Heterozygote Knockout Research Articles

Identification of a dengue 2 virus envelope protein receptor in Aedes aegypti critical for viral midgut infection

The establishment of a productive dengue virus (DENV) infection in the midgut epithelial cells of Aedes aegypti is critical for the viral transmission cycle. The hypothesis that DENV virions interact directly with specific mosquito midgut proteins was explored. We found that DENV serotype 2 (DENV2) pretreated with trypsin interacted with a single 31 kDa protein, identified as AAEL011180 by protein mass spectrometry. This putative receptor is a highly conserved protein and has orthologs in culicine and anopheline mosquitoes. We confirmed that impairing the expression of AAEL011180 in the midgut of Ae. aegypti females abolished the interaction with DENV2, and the virus also bound to immobilized recombinant purified receptor. Furthermore, recombinant DENV2 surface E glycoprotein bound to recombinant AAEL011180 with high affinity (38.2 nM) in binding kinetic analysis using surface plasmon resonance. The gene for this DENV2 E protein receptor (EPrRec) was disrupted, but since the gene is essential in Ae. aegypti, only heterozygote knockout (ΔEPrRec+/-) females could be recovered. Further reducing EPrRec mRNA expression in the midgut of ΔEPrRec+/- females by systemic dsRNA injection significantly reduced the prevalence of DENV2 midgut infection. EPrRec also interacts with heat shock protein 70 cognate 3 (Hsc70-3), and silencing Hsc70-3 expression in ΔEPrRec females also reduced the prevalence of DENV2 midgut infection.

Identification of Phosphodiesterase-7A (PDE7A) as a Novel Target for Reducing Ethanol Consumption in Mice.

Ethanol elicits a rapid stimulatory effect and a subsequent, prolonged sedative response, which are potential predictors of EtOH consumption by decreasing adenosine signaling; this phenomenon also reflects the obvious sex difference. cAMP (cyclic Adenosine Monophosphate)-PKA (Protein Kinase A) signaling pathway modulation can influence the stimulatory and sedative effects induced by EtOH in mice. This study's objective is to clarify the role of phosphodiesterase (PDE) in mediating the observed sex differences in EtOH responsiveness between male and female animals. EtOH was administered i.p. for 7 days to identify the changes in PDE isoforms in response to EtOH treatment. Additionally, EtOH consumption and preference of male and female C57BL/6J mice were assessed using the drinking-in-the-dark and 2-bottle choice tests. Further, pharmacological inhibition of PDE7A heterozygote knockout mice was performed to investigate its effects on EtOH-induced stimulation and sedation in both male and female mice. Finally, Western blotting analysis was performed to evaluate the alterations in cAMP-PKA/Epac2 pathways. EtOH administration resulted in an immediate upregulation in PDE7A expression in female mice, indicating a strong association between PDE7A and EtOH stimulation. Through the pharmacological inhibition of PDE7A KD mice, we have demonstrated for the first time, to our knowledge, that PDE7A selectively attenuates EtOH responsiveness and consumption exclusively in female mice, whichmay be associated with the cAMP-PKA/Epac2 pathway and downstream phosphorylation of CREB and ERK1/2. Inhibition or knockdown of PDE7A attenuates EtOH responsivenessand consumption exclusively in female mice, which is associated with alterations in the cAMP-PKA/Epac2 signaling pathways, thereby highlighting its potential as a novel therapeutic target for alcohol use disorder.

Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen-inducible conditional knockout rats

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.

Transcription factor Fli-1 impacts the expression of CXCL13 and regulates immune cell infiltration into the kidney in MRL/lpr mouse

ObjectiveFriend leukaemia virus integration 1 (Fli-1) regulates chemokine/cytokine expression and thus plays an important role in the development of lupus nephritis. Chemokine CXC ligand 13 (CXCL13) is a chemokine that...

