Anxiolytic‐Like Action of Selected 4‐(Alkylamino)‐3‐nitrocoumarin Derivatives in BALB/c Mice

In this work, we explored the possible polypharmacological potential of the already established antimicrobials against gastrointestinal pathogens, 4‐(alkylamino)‐3‐nitrocoumarins, as antianxiety agents, using a battery of in vivo experiments. Three chosen coumarin derivatives, differing in the substituent (sec‐butylamino, hexadecylamino, or benzylamino) at position 4, at the doses of 25, 50 and 100 mg kg^–1, were evaluated in light/dark, open‐field, horizontal wire and diazepam‐induced sleep models using male BALB/c mice. Depending on the applied dose, all three tested coumarins displayed a noteworthy anxiolytic‐like effect. 4‐(sec‐Butylamino)‐3‐nitro‐2H‐chromen‐2‐one and 4‐(hexadecylamino)‐3‐nitro‐2H‐chromen‐2‐one could be recognized as true anxiolytics in the lowest applied dose, based on three tests, without exerting any sedative effects. Thus, the 3‐nitrocoumarin core deserves further chemical diversity exploration in the ‘antianxiety’ direction.     

Autism‐related behaviors in the cyclooxygenase‐2‐deficient mouse model

Prostaglandin E2 (PGE2) is an endogenous lipid molecule involved in normal brain development. Cyclooxygenase‐2 (COX2) is the main regulator of PGE2 synthesis. Emerging clinical and molecular research provides compelling evidence that abnormal COX2/PGE2 signaling is associated with autism spectrum disorder (ASD). We previously found that COX2 knockout mice had dysregulated expression of many ASD genes belonging to important biological pathways for neurodevelopment. The present study is the first to show the connection between irregular COX2/PGE2 signaling and autism‐related behaviors in male and female COX2‐deficient knockin, (COX)‐2^?, mice at young (4‐6 weeks) or adult (8‐11 weeks) ages. Autism‐related behaviors were prominent in male (COX)‐2^? mice for most behavioral tests. In the open field test, (COX)‐2^? mice traveled more than controls and adult male (COX)‐2^? mice spent less time in the center indicating elevated hyperactive and anxiety‐linked behaviors. (COX)‐2^? mice also buried more marbles, with males burying more than females, suggesting increased anxiety and repetitive behaviors. Young male (COX)‐2^? mice fell more frequently in the inverted screen test revealing motor deficits. The three‐chamber sociability test found that adult female (COX)‐2^? mice spent less time in the novel mouse chamber indicative of social abnormalities. In addition, male (COX)‐2^? mice showed altered expression of several autism‐linked genes: Wnt2, Glo1, Grm5 and Mmp9. Overall, our findings offer new insight into the involvement of disrupted COX2/PGE2 signaling in ASD pathology with age‐related differences and greater impact on males. We propose that (COX)‐2^? mice might serve as a novel model system to study specific types of autism.     

Activity‐regulated cytoskeleton‐associated protein (Arc/Arg3.1) regulates anxiety‐ and novelty‐related behaviors

The activity‐regulated cytoskeleton‐associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)—a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR‐subunit surface levels in the adult NAc, and the Arc‐deficient mice show reductions in anxiety‐like behavior, deficits in social novelty preference, and antidepressive‐like behavior. Viral‐mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety‐like behavior in the elevated plus maze (EPM). Consistent with this finding, viral‐mediated reduction of Arc in the adult NAc reduces anxiety‐like behavior in male, but not female, mice in the EPM. NAc‐specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood‐ and anxiety‐related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.     

Early postnatal behavioral changes in the Mecp2‐308 truncation mouse model of Rett syndrome

In a mouse model of Rett syndrome (RTT) which expresses a truncated form of methyl‐CpG‐binding protein 2 (Mecp2) gene (Mecp2‐308), we performed a neurobehavioral evaluation across the life span, starting from soon after birth till adulthood. A focus was made on those developmental phases and behavioral domains which have not been previously investigated. The results evidenced subtle anomalies on postnatal days (pnds) 3 to 9 (so‐called presymptomatic phase) in spontaneous movements by hemizygous neonatal male mice. Specifically as early as pnd 3, mutant pups exhibited more intense curling and more side responses and on pnd 9 more pivoting and head rising behaviors than wild type (wt) littermates. A significant decrease in ultrasonic vocalization rate, also emerged in Mecp2‐308 pups. The same mice were also characterized by increased anxiety‐like behaviors (open‐field and zero‐maze tests) during the early symptomatic phase, in the absence of changes in cognitive passive‐avoidance task and rotarod performances. Upon the clearly symptomatic stage, 5‐month‐old Mecp2‐308 mice were also associated with reduced spontaneous home‐cage motor activity, motor coordination impairments (rotarod and dowel tests), and a more marked profile of d ‐amphetamine (10 mg/kg) released stereotyped behavioral syndrome than wt mice. Present results provide an interesting timeline of the progression of symptoms in the Mecp2‐308 model and emphasize the need for increased attention to the presymptomatic phase which may be especially informative in mouse models of human neurodevelopmental disorders. This analysis has provided evidence of precocious behavioral markers of RTT and has identified an early developmental window of opportunities on which potential therapies could be investigated.     

