The roles of orphan nuclear receptor 4 group A1 and A2 in fibrosis.

Fibrosis is not a disease but rather an outcome of the pathological tissue repair response. Many myofibroblasts are activated which lead to the excessive accumulation of extracellular matrix components such as collagen and fibronectin with fibrosis. A variety of organs, including kidney, liver, lung, heart and skin, can undergo fibrosis under the stimulation of exogenous or endogenous pathogenic factors. The orphan nuclear receptor 4 group A1 (NR4A1) and nuclear receptor 4 group A2（NR4A2）are belong to the nuclear receptor subfamily and inhibit the occurrence and development of fibrosis. NR4A1 is an inhibitory factor of TGF-ß signaling transduction. Overexpression of NR4A1 in fibroblasts can reduce TGF-ß induced collagen deposition and fibrosis related gene expression. Here, we summarize the current research progress on the NR4A1/2 and fibrosis, providing reference for the treatment of fibrosis.

NR4A2 as a Novel Target Gene for Developmental and Epileptic Encephalopathy: A Systematic Review of Related Disorders and Therapeutic Strategies.

The NR4A2 gene encodes an orphan transcription factor of the steroid-thyroid hormone-retinoid receptor superfamily. This review focuses on the clinical findings associated with the pathogenic variants so far reported, including three unreported cases. Also, its role in neurodegenerative diseases, such as Parkinson's or Alzheimer's disease, is examined, as well as a brief exploration on recent proposals to develop novel therapies for these neurological diseases based on small molecules that could modulate NR4A2 transcriptional activity. The main characteristic shared by all patients is mild to severe developmental delay/intellectual disability. Moderate to severe disorder of the expressive and receptive language is present in at least 42%, while neuro-psychiatric issues were reported in 53% of patients. Movement disorders, including dystonia, chorea or ataxia, are described in 37% patients, although probably underestimated because of its frequent onset in late adolescence-young adulthood. Finally, epilepsy was surprisingly present in 42% of patients, being drug-resistant in three of them. The age at onset varied widely, from five months to twenty-six years, as did the classification of epilepsy, which ranged from focal epilepsy to infantile spasms or Lennox-Gastaut syndrome. Accordingly, we propose that NR4A2 should be considered as a first-tier target gene for the genetic diagnosis of developmental and epileptic encephalopathy.

Bis-Indole Derivatives as Dual Nuclear Receptor 4A1 (NR4A1) and NR4A2 Ligands.

Bis-indole derived compounds such as 1,1-bis(3'-indolyl)-1-(3,5-disubstitutedphenyl) methane (DIM-3,5) and the corresponding 4-hydroxyl analogs (DIM8-3,5) are NR4A1 ligands that act as inverse NR4A1 agonists and are potent inhibitors of tumor growth. The high potency of several DIM-3,5 analogs (IC50 < 1 mg/kg/day), coupled with the >60% similarity of the ligand-binding domains (LBDs) of NR4A1 and NR4A2 and the pro-oncogenic activities of both receptors lead us to hypothesize that these compounds may act as dual NR4A1 and NR4A2 ligands. Using a fluorescence binding assay, it was shown that 22 synthetic DIM8-3,5 and DIM-3,5 analogs bound the LBD of NR4A1 and NR4A2 with most KD values in the low µM range. Moreover, the DIM-3,5 and DIM8-3,5 analogs also decreased NR4A1- and NR4A2-dependent transactivation in U87G glioblastoma cells transfected with GAL4-NR4A1 or GAL4-NR4A2 chimeras and a UAS-luciferase reporter gene construct. The DIM-3,5 and DIM8-3,5 analogs were cytotoxic to U87 glioblastoma and RKO colon cancer cells and the DIM-3,5 compounds were more cytotoxic than the DIM8-3,5 compounds. These studies show that both DIM-3,5 and DIM8-3,5 compounds previously identified as NR4A1 ligands bind both NR4A1 and NR4A2 and are dual NR4A1/2 ligands.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Orphan Nuclear Receptor Family 4A (NR4A) Members NR4A2 and NR4A3 Selectively Modulate Elements of the Monocyte Response to Buffered Hypercapnia.