Hydrogen sulfide mitigates skeletal muscle mitophagy‐led tissue remodeling via epigenetic regulation of the gene writer and eraser function

Ketone bodies (KB) serve as the food for mitochondrial biogenetics. Interestingly, probiotics are known to promote KB formation in the gut (especially those that belong to the Lactobacillus genus). Furthermore, Lactobacillus helps produce folate that lowers the levels of homocysteine (Hcy); a hallmark non‐proteinogenic amino acid that defines the importance of epigenetics, and its landscape. In this study, we decided to test whether hydrogen sulfide (H2S), another Hcy lowering agent regulates the epigenetic gene writer DNA methyltransferase (DNMT), eraser FTO and TET2, and thus mitigates the skeletal muscle remodeling. We treated hyperhomocysteinemic (HHcy, cystathionine beta‐synthase heterozygote knockout; CBS+/−) mice with NaHS (the H2S donor). The results suggested multi‐organ damage by HHcy in the CBS+/−mouse strain compared with WT control mice (CBS+/+). H2S treatment abrogated most of the HHcy‐induced damage. The levels of gene writer (DNMT2) and H3K9 (methylation) were higher in the CBS+/− mice, and the H2S treatment normalized their levels. More importantly, the levels of eraser FTO, TET, and associated GADD45, and MMP‐13 were decreased in the CBS+/− mice; however, H2S treatment mitigated their respective decrease. These events were associated with mitochondrial fission, i.e., an increase in DRP1, and mitophagy. Although the MMP‐2 level was lower in CBS+/− compared to WT but H2S could further lower it in the CBS+/− mice. The MMPs levels were associated with an increase in interstitial fibrosis in the CBS+/− skeletal muscle. Due to fibrosis, the femoral artery blood flow was reduced in the CBS+/− mice, and that was normalized by H2S. The bone and muscle strengths were found to be decreased in the CBS+/− mice but the H2S treatment normalized skeletal muscle strength in the CBS+/− mice. Our findings suggest that H2S mitigates the mitophagy‐led skeletal muscle remodeling via epigenetic regulation of the gene writer and eraser function.

Epigenetic Upregulation of H19 and AMPK Inhibition Concurrently Contribute to S-Adenosylhomocysteine Hydrolase Deficiency-Promoted Atherosclerotic Calcification.

S-adenosylhomocysteine (SAH) is a risk factor of cardiovascular disease; inhibition of SAH hydrolase (SAHH) results in SAH accumulation and induces endothelial dysfunction and atherosclerosis. However, the effect and mechanism of SAHH in atherosclerotic calcification is still unclear. We aimed to explore the role and mechanism of SAHH in atherosclerotic calcification. The relationship between SAHH and atherosclerotic calcification was investigated in patients with coronary atherosclerotic calcification. Different in vivo genetic models were used to examine the effect of SAHH deficiency on atherosclerotic calcification. Human aortic and murine vascular smooth muscle cells (VSMCs) were cultured to explore the underlying mechanism of SAHH on osteoblastic differentiation of VSMCs. The expression and activity of SAHH were decreased in calcified human coronary arteries and inversely associated with coronary atherosclerotic calcification severity, whereas plasma SAH and total homocysteine levels were positively associated with coronary atherosclerotic calcification severity. Heterozygote knockout of SAHH promoted atherosclerotic calcification. Specifically, VSMC-deficient but not endothelial cell-deficient or macrophage-deficient SAHH promoted atherosclerotic calcification. Mechanistically, SAHH deficiency accumulated SAH levels and induced H19-mediated Runx2 (runt-related transcription factor 2)-dependent osteoblastic differentiation of VSMCs by inhibiting DNMT3b (DNA methyltransferase 3b) and leading to hypomethylation of the H19 promoter. On the contrary, SAHH deficiency resulted in lower intracellular levels of adenosine and reduced AMPK (AMP-activated protein kinase) activation. Adenosine supplementation activated AMPK and abolished SAHH deficiency-induced expression of H19 and Runx2 and osteoblastic differentiation of VSMCs. Finally, AMPK activation by adenosine inhibited H19 expression by inducing Sirt1 (sirtuin-1)-mediated histone H3 hypoacetylation and DNMT3b-mediated hypermethylation of the H19 promoter in SAHH deficiency VSMCs. We have confirmed a novel correlation between SAHH deficiency and atherosclerotic calcification and clarified a new mechanism that epigenetic upregulation of H19 and AMPK inhibition concurrently contribute to SAHH deficiency-promoted Runx2-dependent atherosclerotic calcification.

A partial reduction of Drp1 improves cognitive behavior and enhances mitophagy, autophagy and dendritic spines in a transgenic Tau mouse model of Alzheimer disease.

