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Lepidium meyenii (maca) is an annual or biennial herb from South America that is a member of the genus Lepidium L. in the family Cruciferae. This herb possesses antioxidant and antiapoptotic activities, enhances autophagy functions, prevents cell death, and protects neurons from ischemic damage. Macamide B, an effective active ingredient of maca, exerts a neuroprotective effect on neonatal hypoxic-ischemic brain damage (HIBD), but the mechanism underlying its neuroprotective effect is not yet known. The purpose of this study was to explore the effect of macamide B on HIBD-induced autophagy and apoptosis and its potential neuroprotective mechanism. The modified Rice-Vannucci method was used to induce HIBD in 7-day-old (P7) macamide B- and vehicle-pretreated pups. TTC staining was performed to evaluate the cerebral infarct volume in pups, the brain water content was measured to evaluate the neurological function of pups, neurobehavioural testing was conducted to assess functional recovery after HIBD, TUNEL and FJC staining was performed to detect cellular autophagy and apoptosis, and Western blot analysis was used to detect the levels of proteins in the pro-survival phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway and autophagy and apoptosis-related proteins. Macamide B pretreatment significantly decreases brain damage and improves the recovery of neural function after HIBD. At the same time, macamide B pretreatment activates the PI3K/AKT signaling pathway after HIBD, enhances autophagy, and reduces hypoxic-ischemic (HI)-induced apoptosis. In addition, 3-methyladenine (3-MA), an inhibitor of the PI3K/AKT signaling pathway, significantly inhibits the increase in autophagy levels, aggravates HI-induced apoptosis, and reverses the neuroprotective effect of macamide B on HIBD. Our data indicate that a macamide B pretreatment might regulate autophagy through the PI3K/AKT signaling pathway, thereby reducing HIBD-induced apoptosis and exerting neuroprotective effects on neonatal HIBD. Macamide B may become a new drug for the prevention and treatment of HIBD.

Deoxynivalenol is one the of most common mycotoxins in cereals and grains and causes a serious health threat to poultry and farm animals. Our previous study found that DON decreased the production performance of laying hens. It has been reported that DON could exert significant toxic effects on the intestinal barrier and microbiota. However, whether the decline of laying performance is related to intestinal barrier damage, and the underlying mechanisms of DON induced intestine function injury remain largely unclear in laying hens. In this study, 80 Hy-line brown laying hens at 26 weeks were randomly divided into 0, 1, 5 and 10 mg/kg.bw (body weight) DON daily for 6 weeks. The morphology of the duodenum, the expression of inflammation factors and tight junction proteins, and the diversity and abundance of microbiota were analyzed in different levels of DON treated to laying hens. The results demonstrated that the mucosal detachment and reduction of the villi number were presented in different DON treated groups with a dose-effect manner. Additionally, the genes expression of pro-inflammatory factors IL-1β, IL-8, TNF-α and anti-inflammatory factors IL-10 were increased or decreased at 5 and 10 mg/kg.bw DON groups, respectively. The levels of ZO-1 and claudin-1 expression were significantly decreased in 5 and 10 mg/kg.bw DON groups. Moreover, the alpha diversity including Chao, ACE and Shannon indices were all reduced in DON treated groups. At the phylum level, Firmicutes and Actinobacteria and Bacteroidetes, Proteobacteria, and Spirochaetes were decreased and increased in 10 mg/kg.bw DON group, respectively. At the genus levels, the relative abundance of Clostridium and Lactobacillus in 5 and 10 mg/kg.bw DON groups, and Alkanindiges and Spirochaeta in the 10 mg/kg.bw DON were significantly decreased and increased, respectively. Moreover, there were significant correlation between the expression of tight junction proteins and the relative abundance of Lactobacillus and Succinispira. These results indicated that DON exposure to the laying hens can induce the inflammation and disrupt intestinal tight junctions, suggesting that DON can directly damage barrier function, which may be closely related to the dysbiosis of intestinal microbiota.

Objective: Hypoxic-ischemic brain damage (HIBD) is one of the leading causes of permanent neurological dysfunction and acute death in infants and young children. As the only effective treatment for neonatal HIBD, therapeutic hypothermia (TH) can only moderately improve the prognosis. Previous evidence has shown that icariin (ICA) can inhibit apoptosis and exert a neuroprotective effect on neonatal mouse HIBD, but the specific mechanism remains unknown. This study aimed to explore whether ICA played a neuroprotective role in inhibiting apoptosis by regulating autophagy through the estrogen receptor α (ERα) and estrogen receptor β (ERβ) pathways in neonatal mice with HIBD.Method: In vivo experiments, the HIBD model of neonatal mice were constructed according to the modified Rice-Vannucci method. The effects of ICA on the expression levels of autophagy-related proteins as well as ERα and ERβ proteins were detected. At the same time as ICA pretreatment, HIBD newborn mice were injected with the autophagy inhibitor 3-MA into the lateral ventricle to test whether ICA pretreatment might exert the neuroprotective effect of HIBD on newborn mice by promoting autophagy and inhibiting HIBD-induced apoptosis. At the same time as ICA pretreatment, HIBD newborn mice were injected with ERα inhibitor MPP or ERβ inhibitor PHTPP into the lateral ventricle, to further test whether ICA might promote autophagy and inhibit HIBD-induced apoptosis by activating ERα or ERβ pathways, thereby exerting a neuroprotective effect on HIBD in newborn mice. The cerebral infarct volume and the degree of brain injury in the mice were evaluated by TTC staining and brain water content determination. Body weight measurements and neurobehavioral tests were carried out to assess neurological recovery after HIBD in neonatal mice; The expressions of ERα, ERβ, autophagy and apoptosis-related proteins were detected by tissue immunofluorescence and western blot, and the level of apoptosis was further detected by TUNEL and FJC staining. A caspase-3 activity detection kit was used to detect the relative activity level of caspase-3. The glucose and oxygen deprivation (OGD) model of HT22 cells in the mouse hippocampal neuron system was constructed and experimented in vitro.Result: ICA pretreatment significantly promoted the autophagy level of HIBD mice. Treatment with 3-MA significantly inhibited the increase of autophagy induced by ICA pretreatment, reversed the neuroprotective effect of ICA pretreatment, and promoted apoptosis. ICA pretreatment significantly increased the expression levels of ERα and ERβ proteins in HIBD newborn mice. The MPP and PHTPP treatments significantly inhibited the expression levels of ERα and ERβ proteins activated by ICA pretreatment, reversed the neuroprotective effects of ICA pretreatment, inhibited the increase in autophagy induced by ICA pretreatment, and promoted apoptosis.Conclusion: ICA pretreatment may promote autophagy by activating the ERα and ERβ pathway, and thus reduce the apoptosis induced by HIBD, then exert a neuroprotective effect on neonatal mice with HIBD.

Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia. In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP). Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis. We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia., GRAPHICAL ABSTRACT

Severe local acidosis causes tissue damage and pain, and is associated with many diseases, including cerebral and cardiac ischemia, cancer, infection, and inflammation. However, the molecular mechanisms of the cellular response to extracellular acidic environment are not fully understood. We recently identified a novel and evolutionarily conserved membrane protein, PAC (also known as PACC1 or TMEM206), encoding the proton-activated chloride (Cl−) channel, whose activity is widely observed in human cell lines. We demonstrated that genetic deletion of Pac abolished the proton-activated Cl− currents in mouse neurons and also attenuated the acid-induced neuronal cell death and brain damage after ischemic stroke. Here, we show that the proton-activated Cl− currents are also conserved in primary rat cortical neurons, with characteristics similar to those observed in human and mouse cells. Pac gene knockdown nearly abolished the proton-activated Cl− currents in rat neurons and reduced the neuronal cell death triggered by acid treatment. These data further support the notion that activation of the PAC channel and subsequent Cl− entry into neurons during acidosis play a pathogenic role in acidotoxicity and brain injury.

Mycotoxins such as zearalenone (ZEN), deoxynivalenol (DON) and T-2 toxin (T-2) are the most poisonous biological toxins in food pollution. Mycotoxin contaminations are a global health issue. The aim of the current study was to use porcine Leydig cells as a model to explore the toxic effects and underlying mechanisms of ZEN, DON and T-2. The 50% inhibitory concentration (IC50) of ZEN was 49.71 μM, and the IC50 values of DON and T-2 were 2.49 μM and 97.18 nM, respectively. Based on the values of IC50, ZEN, DON and T-2 exposure resulted in increased cell apoptosis, as well as disrupted mitochondria membrane potential and cell cycle distribution. The results also showed that ZEN and DON significantly reduced testosterone and progesterone secretion in Leydig cells, but T-2 only reduced testosterone secretion. Furthermore, the expression of steroidogenic acute regulatory (StAR) protein and 3β-hydroxysteroid dehydrogenase (3β-HSD) were significantly decreased by ZEN, DON and T-2; whereas the protein expression of cholesterol side-chain cleavage enzyme (CYP11A1) was only significantly decreased by ZEN. Altogether, these data suggest that the ZEN, DON and T-2 toxins resulted in reproductive toxicity involving the inhibition of steroidogenesis and cell proliferation, which contributes to the cellular apoptosis induced by mitochondrial injury in porcine Leydig cells.

Decabromodiphenyl ether (BDE-209), the primary constituent of a widely used flame retardant formulation, is often present in high levels in avian derived products and could be transferred to humans through consumption. The purpose of this study was to investigate the toxicokinetics and bioaccumulation patterns of BDE-209 in different tissues of broilers, which would benefit the evaluation of chicken product safety. Male broilers received a single oral administration of BDE-209 at 25 mg/kg.BW and then BDE-209 concentrations in the plasma, liver, leg muscle, breast muscle, and other tissues were measured using gas chromatography-electron capture detection (GC-ECD). The changes of BDE-209 concentrations in the plasma were fitted to a non-compartmental model for kinetic analysis. Peak values were observed at 24 h (t

2,2’,4,4’-tetrabromodiphenyl ether (BDE-47)-induced nephrotoxicity is closely associated with oxidative stresses and mitochondrial abnormalities. Mitochondrial fusion and fission dynamics are crucial for maintaining mitochondrial and cellular physiological homeostasis. However, the detailed mechanisms through which BDE-47 disrupts this dynamic and contributes to renal injuries are still not fully understood. The porcine kidney-15 (PK15) cell line, a well-defined in vitro animal renal toxicological model, was exposed to BDE-47 with concentrations of 12.5, 25, 50, and 100 μM, respectively. Cell viability, the levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP), the mitochondrial membrane potential (MMP), and the expression levels of key mitochondrial fusion and fission proteins were assessed. BDE-47 reduced cell viability and disrupted mitochondrial dynamics by inhibiting mitochondrial fusion and fission simultaneously, leading to MMP decreases, ROS overgeneration, ATP depletion, and cellular disintegration in a dose-dependent manner. Additionally, the mitochondrial division inhibitor (Mdivi-1) with the concentration of 20 μM observed to restore the downregulation of mitochondrial fusion and fission proteins, alleviate damages in mitochondrial morphology and functionality, correct ROS overproduction, and enable cell survival. The antioxidant N-acety- L -cysteine (NAC) with the concentration of 1 mM also simultaneously reversed the imbalance of mitochondrial dynamics, decreased ROS production, and restored mitochondrial morphology in PK15 cells exposed to BDE-47. Our data provide new insights indicating that BDE-47 disrupts mitochondrial fusion/fission dynamics to induce mitochondrial abnormalities, triggering oxidative stresses and thus contributing to PK15 cell dysfunction. ROS-dependent pathways in mitochondrial dynamics may provide a new avenue for developing effective strategies to protect cells against BDE-47-induced nephrotoxicity.

A new protein channel family Exposure of cells to acidic conditions outside the cell activates chloride-conducting channels that influence physiological and pathological processes. Yang et al. used an unbiased RNA interference screen to identify the channel protein that allows such proton-activated ion conductance. The protein, called PAC for proton-activated channel, has two predicted transmembrane domains and is unlike any previously identified channel. Identification of the channel should advance studies of its physiological roles, which range from tissue injury after stroke to adaptation of Tibetan natives to a high-altitude environment. Science , this issue p. 395

Masked mycotoxins are biologically modified phase II metabolites formed by plant defense mechanisms through glucosylation catalyzed by uridine diphosphate -glucosyltransferases. Most of the current reports focus on the occurrence of masked mycotoxins in Europe, America, Africa, and cover other geographic regions, e.g. China and Japan. High proportions of masked mycotoxins co-occurring with their parent forms in various cereal-based food and feedstuff could clearly increase total exposures and pose additional health risks to humans and animals. In contrast to the parent mycotoxins, the data on the toxicity of masked mycotoxins are still scarce, however, the poor existing information showed that masked mycotoxins generally exhibit significant in vitro and in vivo toxicities lower than those of their parent forms, especially for deoxynivalenol-3-glucoside, which is the only thoroughly investigated masked mycotoxin. Although the lower toxicity level of masked mycotoxins, these are probably hydrolyzed into their free forms by intestinal microorganisms in the digestive tract of mammals and thus contribute to unpredicted toxicity. The metabolic characteristics of reported masked mycotoxins are species-specific. The most relevant animal model of human sensitivity, the pig, is most sensitive to masked mycotoxins. This review focuses on updates in the current knowledge on country-specific natural-occurrence data in global surveys, as well as in vitro and in vivo toxicology and metabolic investigations of masked mycotoxins.