Progesterone modulation of α5 nAChR subunits influences anxiety‐related behavior during estrus cycle

Smokers often report an anxiolytic effect of cigarettes. In addition, stress‐related disorders such as anxiety, post‐traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety‐related responses, control and α5 subunit null mice (α5^?/?) were subjected to the open field activity (OFA), light–dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, α5^?/? behaved like wild‐type controls. In the EPM, female α5^?/? mice displayed an anxiolytic‐like phenotype, while male α5^?/? mice were undistinguishable from littermate controls. We studied the hypothalamus–pituitary–adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin‐releasing factor. Consistent with an anxiolytic‐like phenotype, female α5^?/? mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of α5, we treated cultured NTera 2 cells with progesterone and found that α5 protein levels were upregulated. In addition, brain levels of α5 mRNA increased upon progesterone injection into ovariectomized wild‐type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic‐like in wild‐type mice, but no cycle‐dependent fluctuations in anxiety levels were found in α5^?/? females. Thus, α5‐containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone‐dependent modulation of α5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.     

Hippocampal nuclear factor kappa B accounts for stress‐induced anxiety behaviors via enhancing neuronal nitric oxide synthase (nNOS)‐carboxy‐terminal PDZ ligand of nNOS‐Dexras1 coupling

Anxiety disorders are associated with a high social burden worldwide. Recently, increasing evidence suggests that nuclear factor kappa B (NF‐κB) has significant implications for psychiatric diseases, including anxiety and depressive disorders. However, the molecular mechanisms underlying the role of NF‐κB in stress‐induced anxiety behaviors are poorly understood. In this study, we show that chronic mild stress (CMS) and glucocorticoids dramatically increased the expression of NF‐κB subunits p50 and p65, phosphorylation and acetylation of p65, and the level of nuclear p65 in vivo and in vitro , implicating activation of NF‐κB signaling in chronic stress‐induced pathological processes. Using the novelty‐suppressed feeding (NSF) and elevated‐plus maze (EPM) tests, we found that treatment with pyrrolidine dithiocarbamate (PDTC; intra‐hippocampal infusion), an inhibitor of NF‐κB, rescued the CMS‐ or glucocorticoid‐induced anxiogenic behaviors in mice. Microinjection of PDTC into the hippocampus reversed CMS‐induced up‐regulation of neuronal nitric oxide synthase (nNOS), carboxy‐terminal PDZ ligand of nNOS (CAPON), and dexamethasone‐induced ras protein 1 (Dexras1) and dendritic spine loss of dentate gyrus (DG) granule cells. Moreover, over‐expression of CAPON by infusing LV‐CAPON‐L‐GFP into the hippocampus induced nNOS‐Dexras1 interaction and anxiety‐like behaviors, and inhibition of NF‐κB by PDTC reduced the LV‐CAPON‐L‐GFP‐induced increases in nNOS‐Dexras1 complex and anxiogenic‐like effects in mice. These findings indicate that hippocampal NF‐κB mediates anxiogenic behaviors, probably via regulating the association of nNOS‐CAPON‐Dexras1, and uncover a novel approach to the treatment of anxiety disorders.

     

Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Immunoglobin G with α‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies with α‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusively α‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α‐2,6 sialyltransferase produced IgG with significant levels of both α‐2,6 and α‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α‐2,6 sialylation level relative to α‐2,3 sialylation for the α‐2,3 sialyltransferase knockouts when combined with α‐2,6 sialyltransferase overexpression. Indeed, α‐2,3 linked sialic acids were not detected on IgG produced from the α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

Fgf3‐Fgf4‐cis: A new mouse line for studying Fgf functions during mouse development

