Phenotype Of Mice Research Articles

Inhaled xenon modulates microglia and ameliorates disease in mouse models of amyloidosis and tauopathy

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. Antiamyloid antibody treatments modestly slow disease progression in mild dementia due to AD. Emerging evidence shows that homeostatic dysregulation of the brain immune system, especially that orchestrated by microglia, plays an important role in disease onset and progression. Thus, a major question is how to modulate the phenotype and function of microglia to treat AD. Xenon (Xe) gas is a noble gas used in human patients as an anesthetic and a neuroprotectant used for treating brain injuries. Xe penetrates the blood-brain barrier, which could make it an effective therapeutic. To assess the effect of Xe on microglia and AD pathology, we designed a custom Xe inhalation chamber and treated several mouse models of AD with Xe gas. Xe treatment induced mouse microglia to adopt an intermediate activation state that we have termed pre–neurodegenerative microglia (pre-MGnD). This microglial phenotypic transition was observed in mouse models of acute neurodegeneration and amyloidosis (APP/PS1 and 5xFAD mice) and tauopathy (P301S mice). This microglial state enhanced amyloid plaque compaction and reduced dystrophic neurites in the APP/PS1 and 5xFAD mouse models. Moreover, Xe inhalation reduced brain atrophy and neuroinflammation and improved nest-building behavior in P301S mice. Mechanistically, Xe inhalation induced homeostatic brain microglia toward a pre-MGnD state through IFN-γ signaling that maintained the microglial phagocytic response in APP/PS1 and 5xFAD mice while suppressing the microglial proinflammatory phenotype in P301S mice. These results support the translation of Xe inhalation as an approach for treating AD.

Inhibition of IFITM3 in cerebrovascular endothelium alleviates Alzheimer's-related phenotypes.

Interferon-induced transmembrane protein 3 (IFITM3) modulates γ-secretase in Alzheimer's Disease (AD). Although IFITM3 knockout reduces amyloid β protein (Aβ) production, its cell-specific effect on AD remains unclear. Single nucleus RNA sequencing (snRNA-seq) was used to assess IFITM3 expression. Adeno-associated virus-BI30 (AAV-BI30) was injected to reduce IFITM3 expression in the cerebrovascular endothelial cells (CVECs). The effects on AD phenotypes in cells and AD mice were examined through behavioral tests, two-photon imaging, flow cytometry, Western blot, immunohistochemistry, and quantitative polymerase chain reaction assay (qPCR). IFITM3 expression was increased in the CVECs of patients with AD. Overexpression of IFITM3 in primary endothelial cells enhanced Aβ generation through regulating beta-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Aβ further increased IFITM3 expression, creating a vicious cycle. Knockdown of IFITM3 in CVECs decreased Aβ accumulation within cerebrovascular walls, reduced Alzheimer's-related pathology, and improved cognitive performance in AD transgenic mice. Knockdown of IFITM3 in CVECs alleviates AD pathology and cognitive impairment. Targeting cerebrovascular endothelial IFITM3 holds promise for AD treatment. Interferon-induced transmembrane protein 3 (IFITM3) expression was increased in the cerebrovascular endothelial cells (CVECs) of patients with Alzheimer's Disease (AD). Cerebrovascular endothelial IFITM3 regulates amyloid β protein (Aβ) generation through regulating beta-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Knockdown of IFITM3 in CVECs reduces Aβ deposits and improves cognitive impairments in AD transgenic mice. Cerebrovascular endothelial IFITM3 could be a potential target for the treatment of AD.