The purpose of our study is to understand the impact of a partial dynamin-related protein 1 (Drp1) on cognitive behavior, mitophagy, autophagy and mitochondrial and synaptic activities in transgenic Tau mice in Alzheimer's disease (AD). Our laboratory reported increased levels of amyloid-beta (Aβ) and phosphorylated Tau (P-Tau) and reported that abnormal interactions between Aβ and Drp1, P-Tau and Drp1 induced increased mitochondrial fragmentation and reduced fusion and synaptic activities in AD. These abnormal interactions result in the proliferation of dysfunctional mitochondria in AD neurons. Recent research on mitochondria revealed that fission protein Drp1 is largely implicated in mitochondrial dynamics in AD. To determine the impact of reduced Drp1 in AD, we recently crossed transgenic Tau mice with Drp1 heterozygote knockout (Drp1+/-) mice and generated double mutant (P301LDrp1+/-) mice. In the current study, we assessed the cognitive behavior, mRNA and protein levels of mitophagy, autophagy, mitochondrial biogenesis, dynamics and synaptic genes, mitochondrial morphology and mitochondrial function and dendritic spines in Tau mice relative to double mutant mice. When compared with Tau mice, double mutant mice did better on the Morris Maze (reduced latency to find hidden platform, increased swimming speed and time spent on quadrant) and rotarod (stayed a longer period of time) tests. Both mRNA- and protein-level autophagy, mitophagy, mitochondrial biogenesis and synaptic proteins were increased in double mutant mice compared with Tau (P301L) mice. Dendritic spines were significantly increased; mitochondrial number was reduced and length was increased in double mutant mice. Based on these observations, we conclude that reduced Drp1 is beneficial in a symptomatic-transgenic Tau (P301L) mice.

Enhanced Thrombotic Responses Are Associated With Striatin Deficiency and Aldosterone.

BackgroundIn addition to its role on blood pressure, aldosterone (ALDO) also affects the hemostatic system leading to increased experimental thrombosis. Striatin is an intermediate in the rapid, nongenomic actions of ALDO. Striatin heterozygote knockout (Strn +/‐ ) mice have salt sensitivity of blood pressure and mildly chronically increased ALDO levels. In addition, in humans, striatin polymorphic gene variants are associated with increased salt sensitivity of blood pressure. Thus, we hypothesized that striatin deficiency would be associated with an increased prothrombotic response.Methods and Results Strn+/ ‐ mice and wild‐type littermates were maintained on a liberal sodium diet (1.6%). We measured in vivo thrombus formation following laser‐induced injury in cremaster arterioles using intravital microscopy. Mice were randomized to intravenous administration of ALDO or its vehicle. Acutely, ALDO increased thrombotic responses in wild‐type mice (P<0.01) versus controls within minutes as determined by increased platelet accumulation and fibrin deposition at the site of laser injury. We then compared thrombus formation without ALDO administration in Strn+/‐ and wild‐type mice. Strn+/‐ mice showed highly significant increases in laser‐induced thrombosis (P<0.001), as shown by increased platelet accumulation and fibrin deposition. Interestingly, the response in the Strn+/‐ mice basally was far greater than the wild‐type mice with ALDO administration, and ALDO administration produced no additional effect on thrombus responses in Strn+/‐ mice.ConclusionsThese results demonstrate a novel protective role of striatin in experimental thrombosis. Such a protective effect may be reduced in human striatin risk allele carriers, given the similar salt sensitivity of blood pressure in these individuals and Strn+/‐ mice.

Role of endothelial microRNA 155 on capillary leakage in systemic inflammation

BackgroundCapillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage.MethodsSmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance.ResultsEndothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects.ConclusionsWe found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.

Enriched environment ameliorates adult hippocampal neurogenesis deficits in Tcf4 haploinsufficient mice

BackgroundTranscription factor 4 (TCF4) has been linked to human neurodevelopmental disorders such as intellectual disability, Pitt-Hopkins Syndrome (PTHS), autism, and schizophrenia. Recent work demonstrated that TCF4 participates in the control of a wide range of neurodevelopmental processes in mammalian nervous system development including neural precursor proliferation, timing of differentiation, migration, dendritogenesis and synapse formation. TCF4 is highly expressed in the adult hippocampal dentate gyrus – one of the few brain regions where neural stem / progenitor cells generate new functional neurons throughout life.ResultsWe here investigated whether TCF4 haploinsufficiency, which in humans causes non-syndromic forms of intellectual disability and PTHS, affects adult hippocampal neurogenesis, a process that is essential for hippocampal plasticity in rodents and potentially in humans. Young adult Tcf4 heterozygote knockout mice showed a major reduction in the level of adult hippocampal neurogenesis, which was at least in part caused by lower stem/progenitor cell numbers and impaired maturation and survival of adult-generated neurons. Interestingly, housing in an enriched environment was sufficient to enhance maturation and survival of new neurons and to substantially augment neurogenesis levels in Tcf4 heterozygote knockout mice.ConclusionThe present findings indicate that haploinsufficiency for the intellectual disability- and PTHS-linked transcription factor TCF4 not only affects embryonic neurodevelopment but impedes neurogenesis in the hippocampus of adult mice. These findings suggest that TCF4 haploinsufficiency may have a negative impact on hippocampal function throughout adulthood by impeding hippocampal neurogenesis.