Hypercapnia occurs when the partial pressure of carbon dioxide (CO2) in the blood exceeds 45 mmHg. Hypercapnia is associated with several lung pathologies and is transcriptionally linked to suppression of immune and inflammatory signalling through poorly understood mechanisms. Here we propose Orphan Nuclear Receptor Family 4A (NR4A) family members NR4A2 and NR4A3 as potential transcriptional regulators of the cellular response to hypercapnia in monocytes. Using a THP-1 monocyte model, we investigated the sensitivity of NR4A family members to CO2 and the impact of depleting NR4A2 and NR4A3 on the monocyte response to buffered hypercapnia (10% CO2) using RNA-sequencing. We observed that NR4A2 and NR4A3 are CO2-sensitive transcription factors and that depletion of NR4A2 and NR4A3 led to reduced CO2-sensitivity of mitochondrial and heat shock protein (Hsp)-related genes, respectively. Several CO2-sensitive genes were, however, refractory to depletion of NR4A2 and NR4A3, indicating that NR4As regulate certain elements of the cellular response to buffered hypercapnia but that other transcription factors also contribute. Bioinformatic analysis of conserved CO2-sensitive genes implicated several novel putative CO2-sensitive transcription factors, of which the ETS Proto-Oncogene 1 Transcription Factor (ETS-1) was validated to show increased nuclear expression in buffered hypercapnia. These data give significant insights into the understanding of immune responses in patients experiencing hypercapnia.

Relapse to cocaine seeking is regulated by medial habenula NR4A2/NURR1 in mice.

Drugs of abuse can persistently change the reward circuit in ways that contribute to relapse behavior, partly via mechanisms that regulate chromatin structure and function. Nuclear orphan receptor subfamily4 groupA member2 (NR4A2, also known as NURR1) is an important effector of histone deacetylase 3 (HDAC3)-dependent mechanisms in persistent memory processes and is highly expressed in the medial habenula (MHb), a region that regulates nicotine-associated behaviors. Here, expressing the Nr4a2 dominant negative (Nurr2c) in the MHb blocks reinstatement of cocaine seeking in mice. We use single-nucleus transcriptomics to characterize the molecular cascade following Nr4a2 manipulation, revealing changes in transcriptional networks related to addiction, neuroplasticity, and GABAergic and glutamatergic signaling. The network controlled by NR4A2 is characterized using a transcription factor regulatory network inference algorithm. These results identify the MHb as a pivotal regulator of relapse behavior and demonstrate the importance of NR4A2 as a key mechanism driving the MHb component of relapse.

Pathological features and molecular signatures of early olfactory dysfunction in 3xTg-AD model mice.

BACKGROUND: Olfactory dysfunction is known to be an early manifestation of Alzheimer's disease (AD). However, the underlying mechanism, particularly the specific molecular events that occur during the early stages of olfactory disorders, remains unclear. METHODS: In this study, we utilized transcriptomic sequencing, bioinformatics analysis, and biochemical detection to investigate the specific pathological and molecular characteristics of the olfactory bulb (OB) in 4-month-old male triple transgenic 3xTg-AD mice (PS1M146V/APPSwe/TauP301L). RESULTS: Initially, during the early stages of olfactory impairment, no significant learning and memory deficits were observed. Correspondingly, we observed significant accumulation of amyloid-beta (Aß) and Tau pathology specifically in the OB, but not in the hippocampus. In addition, significant axonal morphological defects were detected in the olfactory bulb, cortex, and hippocampal brain regions of 3xTg-AD mice. Transcriptomic analysis revealed a significant increase in the expression of neuroinflammation-related genes, accompanied by a significant decrease in neuronal activity-related genes in the OB. Moreover, immunofluorescence and immunoblotting demonstrated an activation of glial cell biomarkers Iba1 and GFAP, along with a reduction in the expression levels of neuronal activity-related molecules Nr4a2 and FosB, as well as olfaction-related marker OMP. CONCLUSION: In sum, the early accumulation of Aß and Tau pathology induces neuroinflammation, which subsequently leads to a decrease in neuronal activity within the OB, causing axonal transport deficits that contribute to olfactory disorders. Nr4a2 and FosB appear to be promising targets for intervention aimed at improving early olfactory impairment in AD.