Mining functional gene modules by multi-view NMF of phenome-genome association

BackgroundMining functional gene modules from genomic data is an important step to detect gene members of pathways or other relations such as protein-protein interactions. This work explores the plausibility of detecting functional gene modules by factorizing gene-phenotype association matrix from the phenotype ontology data rather than the conventionally used gene expression data. Recently, the hierarchical structure of phenotype ontologies has not been sufficiently utilized in gene clustering while functionally related genes are consistently associated with phenotypes on the same path in phenotype ontologies.ResultsThis work demonstrates a hierarchical Nonnegative Matrix Factorization (NMF) framework, called Consistent Multi-view Nonnegative Matrix Factorization (CMNMF), which factorizes genome-phenome association matrix at consecutive levels of the hierarchical structure in phenotype ontology to mine functional gene modules. CMNMF constrains the gene clusters from the association matrices at two consecutive levels to be consistent since the genes are annotated with both the child-level phenotypes and the parent-level phenotypes in two levels. CMNMF also restricts the identified gene clusters to be densely connected in the phenotype ontology hierarchy. In the experiments on mining functionally related genes from mouse phenotype ontology and human phenotype ontology, CMNMF effectively improves clustering performance over the baseline methods. Gene ontology enrichment analysis is also conducted to verify its practical effectiveness to reveal meaningful gene modules.ConclusionsUtilizing the information in the hierarchical structure of phenotype ontology, CMNMF can identify functional gene modules with more biological significance than conventional methods. CMNMF can also be a better tool for predicting members of gene pathways and protein-protein interactions.

Tissue-Specific Ablation of Liver Fatty Acid-Binding Protein Induces a Metabolically Healthy Obese Phenotype in Female Mice.

Obesity is associated with numerous metabolic complications including insulin resistance, dyslipidemia, and a reduced capacity for physical activity. Whole-body ablation of liver fatty acid-binding protein (LFABP) in mice was shown to alleviate several of these metabolic complications; high fat (HF) fed LFABP knockout (LFABP -/- ) mice developed higher fat mass than their wild-type (WT) counterparts but displayed a metabolically healthy obese (MHO) phenotype with normoglycemia, normoinsulinemia, and reduced hepatic steatosis compared with WT. LFABP is expressed in both liver and intestine, thus in the present study, LFABP conditional knockout (cKO) mice were generated to determine the contributions of LFABP specifically within the liver or the intestine to the whole body phenotype of the global knockout. Female liver-specific LFABP knockout (LFABP liv-/- ), intestine-specific LFABP knockout (LFABP int-/- ), and floxed LFABP (LFABP fl/fl ) control mice were fed a 45% Kcal fat semipurified HF diet for 12 weeks. While not as dramatic as found for whole-body LFABP -/- mice, both LFABP liv-/- and LFABP int-/- mice had significantly higher body weights and fat mass compared with LFABP fl/fl control mice. As with the global LFABP nulls, both LFABP liv-/- and LFABP int-/- mice remained normoglycemic and normoinsulinemic. Despite their greater fat mass, the LFABP liv-/- mice did not develop hepatic steatosis. Additionally, LFABP liv-/- and LFABP int-/- mice had higher endurance exercise capacity when compared with LFABP fl/fl control mice. The results suggest, therefore, that either liver-specific or intestine-specific ablation of LFABP in female mice is sufficient to induce, at least in part, the MHO phenotype observed following whole-body ablation of LFABP.

Enhanced CB1 receptor function in GABAergic neurons mediates hyperexcitability and impaired sensory-driven synchrony of cortical circuits in Fragile X Syndrome model mice.

Electroencephalographic (EEG) recordings in individuals with Fragile X Syndrome (FXS) and the mouse model of FXS ( Fmr1 KO) display cortical hyperexcitability at rest, as well as deficits in sensory-driven cortical network synchrony. A form of circuit hyperexcitability is observed in ex vivo cortical slices of Fmr1 KO mice as prolonged persistent activity, or Up, states. It is unknown if the circuit mechanisms that cause prolonged Up states contribute to FXS-relevant EEG phenotypes. Here we examined the role of endocannabinoids (eCB) in prolonged Up states in slices and resting and sensory-driven EEG phenotypes in awake Fmr1 KO mice. Bidirectional changes in eCB function are reported in the Fmr1 KO that depend on synapse type (excitatory or inhibitory). We demonstrate that pharmacological or genetic reduction of Cannabinoid Receptor 1 (CB1R) in GABAergic neurons rescues prolonged cortical Up states and deficits in sensory-driven cortical synchrony in Fmr1 KO mice. In support of these findings, recordings from Fmr1 KO cortical Layer (L) 2/3 pyramidal neurons revealed enhanced CB1R-mediated suppression of inhibitory synaptic currents. In contrast, genetic reduction of Cnr1 in glutamatergic neurons did not affect Up state duration, but deletion of Fmr1 in the same neurons was sufficient to cause long Up states. These findings support a model where loss of Fmr1 in glutamatergic neurons leads to enhanced CB1R-mediated suppression of GABAergic synaptic transmission, prolonged cortical circuit activation and reduced sensory-driven circuit synchronization. Results suggest that antagonism of CB1Rs as a therapeutic strategy to correct sensory processing deficits in FXS.