The wide usage of decabromodiphenyl ether (BDE-209) results in its increasing occurrence in the environment and increasing attention in regard to human and animal health. BDE-209 is an endocrine disruptor for hypothyroidism, but the toxicity mechanism is unclear. Here, the histopathology and transcriptome sequencing of thyroid tissue from broiler chicks were investigated by supplemental feeding with different concentrations of BDE-209 for 42 days (0–4 g/kg in basal diet), followed by determining the levels of thyroid hormones in serum. The results showed ruptured and even hyperplastic follicular epithelial cells in the thyroid, and a total of 501 differentially expressed genes were screened out: 222 upregulated and 279 downregulated. Based on the Kyoto Encyclopedia of Genes and Genomes database, neuroactive ligand-receptor interaction pathway was significantly enriched, and α1D-adrenergic receptor, follicle-stimulating hormone receptor, thyroid stimulating hormone receptor, and somatostatin receptor type 2 were shown to be candidate biomarkers. Thyroxine was a possible biomarker due to clear reduction in serum and significant correlation with exposure concentrations. These results suggested that oral intake of BDE-209 can cause structural injuries and even hyperplasia, and affect gene transcription involved in the neuroactive ligand-receptor interaction pathway of thyroid, as well as thyroid hormones in serum.

The flame retardant decabrominated diphenyl ether (BDE-209) is a widely used chemical in a variety of products and exists extensively in the environment. BDE-209 has been reported to induce kidney injury and dysfunction. However, the causes and mechanisms of its nephrotoxicity are still under investigation. In this study, 150 male broilers were exposed to BDE-209 concentrations of 0, 0.004, 0.04, 0.4, 4.0 g/kg for 42 days. The relative kidney weight, histopathology, markers of renal injury, oxidative stress, inflammation, apoptosis and the expression of MAPK signaling pathways-related proteins were assessed. The results showed that the concentrations of blood urea nitrogen (BUN), creatinine (CRE) and the neutrophil gelatinase-associated lipocalin (NGAL), significantly increased after exposure to BDE-209 with the doses more than 0.04 g/kg. Similarly, severe damage of renal morphology was observed, including atrophy and necrosis of glomeruli, and swelling and granular degeneration of the renal tubular epithelium. In the renal homogenates, the oxidative stress was evidenced by the elevated concentrations of MDA and NO, and decreased levels of GSH-Px, GSH and SOD. Due to the inflammatory response, the level of NF-κB and the pro-inflammatory cytokines TNF-α, IL-1β, IL-18 were remarkably upregulated, while the content of the anti-inflammatory cytokine IL-10 decreased. Additionally, the apoptotic analysis showed notable upregulations of Bax/Bcl-2 ratio, the relative expression of p-ERK1/2 and p-JNK1/2, and the expression of Bax, cytochrome c and caspase 3. The present study indicates that BDE-209 exposure can cause nephrotoxicity in broilers through oxidative stress and inflammation, which activate the phosphorylation of key proteins of the MAPK signaling pathways, and subsequently induce mitochondria-mediated kidney apoptosis.

Neonatal lipopolysaccharide (LPS) exposure can lead to depressive-like behaviors in mice through inducing pro-inflammatory cytokines including interferon(IFN)-{gamma}. ATP8A2 is a phospholipid transporter located on the cell membrane. Studies have shown that the decrease in ATP8A2 expression in the prefrontal cortex (PFC) is associated with depressive behavior. Moreover, it has been reported that IFN-{gamma} could reduce ATP8A2 expression in non-neuronal cells. These findings prompted us to hypothesize that neonatal LPS exposure might induce ATP8A2 down-regulation in PFC in mice by increasing the IFN-{gamma} level. Mice pups consisting approximately evenly of both sexes were intraperitoneally injected with 3 doses of LPS (50 g/kg body weight for each dose) on postnatal day (PND5), PND7 and PND9. Here, we first found that PFC ATP8A2 expression decreased significantly and transiently till ten days after neonatal LPS exposure with the lowest level at two days after it. Moreover, a negative correlation of PFC ATP8A2 expression was found with the PFC level of IFN-{gamma}, rather than the other LPS-induced pro-inflammatory cytokines. Using anti-IFN-{gamma} neutralizing mAb, IFN-{gamma} was identified as the key mediator of LPS-induced ATP8A2 down-regulation in PFC in mice. Besides, neutralizing IFN-{gamma} partially but significantly rescued the depressive-like behaviors in adulthood induced by neonatal LPS exposure. In sum, the present study showed that neonatal LPS exposure induced ATP8A2 down-regulation in PFC and depressive-like behaviors in mice through increasing the IFN-{gamma} level.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we “clone” the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz (“TcMAC21”). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Glioma-associated oncogene family zinc finger 2 (Gli2), a key component of the hedgehog signaling pathway, has been previously demonstrated to promote the malignant properties of prostate cancer in vitro. However, the role of Gli2 in the development of castration-resistant prostate cancer (CRPC) has yet to be fully elucidated. In the present study, Gli2 expression was knocked down in androgen-responsive prostate cancer cells using an inducible Gli2 short hairpin RNA. Suppression of Gli2 expression resulted in significant reduction of cell viability, increased the proportion of cells in the G0/G1 phases of the cell cycle and reduced the expression of genes associated with cell cycle progression. Gli2 knockdown sensitized both androgen-dependent and -independent prostate cancer cells to the antiandrogen drug Casodex and prevented the outgrowth of LNCaP prostate cancer cells. In addition, Gli2 knockdown significantly suppressed the development of CRPC in a LNCaP xenograft mouse model, which was reversed by the re-expression of Gli2. In conclusion, to the best of our knowledge, the present study was the first occasion in which the essential role of Gli2 in the development of CRPC was demonstrated, providing a potential therapeutic target for the intervention of CRPC.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites’ binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins’ combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups (p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

We have previously verified that neonatal hepatitis B vaccination induced hippocampal neuroinflammation and behavior impairments in mice. However, the exact mechanism of these effects remain unclear. In this study, we observed that neonatal hepatitis B vaccination induced an anti-inflammatory cytokine response lasting for 4–5 weeks in both the serum and the hippocampus, primarily indicated by elevated IL-4 levels. Three weeks after the vaccination schedule, however, hepatitis B vaccine (HBV)-mice showed delayed hippocampal neuroinflammation. In periphery, IL-4 is the major cytokine induced by this vaccine. Correlation analyses showed a positive relationship in the IL-4 levels between serum and hippocampus in HBV-mice. Thus, we investigated whether neonatal over-exposure to systemic IL-4 influences brain and behavior. We observed that mice injected intraperitoneally with recombinant mouse IL-4 (mIL-4) during early life had similar neuroinflammation and cognition impairment similar to those induced by neonatal hepatitis B vaccination. Next, the mechanism underlying the effects of IL-4 on brain in mice was explored using a series of experiments. In brief, these experiments showed that IL-4 mediates the delayed neurobehavioral impairments induced by neonatal hepatitis B vaccination, which involves the permeability of neonatal blood–brain barrier and the down-regulation of IL-4 receptor. This finding suggests that clinical events concerning neonatal IL-4 over-exposure, including neonatal hepatitis B vaccination and allergic asthma in human infants, may have adverse implications for brain development and cognition.