Parallel expression patterns of NR4A nuclear receptor family genes in the pituitary gland of proestrus rats.

The NR4A nuclear receptor family (NR4As), encompassing NR4A1, NR4A2, and NR4A3, exerts pivotal roles in cellular processes through intricate expression patterns and interactions. Despite the influence of some NR4As on anterior pituitary functions regulated by the hypothalamus, their physiological expression patterns remain unclear. In our prior work, we demonstrated the specific upregulation of NR4A3 in the rat anterior pituitary gland during the proestrus afternoon, coinciding with a gonadotropin surge. In this study, we investigated changes in pituitary Nr4a gene expression throughout the estrous cycle in rats and a gonadotropin surge-induced model. Nr4a1 and Nr4a2 gene expression significantly increased during proestrus, aligning with previous observations for Nr4a3. Furthermore, prolactin gene expression increased sequentially with rising Nr4a gene expression, while thyroid-stimulating hormone beta gene expression remained stable. Immunohistochemistry revealed a widespread and differential distribution of NR4A proteins in the anterior pituitary, with NR4A1 and NR4A3 being particularly abundant in thyrotrophs, and NR4A2 in gonadotrophs. In estrogen-treated ovariectomized rats, elevated luteinizing hormone secretion corresponded to markedly upregulated expression of Nr4a1, Nr4a2, and Nr4a3. In gonadotroph and somatomammotroph cell lines, gonadotropin- and thyrotropin-releasing hormones transiently and dose-dependently increased the expression of Nr4a genes. These findings suggest that hypothalamic hormone secretion during proestrus may induce the parallel expression of pituitary Nr4a genes, potentially influencing the pituitary gene expression program related to endocrine functions before and after ovulation.

PROKR1-CREB-NR4A2 axis for oxidative muscle fiber specification and improvement of metabolic function.

Metabolic disorders are characterized by an imbalance in muscle fiber composition, and a potential therapeutic approach involves increasing the proportion of oxidative muscle fibers. Prokineticin receptor 1 (PROKR1) is a G protein-coupled receptor that plays a role in various metabolic functions, but its specific involvement in oxidative fiber specification is not fully understood. Here, we investigated the functions of PROKR1 in muscle development to address metabolic disorders and muscular diseases. A meta-analysis revealed that the activation of PROKR1 upregulated exercise-responsive genes, particularly nuclear receptor subfamily 4 group A member 2 (NR4A2). Further investigations using ChIP-PCR, luciferase assays, and pharmacological interventions demonstrated that PROKR1 signaling enhanced NR4A2 expression by Gs-mediated phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in both mouse and human myotubes. Genetic and pharmacological interventions showed that the PROKR1-NR4A2 axis promotes the specification of oxidative muscle fibers in both myocytes by promoting mitochondrial biogenesis and metabolic function. Prokr1-deficient mice displayed unfavorable metabolic phenotypes, such as lower lean mass, enlarged muscle fibers, impaired glucose, and insulin tolerance. These mice also exhibited reduced energy expenditure and exercise performance. The deletion of Prokr1 resulted in decreased oxidative muscle fiber composition and reduced activity in the Prokr1-CREB-Nr4a2 pathway, which were restored by AAV-mediated Prokr1 rescue. In summary, our findings highlight the activation of the PROKR1-CREB-NR4A2 axis as a mechanism for increasing the oxidative muscle fiber composition, which positively impacts overall metabolic function. This study lays an important scientific foundation for the development of effective muscular-metabolic therapeutics with unique mechanisms of action.

A role of NR4A2 in Graves' disease: regulation of Th17/Treg.