CaMKIIα hub ligands are unable to reverse known phenotypes in Angelman syndrome mice.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of ubiquitin-protein ligase E3A (UBE3A), resulting in marked changes in synaptic plasticity. In AS mice, a dysregulation of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) was previously described. This has been convincingly validated through genetic rescue of prominent phenotypes in mouse cross-breeding experiments. Selective ligands that specifically stabilize the CaMKIIα central association (hub) domain and affect different conformational states in vitro are now available. Two of these ligands, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA), confer neuroprotection after ischemic stroke in mice where CaMKIIα is known to be dysregulated. Here, we sought to investigate whether pharmacological modulation with these prototypical CaMKIIα hub ligands presents a viable approach to alleviate AS symptoms. We performed an in vivo functional evaluation of AS mice treated for a total of 14 days with either HOCPCA or Ph-HTBA (7days pre-treatment and 7days of behavioural assessment). Both compounds were well-tolerated but unable to revert robust phenotypes of motor performance, anxiety, repetitive behaviour or seizures in AS mice. Biochemical experiments subsequently assessed CaMKIIα autophosphorylation in AS mouse brain tissue. Taken together our results indicate that pharmacological modulation of CaMKIIα via the selective hub ligands used here is not a viable treatment strategy in AS.

Neonatal infection with Bordetella pertussis promotes autism-like phenotypes in mice

Neonatal infection with Bordetella pertussis promotes autism-like phenotypes in mice

Shank3 Overexpression Leads to Cardiac Dysfunction in Mice by Disrupting Calcium Homeostasis in Cardiomyocytes.

SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca²⁺ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca²⁺ homeostasis was assessed by analyzing cytosolic Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometry-based identification was employed to identify proteins in the cardiac Shank3 interactome. Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca²⁺ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca²⁺ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca²⁺ homeostasis and contraction, with a notable reduction in troponin I.

LGR4 is essential for maintaining β-cell homeostasis through suppression of RANK.

Loss of functional β-cell mass is a major cause of diabetes. Thus, identifying regulators of β-cell health is crucial for treating this disease. The Leucine-rich repeat-containing G-protein-coupled receptor (GPCR) 4 (LGR4) is expressed in β-cells and is the fourth most abundant GPCR in human islets. Although LGR4 has regenerative, anti-inflammatory, and anti-apoptotic effects in other tissues, its functional significance in β-cells remains unknown. We have previously identified Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) as a negative regulator of β-cell health. In this study, we assessed the regulation of Lgr4 in islets, and the role of LGR4 and LGR4/RANK stoichiometry in β-cell health under basal and stress-induced conditions, invitro and invivo. We evaluated Lgr4 expression in mouse and human islets in response to acute (proinflammatory cytokines), or chronic (high fat fed mice, db/db mice, and aging) stress. To determine the role of LGR4 we employed invitro Lgr4 loss and gain of function in primary rodent and human β-cells and examined its mechanism of action in the rodent INS1 cell line. Using Lgr4fl/fl and Lgr4fl/fl/Rankfl/fl×Ins1-Cre mice we generated β-cell-specific conditional knockout (cko) mice to test the role of LGR4 and its interaction with RANK invivo under basal and stress-induced conditions. Lgr4 expression in rodent and human islets was reduced by multiple stressors. Invitro, Lgr4 knockdown decreased proliferation and survival in rodent β-cells, while overexpression protected against cytokine-induced cell death in rodent and human β-cells. Mechanistically, LGR4 protects β-cells by suppressing RANK- Tumor necrosis factor receptor associated factor 6 (TRAF6) interaction and subsequent activation of NFκB. Lgr4cko mice exhibit normal glucose homeostasis but increased β-cell death in both sexes and decreased β-cell proliferation and maturation only in females. Male Lgr4cko mice under stress displayed reduced β-cell proliferation and a further increase in β-cell death. The impaired β-cell phenotype in Lgr4cko mice was rescued in Lgr4/Rank double ko (dko) mice. Upon aging, both male and female Lgr4cko mice displayed impaired β-cell homeostasis, however, only female mice became glucose intolerant with decreased plasma insulin. These data demonstrate a novel role for LGR4 as a positive regulator of β-cell health under basal and stress-induced conditions, through suppressing the negative effects of RANK.