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause ofamyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism isthe aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells.We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

Activation of the neonatal immune system may contribute to deficits in neuronal plasticity. We have reported that neonatal vaccination with a hepatitis B vaccine (HBV) transiently impairs mood status and spatial memory involving a systemic T helper (Th) 2 bias and M1 microglial activation. Here, an EE induced microglial anti-inflammatory M2 polarization, as evidenced by selectively enhanced expression of the Arginase1 gene (Arg-1) in the hippocampus. Interestingly, knock-down of the Arg-1 gene prevented the effects of EE on restoring the dendritic spine density. Moreover, levels of the Th1-derived cytokine IFN-gamma (IFN-γ) were elevated in the choroid plexus (CP), which is the interface between the brain and the periphery. IFN-γ-blocking antibodies blunted the protective effects of an EE on spine density and LTP. Furthermore, levels of complement proteins C1q and C3 were elevated, and this elevation was associated with synapse loss induced by the HBV, whereas an EE reversed the effects of the HBV. Similarly, blockade of C1q activation clearly prevented synaptic pruning by microglia, LTP inhibition and memory deficits in hepatitis B-vaccinated mice. Together, the EE-induced increase in IFN-γ levels in the CP may disrupt systemic immunosuppression related to HBV via an IFN-γ/Arg-1/complement-dependent pathway.

Transplantation of microbiota from small intestine, not large intestine, of healthy mice exerts obvious effects on healthy recipients, bringing a new perspective on gut flora transplantation., Fecal microbiota transplantation is widely used. Large intestinal microbiota (LIM) is more similar to fecal microbiota than small intestinal microbiota (SIM). The SIM communities are very different from those of LIM. Therefore, SIM transplantation (SIMT) and LIM transplantation (LIMT) might exert different influences. Here, healthy adult male C57Bl/6 mice received intragastric SIMT, LIMT, or sterile PBS administration. Microbiota graft samples were collected from small/large intestine of healthy mice of the same age, sex, and strain background. Compared with PBS treatment, SIMT increased pellet number, stool wet weight, and stool water percentage; induced a fecal microbiota profile shift toward the microbial composition of the SIM graft; induced a systemic anti-inflammatory cytokines profile; and ameliorated depressive-like behaviors in recipients. LIMT, however, induced merely a slight alteration in fecal microbial composition and no significant influence on the other aspects. In sum, SIMT, rather than LIMT, affected defecation features, fecal microbial composition, cytokines profile, and depressive-like behaviors in healthy mice. This study reveals the different effects of SIMT and LIMT, providing an interesting clue for further researches involving gut microbial composition change.

The immune system plays a crucial role in the progression of Alzheimer's disease (AD). Recently, immune-dependent cascade induced by systemic immune activation has been verified to play a beneficial role in AD mouse models. Here, we tested whether Bacillus Calmette-Guerin (BCG) immunization alters AD pathology and cognitive dysfunction in APP/PS1 AD mouse model, and with 4Aβ1-15 vaccination as positive control. It was found that BCG treatment reversed the cognitive decline to the extent observed in 4Aβ1-15 group, but did not reduce the β-amyloid (Aβ) burden in the brain. Then, we demonstrated the enhanced recruitment of inflammation-resolving monocytes across the choroid plexus and perivascular spaces to cerebral sites of plaque pathology in APP/PS1 mice immunized with BCG. Furthermore, elevated splenocyte Foxp3+ regulatory T cell levels in the control APP/PS1 mice were down-regulated back to the wild-type (WT) levels by BCG treatment but not 4Aβ1-15 vaccination. In addition, BCG treatment induced the production of more circulating interferon (IFN)-γ than the controls and 4Aβ1-15 vaccination. Though the similar reductions in brain levels of pro-inflammatory cytokines were observed in the BCG and 4Aβ1-15 groups compared to the controls, only BCG had the great effect in upregulating cerebral anti-inflammatory cytokine levels as well as elevating the expression of neurotrophic factors in the brain of APP/PS1 mice. Thus, it is suggested that BCG exerts a beneficial immunomodulatory effect in APP/PS1 mice through mitigation of systemic immune suppression, induction of IFN-γ response and alleviation of the neuroinflammatory response.

Deoxynivalenol (DON, vomitoxin) is the most common mycotoxin in cereals and grains. DON contamination can cause a serious health threat to humans and farm animals. DON has been reported to exert significant toxicity effects on the male reproductive system. However, the causes and mechanisms underlying efforts of DON on sperm and testicular damage remain largely unclear. In the present study, we thoroughly investigated this issue. Eighty male BALB/c mice were randomly divided into a control group ( n = 40) and DON treatment group (2.4 mg/kg of body weight, n = 40). The ratio of testes and seminal vesicle to body, sperm survival and motility, and morphology of sperm and testis were observed in DON-treated and control mice. In addition, the concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione (GSH), and also the expression levels of JNK/c-Jun signaling and apoptotic factors such as caspase-3, caspase-8, caspase-9, Bim, and Bid were analyzed and compared between the two groups. The results demonstrated that a single topical application of DON significantly increased the percentage of abnormal sperm and decreased the motility of sperm, indicating the sperms are damaged by DON. Additionally, the reduced relative body weight of testis and severe destruction of testicular morphology were observed. Moreover, the increased levels of ROS and MDA levels and decreased activities of SOD and GSH were found in testicular tissues, suggesting that oxidative stress is induced by DON treatment. Furthermore, DON upregulated the expression of stress-induced JNK/c-Jun signaling pathway proteins as well as JNK/c-Jun phosphorylation proteins. In addition, DON could enhance testicular apoptosis by increasing expression levels of apoptotic genes including Bim, cytochrome c, caspase 3, caspase 8, and caspase 9. These results suggest that DON exposure can cause sperm damage, oxidative stress, testicular apoptosis, and phosphorylation of JNK/c-Jun signaling pathway. The underlying mechanisms may be that DON induces sperm damage by exacerbating oxidative stress-mediated testicular apoptosis via JNK/c-Jun signaling pathway.

// Zhao Liu 1,2,4 , Long Wang 2 , Junhua Yang 2 , Abhik Bandyopadhyay 2 , Virginia Kaklamani 3 , Shui Wang 1 and Lu-Zhe Sun 2,3 1 Department of Breast Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China 2 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, Texas, United States of America 3 Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, Texas, United States of America 4 Department of Thyroid and Breast Surgery, Affiliated Hospital of Xuzhou Medical College, Xuzhou, China Correspondence to: Shui Wang, email: // Lu-Zhe Sun, email: // Keywords : estrogen receptor alpha; senescence; transformation; ras; Gerotarget Received : November 18, 2015 Accepted : May 20, 2016 Published : June 01, 2016 Abstract Exposure to estrogen has long been associated with an increased risk of developing breast cancer. However, how estrogen signaling promotes breast carcinogenesis remains elusive. Senescence is known as an important protective response to oncogenic events. We aimed to elucidate the role of estrogen receptor alpha (ERα) on senescence in transformed human mammary epithelial cells and breast cancer cells. Our results show that ectopic expression of oncoprotein H-ras-V12 in immortalized human mammary epithelial cells (HMEC) significantly inhibited the phosphorylation of the retinoblastoma protein (Rb) and increased the activity of the senescence-associated beta-galactosidase (SA-β-Gal). These senescence-like phenotypes were reversed by ectopic expression of ERα. Similar inhibition of the H-ras-V12-induced SA-β-Gal activity by ERα was also observed in the human mammary epithelial MCF-10A cells. Co-expression of ERα and H-ras-V12 resulted in HMEC anchorage-independent growth in vitro and tumor formation in vivo . Furthermore, inhibition of ERα expression induced senescence-like phenotypes in ERα positive human breast cancer cells such as increased activity of SA-β-Gal, decreased phosphorylation of RB, and loss of mitogenic activity. Thus, the suppression of cellular senescence induced by oncogenic signals may be a major mechanism by which ERα promotes breast carcinogenesis.

Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue-induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.

Bacillus Calmette-Guérin (BCG) is administered to neonates worldwide, but it is still unknown whether this neonatal vaccination affects brain development during early postnatal life, despite the close association of the immune system with the brain. Newborn C57BL/6 mice were injected subcutaneously with BCG or phosphate-buffered saline (PBS) and their mood status and spatial cognition were observed at four and eight weeks (w) old. The mice were also subjected to tests at 2 and 6 w to examine BCG's effects on neurogenesis, the hippocampal microglia phenotype and number, and the expression of hippocampal neuroimmune molecules and peripheral cytokines. The BCG-injected mice showed better behavioral performances at 4 w. We observed elevated neurogenesis, M2 microglial activation and a neurotrophic profile of neuroimmune molecules [more interferon (IFN)-γ, interleukin (IL)-4, transforming growth factor (TGF)-β, brain-derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF)-1 and less tumor necrosis factor (TNF)-α and IL-1β] in the hippocampus of the 2-w-old BCG-mice. In the periphery, BCG induced a T helper (Th)-1 serum response. At the individual level, there were positive correlations between the serum IFN-γ/IL-4 ratio and the levels of neurotrophins and neurogenesis in the hippocampus. These findings suggest that neonatal BCG vaccination improved neurogenesis and mouse behavior in early life by affecting the neuroimmune milieu in the brain, which may be associated with a systemic Th1 bias.

T-2 toxin, one of the most toxic mycotoxins, is commonly presented along with its metabolites, HT-2 toxin, neosolaniol (NEO), T-2 triol, and T-2 tetraol in foodstuff and feed. The aim of this study was to evaluate the cytotoxic effects of T-2 toxin alone and in combination with its metabolites on porcine Leydig cells. Based on the determination of cell viability with CCK-8, toxicological interactions were investigated using Combination Index method. The cytotoxic potency of five tested mycotoxins individual and their mixtures all showed with a dose-dependent manner. In view of IC50 values, the decreasing cytotoxicity of mycotoxins was ranking: T-2 toxin > HT-2 toxin > T-2 triol > NEO > T-2 tetraol. Combinations of T-2+HT-2, T-2+NEO, and HT-2+NEO displayed synergism at low doses but antagonism at high doses, while the ternary combination of T-2+HT-2+NEO revealed adverse situation from antagonism to synergism. All binary and ternary combinations of T-2 toxin, T-2 triol, and T-2 tetraol exhibited antagonistic interactions. Our results suggest that the co-occurrence of T-2 toxin and its metabolites might pose a slight threat to reproductive health due to antagonistic interactions. However, the synergy observed should be not ignored especially at low doses of mycotoxins co-occurrence in the diet.

Previous study has demonstrated the neurobeneficial role of BCG and influenza vaccines. Based on this, our study concentrated on the synergistic effects on development of central nervous system by combined vaccination with BCG and influenza vaccines in rats. Our results displayed that pups combinedly vaccinated with BCG and influenza vaccines showed a significant enhance in spatial cognition, induction of LTP, hippocampal neurogenesis and morphology of dendritic spines compared with pups vaccinated with BCG solely. Furthermore, combined vaccination with BCG and influenza vaccines showed higher expression of BDNF, IGF-1, IL-4, IFN-γ and lower IL-1β, TNF-α and IL-6 than BCG. Taken together, combined vaccination with BCG and influenza vaccines presented synergistic effects on spatial cognition and hippocampal plasticity in rats.

A method for the simultaneous determination of six aflatoxins (AFTs) and sterigmatocystin (SMC) in livestock and poultry tissues by QuEChERS-ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The samples were extracted with 10 mL acetonitrile-water (84:16, v/v) after enzymatic hydrolysis with 20 μL β-glucosidase. The extracts were cleaned with 1.0 g each of sodium chloride and anhydrous magnesium sulfate, and then degreased with 5 mL hexane. The residues were re-dissolved in 1 mL acetonitrile-water (80:20, v/v) containing 5 mmol/L ammonium acetate. Methanol and 5 mmol/L ammonium acetate were used as the mobile phases. Six AFTs and SMC were analyzed by multiple reaction monitoring in the positive ion mode. Matrix-matched calibration curves and external standards were used for the accurate quantification of the six AFTs and SMC. Good linear relationships were obtained, the correlation coefficients (

Background Prenatal infection is a substantial risk factor for neurodevelopmental disorders such as autism in offspring. We have previously reported that influenza vaccination (VAC) during early pregnancy contributes to neurogenesis and behavioral function in offspring. Results Here, we probe the efficacy of VAC pretreatment on autism-like behaviors in a lipopolysaccharide (LPS)-induced maternal immune activation (MIA) mouse model. We show that VAC improves abnormal fetal brain cytoarchitecture and lamination, an effect associated with promotion of intermediate progenitor cell differentiation in MIA fetal brain. These beneficial effects are sufficient to prevent social deficits in adult MIA offspring. Furthermore, whole-genome analysis suggests a strong interaction between Ikzf1 (IKAROS family zinc-finger 1) and neuronal differentiation. Intriguingly, VAC rescues excessive microglial Ikzf1 expression and attenuates microglial inflammatory responses in the MIA fetal brain. Conclusions Our study implies that a preprocessed influenza vaccination prevents maternal bacterial infection from causing neocortical lamination impairments and autism-related behaviors in offspring. Electronic supplementary material The online version of this article (10.1186/s12974-018-1252-z) contains supplementary material, which is available to authorized users.

Purpose: We investigated the mechanisms of how TGFβ pathway is activated by chemotherapeutics and whether a novel TGFβ trap called RER can block chemotherapeutics-induced TGFβ pathway activation and enhance their antitumor activity in gynecologic cancer. Patients and Methods: An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFβ signaling and function, and the effects of RER on chemotherapy-induced TGFβ signaling were determined in gynecologic cancer cells. Results: Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFβ signaling activation. Chemotherapy was found to activate TGFβ signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFβ1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial–mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFβ-like effects were due to the stimulation of TGFβ1 expression and secretion, and could all be abrogated by TGFβ inhibitors including a novel TGFβ trap protein called RER both in vitro and in vivo. Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer. Conclusions: Chemotherapeutics can stimulate TGFβ1 production and consequently enhance TGFβ signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFβ inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. Clin Cancer Res; 24(12); 2780–93. ©2018 AACR.