PURPOSE: This study aimed to explore the molecular pathogenesis of Graves' disease (GD). METHODS: The gene expression profile in CD4+ T cells from GD patients and healthy controls were analyzed through mRNA-sequencing. The expression of NR4A2 was determined by quantitative real-time PCR and western blot. The levels of Th17 and Treg were determined by flow cytometry. ELISA was employed to detect the levels of IL-10, IL-17A, IL-17F and IL-22. RESULTS: In the CD4+ T cells from GD patients, there were 128 up-regulated and 510 down-regulated genes. Subsequently, we focused on the role of nuclear receptor 4 group A member 2 (NR4A2) in GD. NR4A2 was lowly expressed in the CD4+ T cells from GD patients. Its expression was negatively correlated with free triiodothyronine and tetraiodothyronine, but positively correlated with thyroid stimulating hormone. NR4A2 knockdown decreased the percentage of Treg cells, with a decreased IL-10 level. While its over-expression augmented the Treg differentiation, with an elevated IL-10 level. In addition, knockdown or over-expression of NR4A2 showed no significant influence on Th17 differentiation. CONCLUSION: These results indicate that the low level of NR4A2 in GD patients may suppress Treg differentiation, but have no influence on Th17 differentiation, leading to the imbalance of Th17/Treg and contributing to the development of GD. Revealing the role of NR4A2 in GD provides a novel insight for the treatment of GD.

Smoke and Spike: Benzo[a]pyrene Enhances SARS-CoV-2 Infection by Boosting NR4A2-Induced ACE2 and TMPRSS2 Expression.

Cigarette smoke aggravates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the underlying mechanisms remain unclear. Here, they show that benzo[a]pyrene in cigarette smoke extract facilitates SARS-CoV-2 infection via upregulating angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). Benzo[a]pyrene trans-activates the promoters of ACE2 and TMPRSS2 by upregulating nuclear receptor subfamily 4 A number 2 (NR4A2) and promoting its binding of NR4A2 to their promoters, which is independent of functional genetic polymorphisms in ACE2 and TMPRSS2. Benzo[a]pyrene increases the susceptibility of lung epithelial cells to SARS-CoV-2 pseudoviruses and facilitates the infection of authentic Omicron BA.5 in primary human alveolar type II cells, lung organoids, and lung and testis of hamsters. Increased expression of Nr4a2, Ace2, and Tmprss2, as well as decreased methylation of CpG islands at the Nr4a2 promoter are observed in aged mice compared to their younger counterparts. NR4A2 knockdown or interferon-λ2/λ3 stimulation downregulates the expression of NR4A2, ACE2, and TMPRSS2, thereby inhibiting the infection. In conclusion, benzo[a]pyrene enhances SARS-CoV-2 infection by boosting NR4A2-induced ACE2 and TMPRSS2 expression. This study elucidates the mechanisms underlying the detrimental effects of cigarette smoking on SARS-CoV-2 infection and provides prophylactic options for coronavirus disease 2019, particularly for the elderly population.

Sodium Butyrate Ameliorates Deoxynivalenol-Induced Oxidative Stress and Inflammation in the Porcine Liver via NR4A2-Mediated Histone Acetylation.

Mycotoxin-induced liver injury is often accompanied by oxidative stress (OS) and inflammation. This research aimed to explore the potential mechanism of sodium butyrate (NaBu) in modulating hepatic anti-oxidation and anti-inflammation pathways in deoxynivalenol (DON)-exposed piglets. The results show that DON induced liver injury, increased mononuclear cell infiltration, and decreased serum total protein and albumin concentrations. Transcriptomic analysis revealed that reactive oxygen species (ROS) and TNF-α pathways were highly activated upon DON exposure. This is associated with disturbed antioxidant enzymes and increased inflammatory cytokines secretion. Importantly, NaBu effectively reversed the alterations caused by DON. Mechanistically, the ChIP-seq result revealed that NaBu strongly depressed DON-increased enrichment of histone mark H3K27ac at the genes involved in ROS and TNF-α-mediated pathways. Notably, we demonstrated that nuclear receptor NR4A2 was activated by DON and remarkably recovered with the treatment of NaBu. In addition, the enhanced NR4A2 transcriptional binding enrichments at the promoter regions of OS and inflammatory genes were hindered by NaBu in DON-exposed livers. Consistently, elevated H3K9ac and H3K27ac occupancies were also observed at the NR4A2 binding regions. Taken together, our results indicated that a natural antimycotic additive, NaBu, could mitigate hepatic OS and inflammatory responses, possibly via NR4A2-mediated histone acetylation.

