interactions. This multifactorial approach is a prerequisite to the use of the HPA axis phenotypes in the genetic selection for more productive and robust animals, with a high level of production of quality products.

Role of phosphorylated Y1252, Y1336 and Y1472 on NR2B subunits in hypoxia tolerance of neuronal cell in vitro.

The N-methyl-D-aspartate (NMDA) receptors are related to the various functioning of the nervous system. It has been shown that the NR2B subunit plays an important role in neurological hypoxic/ischemic diseases by regulating NMDA receptor function. NR2B tyrosine phosphorylation is also an important regulatory mechanism for NMDA receptor function. However, the mechanism of NR2B tyrosine phosphorylation in hypoxic/ischemic injury is still unclear. Therefore, in the present study, we aimed to further clarify the changes in NR2B tyrosine phosphorylation in hypoxic/ischemic damage in the brain and its relationship with neuronal survival under hypoxic/ischemic conditions. Four types of NR2B tyrosine site mutants (Tyr → Phe at 1252, 1336, and 1472, and all three mutations together, named Y1252F, Y1336F, Y1472F, and Triple) and wild-type plasmids were transfected into HT22 cells. The cells were then exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). NR2B, cell apoptosis-related molecules, and neuronal survival factor CREB-related signaling proteins (CaMKII, ERK, Akt) were measured. Cell viability was assessed using the CCK-8 assay. Cell apoptosis and cell cycle were evaluated using flow cytometry. The death ratio of HT22 cells under OGD conditions was further tested using a live cell analysis platform. The viability of HT22 cells in the Y1252F, Y1336F, Y1472F, Triple mutants, and wild-type groups was elevated. Compared to the wild-type, western blotting and real-time PCR showed that Y1252F, Y1336F, Y1472F, and Triple mutants downregulated the expression of apoptosis factors and upregulated anti-apoptosis factors in the OGD/R model. Flow cytometry and cell cycle analysis demonstrated that Y1252F, Y1336F, Y1472F, and Triple mutants reduced the apoptosis rate. The percentage of cells in the S phase decreased significantly. Live cell analysis illustrated that the Y1252F, Y1336F, Y1472F, and Triple mutants contributed to HT22 cell survival under OGD conditions. Additionally, the Y1252F, Y1336F, Y1472F, and Triple mutants activated the survival signaling pathway. Furthermore, compared to the control group (without plasmid), only the Y1336F, Y1472F, and Triple mutants groups showed significant differences in the above tests. The tyrosine phosphorylation of NR2B at Y1336 and Y1472 plays key roles in hypoxic/ischemic injury. These phosphorylation sites may be potential targets for hypoxic/ischemic neural protection.