A rich component of Fructus Aurantii, meranzin hydrate, exerts antidepressant effects via suppressing caspase4 to regulate glial cell and neuronal functions in the hippocampus.

Fructus Aurantii, a Chinese herbal medicine, has been indicated to have antidepressant effects in our previous study. However, the main component and specific mechanisms of the antidepressant effects of Fructus Aurantii still need to be further revealed. This study aimed to explore the main antidepressant component of Fructus Aurantii and the underlying mechanisms of its antidepressant effects in the hippocampus. The results showed that the component of meranzin hydrate (MH) was enrichment in Fructus Aurantii. MH could alleviate depressive phenotypes in LPS-induced mice after a single administration 1 day later. High genetic and proteinic levels of caspase4 in the hippocampus in LPS-induced mice were reversed by MH after a single administration 1 day later. Moreover, MH was capable of relieving inflammatory factors (TNF-a and IL-1β) and LPS in the serum in LPS-induced mice. Subsequently, activation of hippocampal caspase4 blocked MH's antidepressant effects and its effects on suppression of microglia and improvement of astrocyte in the hippocampus. Furthermore, MH could increase long-term potential (LTP) in the hippocampal dentate gyrus (DG) and activation of hippocampal caspase4 blocked MH's enhancement on neuronal activities and synaptic plasticity in the hippocampal DG. To sum up, the antidepressant effects of a rich component MH in Fructus Aurantii suppressed the activation of caspase4 by maintaining glial cells function to promote neuronal activities and synaptic plasticity in the hippocampus.

TMC7 is required for spermiogenesis and male fertility by regulating TGN-derived vesicles.

Infertility affects 10-12 % of couples worldwide, 50 % of which are male. Abnormal spermatogenesis is among the main causes of male infertility. We were curious about the possible role of transmembrane channel-like protein 7 (TMC7) in spermatogenesis because of its aberrant expression in several male infertility patients. In this study, we found that deletion of Tmc7, which is highly expressed during spermiogenesis, causes a human oligoasthenoteratozoospermia (OAT)-like phenotype in male mice. By histological analysis, TEM, RNA-seq and library-free data-independent acquisition mass spectrometry (DIA-MS) of TMC7-null mouse testes, we found that Tmc7 deletion caused abnormal swelling of trans-Golgi network (TGN) vesicles in elongated spermatids. Further immunofluorescence localization analysis revealed that these vesicles were defined by synaptophysin-like 1 (SYPL1). In addition, TMC7 may act as a potential chloride transport channel to regulate the size of transport vesicles. In conclusion, this study demonstrated that TMC7 is essential for male fertility and may be used as a potential protein for the identification and recognition of OAT. On the other hand, TMC7 may be a potential male contraceptive target.

Effect of 2-Aminoethoxydiphenyl Borate on the State of Skeletal Muscles in Dystrophin-Deficient mdx Mice.