Protective effect of Nr4a2 (Nurr1) against LPS-induced depressive-like behaviors via regulating activity of microglia and CamkII neurons in anterior cingulate cortex.

Neuroinflammation is tightly associated with onset of depression. The nuclear receptor related 1 protein (Nurr1, also called Nr4a2), its roles in dopaminergic neurons is well understood, which can alleviate inflammation. Nevertheless, potential effects of Nr4a2 on neuroinflammation associated with depression still remains unclear. Chronic lipopolysaccharides (LPS) stress induced depressive-behaviors were confirmed via behavioral tests. Differentially expressed genes were detected by using RNA-sequencing. The anterior cingulate cortex (ACC) tissues were collected for biochemical experiments. The Golgi-Cox staining and virus labeling were used to evaluate the dendritic spines. We applied fluoxetine (FLX) and amodiaquine dihydrochloride (AQ, a highly selective agonist of Nr4a2) in mice. Overexpression experiments were performed by injecting with AAV-Nr4a2-EGFP into ACC. Chemogenetic activation of CamkII neurons via injecting the hM3Dq virus. Mice treated with LPS displayed depressive- and anxiety-like behaviors. The reduction of Nr4a2 and FosB induced by LPS were rescued by pretreatment with FLX or AQ. More importantly, LPS-induced behavior deficits in mice were also alleviated via fluoxetine treatment and pharmacological activation the expression of Nr4a2. Meanwhile, enhancing the level of Nr4a2 could improve dendritic spines loss of neuron and morphological changes in microglia. Overexpression of Nr4a2 in ACC reversed the depressive- and anxiety-like behaviors caused by LPS administration. Activation of CamkII neurons in ACC could robustly increase the expression of Nr4a2 and improve LPS-induced behavior deficits. Our findings demonstrate that the Nr4a2 may regulate depressive-like behaviors via alleviating the impairment of morphology and function on microglia and CamkII neurons induced by chronic neuroinflammation.

Activity-Dependent Nr4a2 Induction Modulates Synaptic Expression of AMPA Receptors and Plasticity via a Ca2+/CRTC1/CREB Pathway.

Transcription factors have a pivotal role in synaptic plasticity and the associated modification of neuronal networks required for memory formation and consolidation. The nuclear receptors subfamily 4 group A (Nr4a) have emerged as possible modulators of hippocampal synaptic plasticity and cognitive functions. However, the molecular and cellular mechanisms underlying Nr4a2-mediated hippocampal synaptic plasticity are not completely known. Here, we report that neuronal activity enhances Nr4a2 expression and function in cultured mouse hippocampal neurons (both sexes) by an ionotropic glutamate receptor/Ca2+/cAMP response element-binding protein/CREB-regulated transcription factor 1 (iGluR/Ca2+/CREB/CRTC1) pathway. Nr4a2 activation mediates BDNF production and increases expression of iGluRs, thereby affecting LTD at CA3-CA1 synapses in acute mouse hippocampal slices (both sexes). Together, our results indicate that the iGluR/Ca2+/CREB/CRTC1 pathway mediates activity-dependent expression of Nr4a2, which is involved in glutamatergic synaptic plasticity by increasing BDNF and synaptic GluA1-AMPARs. Therefore, Nr4a2 activation could be a therapeutic approach for brain disorders associated with dysregulated synaptic plasticity.SIGNIFICANCE STATEMENT A major factor that regulates fast excitatory synaptic transmission and plasticity is the modulation of synaptic AMPARs. However, despite decades of research, the underlying mechanisms of this modulation remain poorly understood. Our study identified a molecular pathway that links neuronal activity with AMPAR modulation and hippocampal synaptic plasticity through the activation of Nr4a2, a member of the nuclear receptor subfamily 4. Since several compounds have been described to activate Nr4a2, our study not only provides mechanistic insights into the molecular pathways related to hippocampal synaptic plasticity and learning, but also identifies Nr4a2 as a potential therapeutic target for pathologic conditions associated with dysregulation of glutamatergic synaptic function.