The fibroblast growth factor (FGF) family consists of 22 ligands in mice and humans. FGF signaling is vital for embryogenesis and, when dysregulated, can cause disease. Loss‐of‐function genetic analysis in the mouse has been crucial for understanding FGF function. Such analysis has revealed that multiple Fgfs sometimes function redundantly. Exploring such redundancy between Fgf3 and Fgf4 is currently impossible because both genes are located on chromosome 7, about 18.5 kb apart, making the frequency of interallelic cross‐over between existing mutant alleles too infrequent to be practicable. Therefore, we retargeted Fgf3 and Fgf4 in cis, generating an Fgf3 null allele and a conditional Fgf4 allele, subject to Cre inactivation. To increase the frequency of cis targeting, we used an F1 embryonic stem cell line that contained 129/SvJae (129) and C57BL/6J (B6) chromosomes and targeting constructs isogenic to the 129 chromosome. We confirmed cis targeting by assaying for B6/129 allele‐specific single‐nucleotide polymorphisms. We demonstrated the utility of the Fgf3^Δ‐Fgf4^flox‐cis mouse line by showing that the caudal axis extension defects found in the Fgf3 mutants worsen when Fgf4 is also inactivated. This Fgf3^Δ‐Fgf4^flox‐cis line will be useful to study redundancy of these genes in a variety of tissues and stages in development. genesis 54:91–98, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA.     

Mania‐ and anxiety‐like behavior and impaired maternal care in female diacylglycerol kinase eta and iota double knockout mice

Genome‐wide association studies linked diacylglycerol kinase eta and iota to mood disorders, including bipolar disorder and schizophrenia, and both genes are expressed throughout the brain. Here, we generated and behaviorally characterized female mice lacking Dgkh alone, Dgki alone, and double Dgkh/Dgki‐knockout (dKO) mice. We found that fewer than 30% of newborn pups raised by dKO females survived to weaning, while over 85% of pups survived to weaning when raised by wild‐type (WT) females. Poor survival under the care of dKO mothers was unrelated to pup genotype. Moreover, pups from dKO dams survived when fostered by WT dams, suggesting the poor survival rate of dKO‐raised litters was related to impaired maternal care by dKO dams. Nest building was similar between WT and dKO dams; however, some dKO females failed to retrieve any pups in a retrieval assay. Pups raised by dKO dams had smaller or absent milk spots and reduced weight, indicative of impaired nursing. Unlike WT females, postpartum dKO females showed erratic, panicked responses to cage disturbances. Virgin dKO females showed behavioral signs of anxiety and mania, which were not seen in mice lacking either Dgkh or Dgki alone. Our research indicates that combined deletion of Dgkh and Dgki impairs maternal behavior in the early postpartum period, and suggests female dKO mice model symptoms of mania and anxiety.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

Inducible gene modification in the gastric epithelium of Tff1‐CreERT2, Tff2‐rtTA,Tff3‐luc mice

Temporal and spatial regulation of genes mediated by tissue‐specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium‐specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain confers tamoxifen‐inducible irreversible somatic recombination and allows simultaneous doxycycline‐dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1‐CreERT2 and Tff2‐rtTA transgene activity in a partially overlapping subset of long‐term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626–635, 2016. © 2016 Wiley Periodicals, Inc.     

Hyperphagia and Obesity in Na,K‐ATPase α2 Subunit‐Defective Mice

Objective: The Na,K‐ATPase α2 subunit gene (Atp1a2) is expressed in the brain, skeletal muscles, heart, and adipocytes. Specific function of the α2 subunit, such as involvement in differentiation and function of adipocytes, has not been addressed. The aim of this study was to examine whether Atp1a2‐defective heterozygous mice show obesity and reveal the mechanisms underlying the obesity. Research Methods and Procedures: We measured the differentiation and glucose uptake function of in vitro‐differentiated adipocytes derived from embryonic fibroblasts of Atp1a2‐defective mice. Food intake, body temperature, metabolic rate, and spontaneous activity and mRNA levels of neuropeptide genes were compared between the heterozygous and wild‐type adult mice. Results: Atp1a2 heterozygous female mice developed obesity after middle age. The time course of in vitro adipocyte differentiation of embryonic fibroblasts isolated from wild type, heterozygous, and homozygous mice was not different, glucose and Rb uptake activities of the in vitro‐differentiated adipocytes were not altered, and the effects of insulin on glucose uptake and those of monensin and ouabain on Rb uptake were similar among the genotypes. However, food intake in the light phase was significantly greater in the heterozygous mice than the wild type in the 24‐hour dark‐light cycle, whereas it was similar under constant‐light condition. Body temperature, metabolic rate at rest, and spontaneous motor activity of the heterozygous mice were similar to those of the wild type. Orexin mRNA level was lower in heterozygous than wild‐type mice. Discussion: The Na,K‐ATPase α2 subunit is not involved in the differentiation or in glucose and Rb uptake function of in vitro‐differentiated adipocytes. Hyperphagia is the likely primary cause of obesity in Atp1a2 heterozygous mice.