Adipocyte-specific deletion of Tcf7l2 induces dysregulated lipid metabolism and impairs glucose tolerance in mice

Aims/hypothesisTranscription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/β-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues.MethodsTcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays.ResultsReduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p < 0.0001, respectively). Circulating levels of plasma NEFA and fatty acid binding protein 4 were increased by 1.3 ± 0.1-fold and 1.8 ± 0.3-fold vs control mice (p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02).Conclusions/interpretationLoss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk.Graphical abstract

BAG3P215L/KO Mice as a Model of BAG3P209L Myofibrillar Myopathy

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

Dysbiotic 1-carbon metabolism in cardiac muscle remodeling.

Unless there is a genetic defect/mutation/deletion in a gene, the causation of a given disease is chronic dysregulation of gut metabolism. Most of the time, if not always, starts within the gut; that is what we eat. Recent research shows that the imbalance between good versus bad microbial population, especially in the gut, causes systemic diseases. Thus, an appropriate balance of the gut microbiota (eubiosis over dysbiosis) needs to be maintained for normal health (Veeranki and Tyagi, 2017, Journal of Cellular Physiology, 232, 2929-2930). However, during various diseases such as metabolic syndrome, inflammatory bowel disease, diabetes, obesity, and hypertension the dysbiotic gut environment tends to prevail. Our research focuses on homocysteine (Hcy) metabolism that occupies a center-stage in many biochemically relevant epigenetic mechanisms. For example, dysbiotic bacteria methylate promoters to inhibit gene activities. Interestingly, the product of the 1-carbon metabolism is Hcy, unequivocally. Emerging studies show that host resistance to various antibiotics occurs due to inverton promoter inhibition, presumably because of promoter methylation. This results from modification of host promoters by bacterial products leading to loss of host's ability to drug compatibility and system sensitivity. In this study, we focus on the role of high methionine diet (HMD), an ingredient rich in red meat and measure the effects of a probiotic on cardiac muscle remodeling and its functions. We employed wild type (WT) and cystathionine beta-synthase heterozygote knockout (CBS+/- ) mice with and without HMD and with and without a probiotic; PB (Lactobacillus) in drinking water for 16 weeks. Results indicate that matrix metalloproteinase-2 (MMP-2) activity was robust in CBS+/- fed with HMD and that it was successfully attenuated by the PB treatment. Cardiomyocyte contractility and ECHO data revealed mitigation of the cardiac dysfunction in CBS+/- + HMD mice treated with PB. In conclusion, our data suggest that probiotics can potentially reverse the Hcy-meditated cardiac dysfunction.

Probiotics Ameliorate Gut‐Microbial Dysbiosis, Intestinal Permeability, Systemic Inflammation, and Skeletal Muscle Dysfunction in Cystathionine‐β‐synthase‐Deficient Mice