The Volume-Regulated Anion Channel (VRAC) is activated by cell swelling and plays a key role in cell volume regulation. VRAC is ubiquitously expressed in vertebrate cells and also implicated in many other physiological and cellular processes including fluid secretion, glutamate release, membrane potential regulation, cell proliferation, migration, and apoptosis. Although its biophysical properties have been well characterized, the molecular identity of VRAC remained a mystery for almost three decades. The field was transformed by recent discoveries showing that the leucine-rich repeat-containing protein 8A (LRRC8A, also named SWELL1) and its four other homologs form heteromeric VRAC channels. The composition of LRRC8 subunits determines channel properties and substrate selectivity of a large variety of different VRACs. Incorporating purified SWELL1-containing protein complexes into lipid bilayers is sufficient to reconstitute channel activities, a finding that supports the decrease in intracellular ionic strength as the mechanism of VRAC activation during cell swelling. Characterization of Swell1 knockout mice uncovers the important role of VRAC in T cell development, pancreatic β-cell glucose-stimulated insulin secretion, and adipocyte metabolic function. The ability to permeate organic osmolytes and metabolites is a major feature of VRAC. The list of VRAC substrates is expected to grow, now also including some cancer drugs and antibiotics even under non-cell swelling conditions. Therefore, a critical role of VRAC in drug resistance and cell–cell communication is emerging. This review summarizes the exciting recent progress on the structure-function relationship and physiology of VRAC and discusses key future questions to be solved.

Immune activation can exert multiple effects on synaptic transmission. Our study demonstrates the influence of neonatal vaccination on hippocampal synaptic plasticity in rats under normal physiological conditions. The results revealed that neonatal BCG vaccination enhanced synaptic plasticity. In contrast, HBV hampered it. Furthermore, we found that the cytokine balance shifted in favour of the T helper type 1/T helper type 2 immune response in BCG/HBV-vaccinated rats in the periphery. The peripheral IFN-γ:IL-4 ratio was positively correlated with BDNF and IGF-1 in the hippocampus. BCG raised IFN-γ, IL-4, BDNF and IGF-1 and reduced IL-1β, IL-6, and TNF-α in the hippocampus, whereas, HBV triggered the opposite effects.

Summary By releasing glutamate, astrocytes actively regulate synaptic transmission and contribute to excitotoxicity in neurological diseases. However, the mechanisms of astrocytic glutamate release have been debated. Here, we report non-vesicular release of glutamate through the glutamate-permeable volume-regulated anion channel (VRAC). Both cell swelling and receptor stimulation activated astrocytic VRAC, which requires its only obligatory subunit, Swell1. Astrocyte-specific Swell1 knockout mice exhibited impaired glutamatergic transmission due to the decreases in presynaptic release probability and ambient glutamate level. Consistently, the mutant mice displayed hippocampal-dependent learning and memory deficits. During pathological cell swelling, deletion of astrocytic Swell1 attenuated glutamate-dependent neuronal excitability and protected mice from brain damage after ischemic stroke. Our identification of a new molecular mechanism for channel-mediated glutamate release establishes a role for astrocyte-neuron interactions in both synaptic transmission and brain ischemia. It provides a rationale for targeting VRAC for the treatment of stroke and other neurological diseases associated with excitotoxicity.

Immunization with synthetic, preaggregated β-amyloid (Aβ) was the first treatment approach able to dramatically reduce brain Aβ pathology in Alzheimer's disease (AD) animal models. For the development of a safe vaccine, we investigated whether 4Aβ1-15 (four tandem repeats of GPGPG-linked Aβ1-15 sequences) had therapeutic effects in the APP/PS1 transgenic mice model of AD. We described the production of anti-Aβ antibodies in APP/PS1 mice immunized with 4Aβ1-15 mixed with MF59 adjuvant. The anti-Aβ antibody concentrations were increased which bound to AD plaques, markedly reduced Aβ pathology in transgenic AD mice and levels of intracerebral Aβ (soluble and insoluble), whereas increased serum Aβ levels. Immunization via 4Aβ1-15 (mainly of the IgG1 Class) may induce a non-inflammatory Th2 reaction. Immunohistochemistry analysis of MHC Class II and CD45 revealed that microglial cells were in a less activated state. Of note, 4Aβ1-15-immunized mice showed improved acquisition of memory compared with controls in a reference-memory Morris water-maze behavior test. The data identify the novel immunogen 4Aβ1-15 as a promising new tool for AD immunotherapy.

Neonatal Bacillus Calmette-Guerin (BCG) vaccination results in a positive effect on hippocampal neurogenesis and cognition. Serum cytokines are considered to be the chief culprit. In this study, serum from BCG-treated mice was identified as Th1 polarized serum. The serum showed an increased ratio of IFN-γ to IL-4 and decreased levels of TNF-α and IL-6. After Th1 polarized serum was injected intraperitoneally into postnatal mice, the levels of cytokines and ratio of IFN-γ to IL-4 in the serum and hippocampus of postnatal mice showed a similar alteration as those in Th1 polarized serum. This result indicated that the immune homeostatic milieu in postnatal mice was broken and the Th1 polarized systemic environment in the BCG-serum group was remodeled. The BCG-serum group displayed more BrdU+/DCX+ cells, BrdU+/NeuN+ cells, Nestin+ cells and better cognitive abilities. In neural stem cells, the Wnt7a/β-catenin signaling pathway was activated and exposure to the Wnt7a antagonist Dickkopf-1 inhibited BCG-serum-induced Wnt7a/β-catenin signaling, neurogenesis and cognitive function. Additionally, BCG-serum was associated with elevations in hippocampal brain-derived neurotrophic factor (BDNF) levels, and BDNF expression in the BCG-serum group was offset by Dickkopf-1 treatment. By rebalancing the Th1 polarized systemic environment in neonatal mice, it is possible that treatment with BCG-serum promotes hippocampal neurogenesis and improves cognitive functions, which are associated with Wnt7a/β-catenin-BDNF signaling.

Ectopic expression of lineage-specific transcription factors facilitates the conversion of mammalian somatic cells into dopaminergic (DA) neurons, which is a promising strategy for cell therapy of Parkinson's disease (PD). However, this approach still has some drawbacks limiting its clinical application due to the potential risks of integrating vectors into the host genome. Therefore, it is critical to seek a more desired approach to generate DA neurons derived from mammalian somatic cells. Here, we report that mouse embryonic fibroblasts (MEFs) can be efficiently converted into DA neurons by using small molecules along with specific growth factors. These neuron-like cells generate DA neuronal morphology, and acquire immunocytochemical and calcium imaging special for neuronal electrophysiological profile. More importantly, these converted cells can secrete dopamine, indicating that they are functionally similar to DA neurons. Taken together, our study might provide a promising cell source for treating PD by using chemical approach without introduction of exogenous transcription factors.