Ca2+ overload of muscle fibers is one of the factors that secondarily aggravate the development of Duchenne muscular dystrophy (DMD). The purpose of this study is to evaluate the effects of the Ca2+ channel modulator 2-aminoethoxydiphenyl borate (APB) on skeletal muscle pathology in dystrophin-deficient mdx mice. Mice were randomly divided into six groups: wild type (WT), WT+3 mg/kg APB, WT+10 mg/kg APB, mdx, mdx+3 mg/kg APB, mdx+10 mg/kg APB. APB was administered intraperitoneally daily for 28 days. Finally, we assessed the grip strength and hanging time of mice, the histology and ultrastructure of the quadriceps, as well as the parameters reflecting quadricep mitochondrial function. 3 mg/kg APB was shown to reduce creatine kinase activity in the serum, intensity of degeneration and the level of fibrosis in the quadriceps of mdx mice, and improved tissue ultrastructure. However, this effect of APB was not sufficient to improve grip strength and hanging time of mdx mice. The effect of 3 mg/kg APB may be due to improve Ca2+ homeostasis in skeletal muscles, as evidenced by a trend toward decreased Ca2+ overload of quadricep mitochondria. High dose of APB (10 mg/kg body weight) showed less pronounced effect on the pathological phenotype of mdx mice. Moreover, 10 mg/kg APB disrupted the ultrastructure of the quadriceps and caused a decrease in grip strength in WT mice. APB is able to improve the phenotype in mdx mouse DMD model. However, the effect of APB is quite limited, which may be due to its multitargeting of Ca2+ channels in the membranes of muscle fibers and intracellular organelles, differentially expressed in DMD.

Microglia‐Derived Vitamin D Binding Protein Mediates Synaptic Damage and Induces Depression by Binding to the Neuronal Receptor Megalin

AbstractVitamin D binding protein (VDBP) is a potential biomarker of major depressive disorder (MDD). This study demonstrates for the first time that VDBP is highly expressed in core emotion‐related brain regions of mice susceptible to chronic unpredictable mild stress (CUMS). Specifically, the overexpression of microglia (MG)‐derived VDBP in the prelimbic leads to depression‐like behavior and aggravates CUMS‐induced depressive phenotypes in mice, whereas conditional knockout of MG‐derived VDBP can reverse both neuronal damage and depression‐like behaviors. Mechanistically, the binding of MG‐derived VDBP with the neuronal receptor megalin mediates the downstream SRC signaling pathway, leading to neuronal and synaptic damage and depression‐like behaviors. These events may be caused by biased activation of inhibitory neurons and excitatory–inhibitory imbalance. Importantly, this study has effectively identified MG‐derived VDBP as a pivotal mediator in the interplay between microglia and neurons via its interaction with the neuronal receptor megalin and intricate downstream impacts on neuronal functions, thus offering a promising therapeutic target for MDD.

Regulatory Roles of Quercetin in Alleviating Fructose-Induced Hepatic Steatosis: Targeting Gut Microbiota and Inflammatory Metabolites.

While fructose is a key dietary component, concerns have been raised about its potential risks to the liver. This study aimed to assess quercetin's protective effects against fructose-induced mouse hepatic steatosis. Thirty-two male C57BL/6J mice were randomly allocated into four groups: control, high fructose diet (HFrD), HFrD supplemented with low-dose quercetin (HFrD+LQ), and HFrD supplemented with high-dose quercetin (HFrD+HQ). Biochemical, pathological, immune, and metabolic parameters were assessed. Quercetin treatment significantly reduced liver fat percentages in mice on a high fructose diet, with the most notable reduction observed in the HFrD+HQ group. Histological examination confirmed this reduction, revealing diminished lipid droplets and decreased inflammation and steatosis in hepatocytes. Compared to the high fructose group, interleukin-1 β and tumor necrosis factor alpha were significantly decreased, serum aspartate aminotransferase concentrations were markedly reduced, and blood high-density lipoprotein concentrations were substantially elevated after quercetin intervention (p < 0.05). Total bilirubin and triglyceride levels, which were significantly altered following high fructose intervention and reversed after quercetin intervention. Following the administration of 100 mg/kg quercetin, the Firmicutes/Bacteroidetes ratio was significantly reduced compared to the high fructose group. At the genus level, Erysipelotrichaceae_uncultured, Faecalibaculum, Odoribacter, and Allobaculum were significantly decreased (p < 0.05), Lacnospiraceae NK4A136 group, Parabacteroides, and Alloprevotella significantly increased (p < 0.05). However, the 50 mg/kg quercetin treatment only decreased the abundance of Erysipelotrichaceae_uncultured (p < 0.05). In addition, quercetin significantly enhanced the content of propionic acid and total acid (p < 0.05). Moreover, the intestinal flora showed a significant correlation with the hepatic health-related phenotype in mice. Both 50 and 100 mg/kg quercetin treatments significantly mitigated liver fat deposition in mice with fructose-induced hepatic steatosis. However, the higher dose of quercetin (100 mg/kg) demonstrated a more pronounced effect in reducing liver inflammation, likely due to its impact on gut microbiota regulation. This suggests quercetin's potential as a therapeutic agent for fructose-related hepatic steatosis, emphasizing the importance of dose considerations.