Recent studies have reported that dietary supplementation of probiotics is a novel approach for enhancing skeletal development and metabolism in both humans and animals. However, the precise role of probiotics in regulating skeletal muscle mass at the cellular and molecular levels in mice still needs to be clarified. Here we show that altered gut‐microbiota eubiosis, systemic inflammation and skeletal muscle dysfunction caused by hydrogen sulfide deficiency and whether the intragastric administration of probiotics (GSL#5) could retard H2S deficiency‐related skeletal muscle defects. To test this hypothesis, we employed 8‐week‐old female wild‐type (WT), cystathionine‐beta‐synthase heterozygote knockout (CBS+/−), and CBS+/− mice supplemented with probiotics (GSL#5) at the dosage of 1×109 CFU every other day through oral gavage for 6 weeks. In the murine model, H2S deficiency (CBS+/− mice) increased gut permeability, and upregulated the inflammatory cytokines TNFα (TNF), and IL‐17 in the blood and the skeletal muscle. Further, the gut microbiota is significantly altered in CBS‐deficient mice as assessed by the Denaturing gradient gel electrophoresis (DGGE) analysis. We documented that long non‐coding RNA‐PVT1 (lncRNA‐PVT1) is, indeed, upregulated in the soleus muscles of CBS‐deficient mice (4.2 fold), as assessed by RT2 lncRNA qPCR Assay. The mechanistic study shows that depressed H2S signaling stimulates the transcription factor C/EBP‐α binding to the promoter of lncRNA‐PVT1 and promotes the expression of lncRNA‐PVT1. Using an electrophoretic mobility shift assay and chromatin immunoprecipitations, we identified that lncRNA‐PVT1 interacts with TNFAIP3 interacting protein 1, TNIP1, leading to the acetylation of NF‐κB (P65 subunit) activity in the soleus muscle of CBS‐deficient mice. Further, activation of P65 region of NF‐κB transcriptionally upregulates atrogin‐1, MuRF‐1 expression. Overexpression of lncRNA‐PVT1 in cultured C2C12 muscle cell lines stimulates an increase in MuRF‐1 (2.3‐fold) and atrogin‐1 (3.7‐fold) expression. In addition, systemic delivery of synthetic lncRNA‐PVT1 in control mice causes the myofibers to undergo atrophy. Gene knockout of lncRNA‐PVT1 in CBS‐deficient mice attenuates atrophy of the myofibers and muscle wasting. Overall, administration of probiotics in CBS‐deficient mice reverses the gut‐microbiota dysbiosis and preserves skeletal muscle mass. In conclusion, our findings provide novel evidence that the administration of probiotics could balance the gut‐microbiota homeostasis as well as skeletal muscle function by modulating systemic inflammation in H2S‐deficient miceSupport or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockout of dihydrofolate reductase in mice induces hypertension and abdominal aortic aneurysm via mitochondrial dysfunction

Hypertension and abdominal aortic aneurysm (AAA) are severe cardiovascular diseases with incompletely defined molecular mechanisms. In the current study we generated dihydrofolate reductase (DHFR) knockout mice for the first time to examine its potential contribution to the development of hypertension and AAA, as well as the underlying molecular mechanisms. Whereas the homozygote knockout mice were embryonically lethal, the heterozygote knockout mice had global reduction in DHFR protein expression and activity. Angiotensin II infusion into these animals resulted in substantially exaggerated elevation in blood pressure and development of AAA, which was accompanied by excessive eNOS uncoupling activity (featured by significantly impaired tetrahydrobiopterin and nitric oxide bioavailability), vascular remodeling (MMP2 activation, medial elastin breakdown and adventitial fibrosis) and inflammation (macrophage infiltration). Importantly, scavenging of mitochondrial reactive oxygen species with Mito-Tempo in vivo completely abrogated development of hypertension and AAA in DHFR knockout mice, indicating a novel role of mitochondria in mediating hypertension and AAA downstream of DHFR deficiency-dependent eNOS uncoupling. These data for the first time demonstrate that targeting DHFR-deficiency driven mitochondrial dysfunction may represent an innovative therapeutic option for the treatment of AAA and hypertension.

Hydrogen Sulfide Promotes Bone Homeostasis by Balancing Inflammatory Cytokine Signaling in CBS-Deficient Mice through an Epigenetic Mechanism

Previously, we have shown hyperhomocysteinemia (HHcy) to have a detrimental effect on bone remodeling, which is associated with osteoporosis. During transsulfuration, Hcy is metabolized into hydrogen sulfide (H2S), a gasotransmitter molecule known to regulate bone formation. Therefore, in the present study, we examined whether H2S ameliorates HHcy induced epigenetic and molecular alterations leading to osteoporotic bone loss. To test this mechanism, we employed cystathionine-beta-synthase heterozygote knockout mice, fed with a methionine rich diet (CBS+/− +Met), supplemented with H2S-donor NaHS for 8 weeks. Treatment with NaHS, normalizes plasma H2S, and completely prevents trabecular bone loss in CBS+/− mice. Our data showed that HHcy caused inhibition of HDAC3 activity and subsequent inflammation by imbalancing redox homeostasis. The mechanistic study revealed that inflammatory cytokines (IL-6, TNF-α) are transcriptionally activated by an acetylated lysine residue in histone (H3K27ac) of chromatin by binding to its promoter and subsequently regulating gene expression. A blockade of HDAC3 inhibition in CBS+/− mice by HDAC activator ITSA-1, led to the remodeling of histone landscapes in the genome and thereby attenuated histone acetylation-dependent inflammatory signaling. We also confirmed that RUNX2 was sulfhydrated by administration of NaHS. Collectively, restoration of H2S may provide a novel treatment for CBS-deficiency induced metabolic osteoporosis.