The immune system plays a vital role in brain development. The hepatitis B vaccine (HBV) is administered to more than 70% of neonates worldwide. Whether this neonatal vaccination affects brain development is unknown. Newborn C57BL/6 mice were injected intraperitoneally with HBV or phosphate-buffered saline. HBV induced impaired behavioral performances and hippocampal long-term potentiation at 8 weeks (w) of age without influence at 4 or 12w. At 6w, there was decreased neurogenesis, M1 microglial activation and a neurotoxic profile of neuroimmune molecule expression [increased tumor necrosis factor-α and reduced interferon (IFN)-γ, brain-derived neurotrophic factor and insulin-like growth factor-1] in the hippocampus of the HBV-vaccinated mice. In the serum, HBV induced significantly higher levels of interleukin (IL)-4, indicating a T helper (Th)-2 bias. Moreover, the serum IFN-γ/IL-4 ratio was positively correlated with the levels of neurotrophins and neurogenesis in the hippocampus at the individual level. These findings suggest that neonatal HBV vaccination of mice results in neurobehavioral impairments in early adulthood by inducing a proinflammatory and low neurotrophic milieu in the hippocampus, which follows the HBV-induced systemic Th2 bias.

Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2−/−) mice to specifically disturb somatic Ca2+ signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2−/− mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1−/− mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2−/− mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination. DOI: http://dx.doi.org/10.7554/eLife.15043.001, eLife digest Neighbouring neurons connect to each other and share information through structures known as synapses. As the brain develops, many synapses turn out to be redundant. Just like trees in a garden that need to be trimmed, these redundant synapses must be pruned in order to form the right pattern of connections between different neurons. Brain cells called astrocytes play a key role in synaptic pruning, but it is unclear exactly how astrocytes coordinate this process. One important way in which astrocytes communicate with neurons is through a process called calcium signaling, in which the movement of calcium ions into or out of the cell sets off a cascade of activity inside the astrocytes. Yang et al. have now studied developing mice that lacked a gene that is essential for calcium signaling in astrocytes. Two weeks after they were born, these mice still had redundant synapses that are normally lost after birth. However, injecting the developing brain with a substance called ATP prevented this defect and allowed synapses to be correctly pruned. This is likely to be because astrocytes also use ATP to communicate with neurons, and ATP compensated for the missing calcium signaling. The experiments also uncovered the specific structure – called the P2Y1 receptor – on the outer surface of a neuron that ATP latches on to in order to help remove synapses. Further work is now needed to reveal how activating the P2Y1 receptor coordinates synaptic removal. DOI: http://dx.doi.org/10.7554/eLife.15043.002

Mono-ADP-ribosyltransferase 2 (ART2) is found in mouse T cells and has mediated NAD-induced cell death (NICD) alongside the P2X7 pathway. We determined whether ART2 was expressed in mouse brain astrocytes and the possible function of the NAD-ART2-P2X7 pathway in astrocytes. Our results demonstrate that ART2 existed both in cultured mouse astrocytes and mouse brain slices. Exposure of astrocytes to the ART2 substrate, NAD, induced calcium elevation, which was blocked by ART2 and P2X7 inhibitors. ATP and NAD had an additive effect on calcium elevation. NICD in low-calcium conditions was blocked by ART2 and P2X7 inhibitors. The harmful effect of ATP on astrocytes was inhibited by P2X7 and ART2 inhibitors, meaning that endogenous NAD release may occur. Both NICD function and oxygen-glucose deprivation injury in mouse brain slices were also involved in the ART2-P2X7 pathway. Collectively, to our knowledge, our study provides the first evidence that ART2 exists in mouse brain astrocytes and NAD induces calcium elevation and astrocyte death by an ART2 and P2X7-mediated mechanism. The results suggest a novel approach for manipulating astrocyte death.

Biomarkers are lacking for identifying the switch of transforming growth factor-β (TGF-β) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-β to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-β. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-β-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-β-induced cell migration. Because TGF-β is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-β during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-β in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-β and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-β.

Dendritic structure is sensitive to changes in the environment during brain development. Accumulating evidence has demonstrated that early immune activation can significantly affect neuronal development. Our study concentrated on the morphological study of neural dendrites and spines in the hippocampal CA1 area using Diolistic labeling with Sholl analysis and fractal analysis. The results revealed that Bacille Calmette-Guerin (BCG) vaccination enhanced dendritic complexity, as reflected by the increased number of intersections, number of branch points and fractal dimension, and promoted neurite outgrowth. In addition, BCG increased the density and promoted the maturation of dendritic spines. The alterations in dendritic structure and spine morphology were observed at 2 and 4 w, but the differences were more apparent at 4 w than at 2 w. However, no significant difference was observed at 8 w. Furthermore, we observed that BCG increased the expression of hippocampal brain derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1). Hippocampal BDNF/IGF-1 was positively correlated with apical dendritic length, fractal dimension, and spine density. Taken together, we show in this study that neonatal BCG vaccination promotes dendritic development in developing hippocampal CA1 neurons, most likely by increasing the expression of BDNF and IGF-1 in the hippocampus.

The Bacillus Calmette-Guerin (BCG) vaccine is routinely administered to human neonates worldwide. BCG has recently been identified as a neuroprotective immune mediator in several neuropathological conditions, exerting neuroprotection in a mouse model of Parkinson's disease and slowing the progression of clinically isolated syndrome in patients with multiple sclerosis. The immune system is significantly involved in brain development, and several types of neonatal immune activations exert influences on the brain and behavior following a secondary immune challenge in adulthood. However, whether the neonatal BCG vaccination affects the brain in adulthood remains to be elucidated. In the present study, newborn C57BL/6 mice were injected subcutaneously with BCG (105 colony forming units) or phosphate-buffered saline (PBS). A total of 12 weeks later, the mice were injected intraperitoneally with 330 µg/kg lipopolysaccharide (LPS) or PBS. The present study reported that the neonatal BCG vaccination alleviated sickness, anxiety and depression-like behavior, lessened the impairments in hippocampal cell proliferation and downregulated the proinflammatory responses in the serum and brain that were induced by the adult LPS challenge. However, BCG vaccination alone had no evident influence on the brain and behavior in adulthood. In conclusion, the neonatal BCG vaccination alleviated the neurobehavioral impairments and neuroinflammation induced by LPS exposure in adult mice, suggesting a potential neuroprotective role of the neonatal BCG vaccination in adulthood.