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Moxonidine ameliorates cardiac injury in rats with metabolic syndrome by regulating autophagy.

AIMS: Reduced cardiac autophagy, ischemic injury, sympathetic overactivity, and apoptosis all contribute to metabolic syndrome (MetS)-associated cardiovascular risks. NR4A2, an orphan nuclear receptor NR4A family member, induces autophagy while suppressing apoptosis in myocardial infarction. Moxonidine, a sympathoinhibitor imidazoline1 receptor (I1R) agonist, has beneficial metabolic and hemodynamic effects; however, whether autophagy and/or NR4A2 signaling are involved in moxonidine's cardiovascular effects via I1R activation, is unknown, and is the aim of this study. MATERIALS AND METHODS: To induce MetS, rats were fed 3 % salt in their diet and 10 % fructose in their drinking water for 12 weeks. MetS-rats were given either moxonidine (6 mg/kg/day, gavage), efaroxan (I1R antagonist, 0.6 mg/kg/day, i.p), both treatments, or vehicles for the last two weeks. Blood pressure, lipid profile, and glycemic control were evaluated. Histopathological examination, circulating cardiac troponin I (c-TnI), proinflammatory interleukin-6 (IL-6), apoptosis (active caspase-3 and Fas-immunostaining), interstitial fibrosis [transforming growth factor-ß1 (TGF-ß1), Mallory's trichrome staining], and extracellular matrix remodeling [matrix metalloproteinase-9 (MMP-9)], were used to assess cardiac pathology. Cardiac NR4A2 and its downstream factor, p53, as well as autophagic flux markers, SQSTM1/p62, LC3, and Beclin-1 were also determined. KEY FINDINGS: Moxonidine significantly ameliorated MetS-induced metabolic and hemodynamic derangements and the associated cardiac pathology. Moxonidine restored NR4A2 and p53 myocardial levels and enhanced autophagic flux via modulating SQSTM1/p62, LC3, and Beclin-1. Efaroxan reversed the majority of the moxonidine-induced improvements. SIGNIFICANCE: The current study suggests that autophagy modulation via I1R activation is involved in moxonidine-mediated cardiac beneficial effects in MetS.

Temporal origin of mouse claustrum and development of its cortical projections.

The claustrum is known for its extensive connectivity with many other forebrain regions, but its elongated shape and deep location have made further study difficult. We have sought to understand when mouse claustrum neurons are born, where they are located in developing brains, and when they develop their widespread connections to the cortex. We established that a well-characterized parvalbumin plexus, which identifies the claustrum in adults, is only present from postnatal day (P) 21. A myeloarchitectonic outline of the claustrum can be derived from a triangular fiber arrangement from P15. A dense patch of Nurr1+ cells is present at its core and is already evident at birth. Bromodeoxyuridine birth dating of forebrain progenitors reveals that the majority of claustrum neurons are born during a narrow time window centered on embryonic day 12.5, which is later than the adjacent subplate and endopiriform nucleus. Retrograde tracing revealed that claustrum projections to anterior cingulate (ACA) and retrosplenial cortex (RSP) follow distinct developmental trajectories. Claustrum-ACA connectivity matures rapidly and reaches adult-like innervation density by P10, whereas claustrum-RSP innervation emerges later over a protracted time window. This work establishes the timeline of claustrum development and provides a framework for understanding how the claustrum is built and develops its unique connectivity.

Overexpression of NR4A2 alleviates renal and myocardial injury in diabetes nephropathy rats through the HDAC11/SPRY1 pathway.