Cerebrovascular Hyper‐permeability and Memory Impairment after Cortical Contusion Injury during Hyperhomocysteinemia in Mice

Traumatic brain injury (TBI) is the devastating public health problem worldwide. It is accompanied with inflammation, cerebrovascular dysfunction, and memory impairment. The latter is known to be greatly affected by cellular prion protein (PrPC). Elevated blood level of Homocysteine (Hcy), called hyperhomocysteinemia (HHcy) is considered to be an independent inflammatory and high risk factor for many cerebrovascular diseases. HHcy was found in TBI patients. High level of Hcy is associated with increased levels of another inflammatory marker fibrinogen (Fg). In the present study, we tested the hypothesis that HHcy exacerbates TBI‐induced macromolecular protein extravasation resulting in enhanced Fg‐PrPC complex formation leading to reduction in short‐term memory.Cerebrovascular permeability was studied in cystathionine‐β‐synthase heterozygote knockout (CBS+/−) mice, a well‐known model of HHcy. As a control we used wild type (WT, C57BL/6J) mice. Mice were subjected to cortical contusion injury (CCI) or sham operation (sham). Cerebrovascular permeability was assessed by measuring the fluorescence intensity of Alexaflour 647‐labeled bovine serum albumin (647‐BSA) in extravascular space using an intravital fluorescence microscopy. Formation of Fg‐PrPC complex in brain sample cryosections was studied using immunohistochemistry and confocal microscopy. Short‐term memory changes were evaluated by a novel object recognition test (NORT) and Y maze spontaneous alternation and two trial recognition tests.Pial venular permeability to 647‐BSA was greater in all experimental animals with CCI compared to that in respective sham‐operated mice. However, in injured CBS+/− mice the protein leakage was greater (201±6 % of baseline) than that (166±9 % of baseline) in WT animals with CCI. Enhanced Fg‐PrPC complex formation was found in WT animals after CCI compared to that in sham mice. Increased depositions of Fg and PrPC and their complex formations were detected in CBS+/− mice compared to those in control WT group. Short‐term memory deficiency after CCI was noted in both mice groups. However, reduction in short‐term memory (difference in discrimination ratios between sham and CCI groups) defined by NORT was greater (0.4±0.05) in CBS+/− mice compared to that (0.2±0.04) in control WT mice.Thus, our study reveals a novel, additive effect of HHcy in TBI‐induced memory impairment, which can be therapeutically targeted in future.Support or Funding InformationSupported in part by NIH grant NS084823, AHA grant 17SDG33670372, and UofL SOM Basic grant 57139This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Probiotic Treatment Induces Neuroprotection in Hyperhomocysteinemia Mice after Ischemic Stroke