We previously demonstrated that A(H1N1) influenza vaccine (AIV) promoted hippocampal neurogenesis and working memory in pregnant mice. However, the underlying mechanism of flu vaccination in neurogenesis and memory has remained unclear. In this study, we found that T lymphocytes were recruited from the periphery to the choroid plexus (CP) of the lateral and third (3rd) ventricles in pregnant mice vaccinated with AIV (Pre+AIV). Intracerebroventricular delivery of anti-TCR antibodies markedly decreased neurogenesis and the working memory of the Pre+AIV mice. Similarly, intravenous delivery of anti-CD4 antibodies to the periphery also down-regulated neurogenesis. Furthermore, AIV vaccination caused microglia to skew toward an M2-like phenotype (increased Arginase-1 and Ym1 mRNA levels), and elevated levels of brain-derived growth factor (BDNF) and insulin-like growth factor-1 (IGF-1) were found in the hippocampus, whereas these effects were offset by anti-TCR antibody treatment. Additionally, in the CP, the expression level of adhesion molecules and chemokines, which assist leukocytes in permeating into the brain, were also elevated after AIV vaccination of pregnant mice. Collectively, the results suggested that the infiltrative T lymphocytes in the CP contribute to the increase in hippocampal neurogenesis and working memory caused by flu vaccination, involving activation of the brain’s CP, M2 microglial polarization and neurotrophic factor expression.

The basal forebrain (BF) plays a crucial role in cortical activation. Our previous study showed that activation of cholinergic BF neurons alone is sufficient to suppress slow-wave sleep (SWS) and promote wakefulness and rapid-eye-movement (REM) sleep. However, the exact role of silencing cholinergic BF neurons in the sleep-wake cycle remains unclear. We inhibitied the cholinergic BF neurons genetically targeted with archaerhodopsin (Arch) with yellow light to clarify the role of cholinergic BF neurons in the sleep-wake cycle. Bilateral inactivation of cholinergic BF neurons genetically targeted with archaerhodopsin prolonged SWS and decreased the probability of awakening from SWS in mice. However, silencing these neurons changed neither the duration of wakefulness or REM sleep, nor the probability of transitions to other sleep-wake episodes from wakefulness or REM sleep. Furthermore, silencing these neurons for 6 h within the inactive or active period increased the duration of SWS at the expense of the duration of wakefulness, as well as increasing the number of prolonged SWS episodes (120-240 s). The lost wakefulness was compensated by a delayed increase of wakefulness, so the total duration of SWS and wakefulness during 24 h was kept stable. Our results indicate that the main effect of these neurons is to terminate SWS, whereas wakefulness or REM sleep may be determined by co-operation of the cholinergic BF neurons with other arousal-sleep control systems.

Hypertension is the most common cardiovascular disease, afflicting >30% of adults1. The cause of hypertension in most individuals remains unknown2,3, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure4. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia5–9. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

The common pathological hallmark of Alzheimer's disease (AD) is β-amyloid plaque deposition. The ideal therapy would reduce the Aβ burden with a low inflammatory immune response. Passive immunotherapy is an advanced treatment that dramatically reduces brain Aβ pathologies in AD animal models. The objective of our study was to observe the effects of 5C8H5, a novel monoclonal antibody derived from 4Aβ1-15, on brain Aβ pathology in an APP/PS1 mouse model of AD. Six-month-old transgenic mice were administered 5C8H5, 4Aβ1-15 or IgG, and same-aged wild-type untreated C57Bl/6J mice were employed as controls. Inflammatory factors and Aβ40/42 levels were detected by ELISA, while Aβ plaques, microglial cell activation, microhemorrhages and neurogenesis were evaluated by immunohistochemical staining. Compared with 4Aβ1-15-treated mice, the mice in the 5C8H5 group induced more Aβ clearance with less microglial cell activation in a niche of Th2-polarized immune response. The levels of proinflammatory factors, including IL-1β, IL-6, TNF-α and IFN-γ, were significantly decreased in the CNS, while the level of antiinflammatory IL-4 was increased. Moreover, the mice in the 5C8H5 group induced more neurogenesis without microhemorrhage exacerbation and thereby performed better in behavioral assays than did the 4Aβ1-15 group. In conclusion, the novel monoclonal antibody induces more Aβ clearance and less microglial cell activation in the absence of inflammation, accompanied by an increased Th2-polarized immune response, which makes it a more promising therapeutic strategy. These data provide evidence that passive immunity could alleviate pathologic Aβ alterations by modulating inflammation and should be pursued further for the treatment of AD.

TGF[beta]1, [beta]2, and [beta]3 are 25 kDa homodimeric polypeptides that play crucial non-overlapping roles in development, tumor suppression, and wound healing. They exhibit 70-82% sequence identity and transduce their signals by binding and bringing together the TGF[beta] type I and type II receptors, T[beta]RI and T[beta]RII. TGF[beta]2 differs from the other isoforms in that it binds T[beta]RII weakly and is dependent upon the co-receptor betaglycan for function. To explore the physicochemical basis underlying these differences, we generated a series of single amino acid T[beta]RII variants based on the crystal structure of the T[beta]RII:TGF[beta]3 complex and examined these in terms of their TGF[beta] isoform binding affinity and their equilibrium stability. The results showed that T[beta]RII Ile53 and Glu119, which contact TGF[beta]3 Val92 and Arg25, respectively, together with T[beta]RII Asp32, Glu55, and Glu75, which contact TGF[beta]3 Arg94, each contribute significantly, between 1 kcal mol-1 to 1.5 kcal mol-1, to ligand binding affinities. These contacts likely underlie the estimated 4.1 kcal mol-1 lower affinity with which T[beta]RII binds TGF[beta]2 as these three ligand residues are unchanged in TGF[beta]1 but are conservatively substituted in TGF[beta]2 (Lys25, Ile92, and Lys94). To test this hypothesis, a TGF[beta]2 variant was generated in which these three residues were changed to those in TGF[beta]s 1 and 3. This variant exhibited receptor binding affinities comparable to those of TGF[beta]s 1 and 3. Together, these results show that these three residues underlie the lowered affinity of TGF[beta]2 for T[beta]RII and that all isoforms likely induce assembly of the TGF[beta] signaling receptors in the same overall manner

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.

The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

Previous studies have suggested that TGF-β functions as a tumor promoter in metastatic, mesenchymal-like breast cancer cells and that TGF-β inhibitors can effectively abrogate tumor progression in several of these models. Here we report a novel observation with the use of genetic and pharmacological approaches, and murine mammary cell injection models in both syngeneic and immune compromised mice. We found that TGF-β receptor II (TβRII) knockdown in the MMTV-PyMT derived Py8119, a mesenchymal-like murine mammary tumor cell line, resulted in increased orthotopic tumor growth potential in a syngeneic background and a similar trend in an immune compromised background. Systemic treatment with a small-molecule TGF-β receptor I kinase inhibitor induced a trend towards increased metastatic colonization of distant organs following intracardiac inoculation of Py8119 cells, with little effect on the colonization of luminal-like Py230 cells, also derived from MMTV-PyMT tumors. Taken together, our data suggest that the attenuation of TGF-β signaling in mesenchymal-like mammary tumors does not necessarily inhibit their malignant potential, and anti-TGF-β therapeutic intervention requires greater precision in identifying molecular markers in tumors with an indication of functional TGF-β signaling. © 2013 Elsevier Ireland Ltd.