BACKGROUND: Diabetic nephropathy (DN) remains the most prevalent cause of end-stage renal disease. Nuclear receptor subfamily 4 group A member 2 (NR4A2) is a nuclear receptor with unique physiological characteristics. OBJECTIVE: This study explored the molecular mechanism of NR4A2 in renal and cardiac functions of DN rats. METHODS: A rat model of DN was established by intraperitoneal injection of streptozocin. NR4A2, histone deacetylase 11 (HDAC11), and sprouty 1 (SPRY1) expressions were detected. The fasting blood glucose (FBG), urinary albumin (UAlb), serum creatinine (Cr), and blood urea nitrogen (BUN) were determined. The pathological injury of renal and myocardial tissues was evaluated. The mitral early to late diastolic flow velocity ratio (E/A ratio), left ventricular ejection fraction (LVEF), left ventricular systolic function (LVSF), left ventricular internal dimension systole (LVIDs), and left ventricular internal diameter diastole (LVIDd) were tested, and the levels of serum cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) were examined. The enrichment of NR4A2 in HDAC11 promoter and enrichment of H3K27 acetylation in SPRY1 promoter were measured. RESULTS: NR4A2 and SPRY1 were downregulated while HDAC11 was upregulated in renal and myocardial tissues of DN rats. NR4A2 overexpression reduced FBG, UAlb, Cr, and BUN, alleviated pathological injury of renal and myocardial tissues, elevated the E/A ratio, LVEF, and LVFS, but reduced LVIDs, and decreased serum cTnI and CK-MB. NR4A2 depressed HDAC11 expression by binding to the HDAC11 promoter. HDAC11 repressed SPRY1 transcription by suppressing the H3K27ac level. HDAC11 overexpression or SPRY1 inhibition reversed the alleviating effect of NR4A2 overexpression on DN rats. CONCLUSION: NR4A2 was poorly expressed in DN rats. NR4A2 overexpression suppressed HDAC11 expression by binding to the HDAC11 promoter and enhanced SPRY1 transcription by enhancing H3K27 acetylation, thereby alleviating the renal and myocardial injury of DN rats.

Neuropilin-1 (NRP1) expression distinguishes self-reactive helper T cells in systemic autoimmune disease.

Pathogenic T helper cells (Th cells) that respond to self-antigen cannot be easily distinguished from beneficial Th cells. These cells can generate systemic autoimmune disease in response to widely expressed self-antigens. In this study, we have identified neuropilin-1 (NRP1) as a cell surface marker of self-reactive Th cells. NRP1+ Th cells, absent in non-regulatory T cell subsets in normal mice, appeared in models of systemic autoimmune disease and strongly correlated with disease symptoms. NRP1+ Th cells were greatly reduced in Nr4a2 cKO mice, which have reduced self-reactive responses but showed normal responses against exogenous antigens. Transfer of NRP1+ Th cells was sufficient to initiate or accelerate systemic autoimmune disease, and targeting NRP1-expressing Th cells therapeutically ameliorated SLE-like autoimmune symptoms in BXSB-Yaa mice. Peripheral NRP1+ Th cells were significantly increased in human SLE patients. Our data suggest that self-reactive Th cells can be phenotypically distinguished within the Th cell pool. These findings offer a novel approach to identify self-reactive Th cells and target them to treat systemic autoimmune disease.

Two novel heterozygous truncating variants in NR4A2 identified in patients with neurodevelopmental disorder and brief literature review.

Pathogenic variants in the nuclear receptor superfamily 4 group A member 2 (NR4A2) cause an autosomal dominant neurodevelopmental disorder with or without seizures. Here, we described two patients presenting with developmental delay, language impairment, and attention-deficit hyperactivity disorder. Trio-based whole exome sequencing revealed two novel heterozygous variants, c.1541-2A > C and c.915C > A, in NR4A2. Both variants were identified as de novo and confirmed by Sanger sequencing. In vitro functional analyses were performed to assess their effects on expression of mRNA or protein. The canonical splicing variant c.1541-2A > C caused aberrant splicing, leading to the retention of intron 7 and a truncated protein due to an early termination codon within intron 7 with decreased protein expression, while the variant c.915C > A was shown to result in a shorter protein with increased expression level unexpectedly. The clinical and genetic characteristics of the previously published patients were briefly reviewed for highlighting the potential link between mutations and phenotypes. Our research further confirms that NR4A2 is a disease-causing gene of neurodevelopmental disorders and suggests alterations in different domains of NR4A2 cause various severity of symptoms.