Hyperhomocysteinemia (HHcy) is characterized by an abnormally high level of homocysteine (Hcy) and is assumed as an independent risk factor for ischemic stroke. The severity of oxidative stress and neuronal damage are common observable facts during stroke. Studies have shown that the microbiome plays a significant role in brain functions like stress response and emotion‐driven behavior. Probiotic is the ‘good’ bacteria that are part of the microbiome, but the effect of probiotic treatment on ischemic stroke is unclear. Moreover, probiotics have been considered as an emerging source of antioxidants. Therefore, the objective of this study was to determine whether probiotic treatment can induce neuroprotection during ischemic stroke. In our study, we employed 8–10‐week‐old male/female cystathionine‐beta‐synthase heterozygote knockout (CBS+/−) mice (genetically hyperhomocystemic mice). The experimental groups were: (CBS+/−), (CBS +/− +Probiotics); (CBS+/− /I/R), and (CBS+/− /I/R+Probiotics). Mice in stroke (I/R) group were subjected to middle cerebral artery occlusion (MCAO) for 40 min, followed by reperfusion for 72 hours. Experimental mice were intragastrically treated with vehicle or the probiotics (GSL#5) for a period of 3 consecutive days after 3 hrs of stroke. We found that infarct size, brain edema, and permeability were increased in CBS+/− /I/R mice compared to control; however, probiotics treatment in ischemic mice reduces ischemic effects. The levels of NOX‐4 and MDA were significantly upregulated in CBS+/− /I/R mice when compared to controls. On the other hand, the levels of CAT, GPx, and endogenous H2S were significantly reduced in CBS+/− /I/R compared to control. Probiotic treatment ameliorated oxidative stress. Neuronal dysfunction was evaluated by measuring the levels of Neuronal nuclear antigen (Neun), Neuron‐specific‐enolase (NSE) and Fluoro‐jade C(FJC) fluorescence; while cerebrovascular disruption was evaluated by assessing levels of VE‐Cadherin and Claudin‐5 in harvested brain tissue. Cerebrovascular permeability was assessed by evaluating microvascular leakage of fluorescently‐labeled albumin in vivo. Nitric oxide synthase regulation (eNOS, nNOS) and vascular inflammation (ICAM) were also evaluated in brain tissue. Our experimental data showed that neuronal dysfunction (decreased NeuN levels and increased FJC positive neurons in the brain) was more prominent in CBS+/− /I/R mice compared to controls. In CBS+/− /I/R mice, the cerebrovascular disruption was found to be elevated as evident from increased microvascular leakage and decreased in VE‐Cadherin expression compared to controls. The decrease in nNOS and increase in eNOS suggest a trend towards the decrease in potential for neuronal development in stroke mice. In conclusion, probiotics treatment reduces oxidative stress, induces neuroprotection in hyperhomocysteinemia mice after ischemic stroke. This study suggests a potential therapeutic role of dietary probiotics in ischemia stroke.Support or Funding InformationThis work is financially supported by National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledgedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 020: Heterozygote Knockout of TMEM16B in Intestinal Vagal Afferents Causes Cholecystokinin Insensitivity and Obesity in Male but Not Female Mice

Cholecystokinin (CCK) is a well-known satiety peptide that inhibits food intake. The CCK induced satiety signal in intestinal vagal afferents is attenuated in mice on a High Fat Diet (HFD). We have shown that down regulation of a Ca 2+ -activated Cl - channel (CaCC) downstream of CCK receptors is responsible for the neuronal insensitivity to CCK. We also found that the CaCC subunit Ano2/TMEM16B is essential for the CCK-induced current in nodose neurons. In this study we tested the hypothesis that reduction of this subunit in vivo contributes to weight gain. One allele of the Ano2/TMEM16B was knocked out in sensory neurons by crossing the ano2 fl/ fl mice with Nav1.8Cre mice to generate the Nav1.8Cre;ano2 fl /wt mice. The Cre negative littermates were used as control. We found that CCK-induced suppression of food intake is eliminated in male Nav1.8Cre;ano2 fl /wt mice. Food intake measured over 4 hours was 1.21±0.11g (n=5) in male wild type (wt) mice injected with saline and was reduced to 0.77±0.18 g (n=7, p&lt;0.05) in mice injected with CCK-8 (3μg/kg). Those values were 0.63±0.15 g (n=6) in saline injected and 0.91±0.13 g (n=7, p&gt;0.05) in CCK injected male Nav1.8Cre;ano2 fl /wt mice. However, the CCK injection did not affect food intake in either female wt or Nav1.8Cre;ano2 fl /wt mice. The male Nav1.8/ano2 fl/wt mice were on the average 5.5g heavier than wt mice at 40 weeks of age (39.8±1.4 g, n=13 vs 34.3±1.4 g, n=14, p&lt;0.01). Body weight of females was significantly lower than in males but was not different between wt and Nav1.8Cre;ano2 fl /wt mice (30.4±1.0 g, n=14 vs 28.5±1.0 g, n=11, p=0.20 respectively). Single cell mRNA level of Ano2 and CCK-induced TMEM16 currents in nodose neurons were reduced significantly in male Nav1.8Cre;ano2 fl/wt mice compared to Cre negative controls, but such changes were not seen in female mice. We conclude that heterozygote knockout of Ano2/TMEM16B specifically in sensory neurons causes neuronal insensitivity to CCK and excessive weight gain in male but not female mice. Reduction of this subunit may contribute to the HFD induced obesity. The reason for the phenotype and allele expression variability between sexes is unclear.

TPT1 (tumor protein, translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

ABSTRACTTPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.