Negative feedback between PTH1R and IGF1 through the Hedgehog pathway in mediating craniofacial bone remodeling.

Regeneration of orofacial bone defects caused by inflammatory-related diseases or trauma remains an unmet challenge. Parathyroid hormone 1 receptor (PTH1R) signaling is a key mediator of bone remodeling whereas the regulatory mechanisms of PTH1R signaling in oral bone under homeostatic or inflammatory conditions have not been demonstrated by direct genetic evidence. Here we observed that deletion of PTH1R in Gli1+-progenitors led to increased osteogenesis and osteoclastogenesis. Single-cell and bulk RNA-seq analysis revealed that PTH1R suppresses the osteogenic potential of Gli1+-progenitors during inflammation. Moreover, we identified upregulated IGF1 expression upon PTH1R deletion. Dual deletion of IGF1 and PTH1R ameliorated the bone remodeling phenotypes in PTH1R-defienct mice. Furthermore, in vivo evidence revealed an inverse relationship between PTH1R and Hedgehog signaling, which was responsible for the upregulated IGF1 production. Our work underscored the negative feedback between PTH1R and IGF1 in craniofacial bone turnover, and revealed mechanisms modulating orofacial bone remodeling.

Physiological significance of antithrombin D-helix interaction with vascular GAGs.

Antithrombin (AT) is an anticoagulant serpin involved in the regulation of proteolytic activities of coagulation proteases. AT also possesses a direct anti-inflammatory function. The anticoagulant function of AT is mediated through its reactive-center loop (RCL)-dependent inhibition of coagulation proteases, but anti-inflammatory function of AT is mediated via its D-helix-dependent interaction with vascular glycosaminoglycans (GAGs). In vitro assays have established that therapeutic heparins promote the anticoagulant function of AT by binding D-helix and activating the serpin, however, the contribution of vascular GAGs to D-helix-dependent anticoagulant function of AT has remained poorly understood in vivo. Here, we explored this question by utilizing two AT mutants, (AT-4Mut) which exhibits neither affinity for heparin nor D-helix-dependent anti-inflammatory signaling but possesses normal protease-inhibitory function and an inactive signaling-selective AT mutant in which its P1-Arg425 is deleted (AT-R425del). In vivo properties of mutants were compared to wildtype AT (AT-WT) in an siRNA-mediated AT-deficient mouse model. The siRNA knockdown efficiently reduced expression of AT and induced robust procoagulant and pro-inflammatory phenotypes in mice. Infusion of both AT-WT and AT-4Mut rescued the procoagulant phenotype of AT-deficient mice as evidenced by restoration of the plasma clotting time and inhibition of fibrin deposition. AT-WT also attenuated inflammation as evidenced by reduced VCAM-1 expression and leukocyte infiltration in the liver and lungs; however, AT-4Mut failed to attenuate inflammation. Interestingly, AT-R425del also effectively attenuated inflammation in AT-depleted mice. These results suggest interaction of AT D-helix with vascular GAGs may primarily be responsible for anti-inflammatory signaling rather than protease-inhibitory function of the serpin.

Anxio-depressive phenotype and impaired memory in mice with a conditional knockout of brain-derived neurotrophic factor in endothelial cells.

The present study investigated the role of endothelial brain-derived neurotrophic factor (BDNF) in cognition. Male adult mice with a selective knockout of BDNF in endothelial cells (BDNFECKO) and their wild-type (WT) littermates were subjected to tests for detection of anxiety- and depression-like behaviors and impaired recognition memory. Neuronal activity and synaptogenesis were assessed from hippocampal levels of c-fos and synaptophysin, respectively, and cerebral capillary density from forebrain levels of CD31. BDNF/TrkB (tropomyosin-related kinase type B) receptor signaling was investigated through hippocampal levels of BDNF and activated TrkB receptors coupled with their immunolabeling by neurons and endothelial cells from both cerebrovascular fractions enriched in capillaries and hippocampal arterioles. Endothelial nitric oxide (NO) production was assessed from the expression of endothelial NO synthase phosphorylated at serine 1177. BDNFECKO mice exhibited anxio-depressive phenotype, impaired memory, and reduced synaptogenesis. Neither neuronal activity, neuronal BDNF/TrkB signaling, nor capillary density differed between BDNFECKO and WT mice. However, endothelial-activated TrkB receptors as well as endothelial NO production and hippocampal BDNF levels were lower in BDNFECKO than those in WT mice. We conclude that endothelial BDNF is involved in cognition through mechanisms independent of neuronal BDNF/TrkB signaling and that endothelial NO might be a driver of the procognitive effect of endothelial BDNF.NEW & NOTEWORTHY The study provides the proof of concept that endothelial brain-derived neurotrophic factor (BDNF) plays a crucial role in postnatal synaptogenesis and development of behavior/memory. It also shows that neuronal tropomyosin-related kinase type B (TrkB) receptors are not a target of endothelium-derived BDNF.

Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease.

Angiopoietin-like 4 (ANGPTL4), a key protein involved in lipoprotein metabolism, has diverse effects. There is an association between Angptl4 and diabetic kidney disease; however, this association has not been well investigated. We show that both podocyte- and tubule-specific ANGPTL4 are crucial fibrogenic molecules in diabetes. Diabetes accelerates the fibrogenic phenotype in control mice but not in ANGPTL4 mutant mice. The protective effect observed in ANGPTL4 mutant mice is correlated with a reduction in stimulator of interferon genes pathway activation, expression of pro-inflammatory cytokines, reduced epithelial-to-mesenchymal transition and endothelial-to-mesenchymal transition, lessened mitochondrial damage, and increased fatty acid oxidation. Mechanistically, we demonstrate that podocyte- or tubule-secreted Angptl4 interacts with Integrin β1 and influences the association between dipeptidyl-4 with Integrin β1. We demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the kidneys and protects diabetic kidneys from proteinuria and fibrosis. Together, these data demonstrate that podocyte- and tubule-derived Angptl4 is fibrogenic in diabetic kidneys.

POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons.

Inactivating mutations in the melanocortin 4 receptor ( MC4R ) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1 ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1 AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons regulates fertility in females. In vivo , deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro , we dissect the specific action of MC4R on Kiss1 ARH vs Kiss1 AVPV/PeN neurons and show that MC4R activation excites Kiss1 ARH neurons through direct synaptic actions. In contrast, Kiss1 AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1 AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMC ARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1 ARH neurons and restricting the activation of Kiss1 AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

Lithium normalizes ASD-related neuronal, synaptic, and behavioral phenotypes in DYRK1A-knockin mice.

Dyrk1A deficiency is linked to various neurodevelopmental disorders, including developmental delays, intellectual disability (ID) and autism spectrum disorders (ASD). Haploinsufficiency of Dyrk1a in mice reportedly leads to ASD-related phenotypes. However, the key pathological mechanisms remain unclear and human DYRK1A mutations remain uncharacterized in mice. Here, we generated and studied Dyrk1a-knockin mice carrying a human ASD patient mutation (Ile48LysfsX2; Dyrk1a-I48K mice). These mice display severe microcephaly, social and cognitive deficits, dendritic shrinkage, excitatory synaptic deficits, and altered phospho-proteomic patterns enriched for multiple signaling pathways and synaptic proteins. Early chronic lithium treatment of newborn mutant mice rescues the brain volume, behavior, dendritic, synaptic, and signaling/synapse phospho-proteomic phenotypes at juvenile and adult stages. These results suggest that signaling/synaptic alterations contribute to the phenotypic alterations seen in Dyrk1a-I48K mice, and that early correction of these alterations by lithium treatment has long-lasting effects in preventing juvenile and adult-stage phenotypes.