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Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe developmental delay, motor impairment, language and cognition deficits, and often with increased seizure activity. AS is caused by deficiency of UBE3A, which is both an E3 ligase and a cofactor for transcriptional regulation. We previously showed that the small conductance potassium channel protein SK2 is a UBE3A substrate, and that increased synaptic SK2 levels contribute to impairments in synaptic plasticity and fear-conditioning memory, as inhibition of SK2 channels significantly improved both synaptic plasticity and fear memory in male AS mice. In the present study, we investigated UBE3a-mediated regulation of synaptic plasticity and fear-conditioning in female AS mice. Results from both western blot and immunofluorescence analyses showed that synaptic SK2 levels were significantly increased in hippocampus of female AS mice, as compared to wild-type (WT) littermates. Like in male AS mice, long-term potentiation (LTP) was significantly reduced while long-term depression (LTD) was enhanced at hippocampal CA3-CA1 synapses of female AS mice, as compared to female WT mice. Both alterations were significantly reduced by treatment with the SK2 inhibitor, apamin. The shunting effect of SK2 channels on NMDA receptor was significantly larger in female AS mice as compared to female WT mice. Female AS mice also showed impairment in both contextual and tone memory recall, and this impairment was significantly reduced by apamin treatment. Our results indicate that like male AS mice, female AS mice showed significant impairment in both synaptic plasticity and fear-conditioning memory due to increased levels of synaptic SK2 channels. Any therapeutic strategy to reduce SK2-mediated inhibition of NMDAR should be beneficial to both male and female patients.

Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca

Icariin (ICA), extracted from Epimedium, is a flavonoid used in traditional Chinese medicine. Di(2-ethylhexyl) phthalate (DEHP) is a phthalate used in commercial products as a plasticizer that can influence the human endocrine and reproduction system. We previously found that ICA reversed DEHP-induced damage through the prevention of reactive oxygen species accumulation and promotion of testosterone secretion. Here we investigated the mechanisms of ICA in promoting testosterone secretion from murine Leydig cells. We used ICA, DEHP, the Akt agonist SC-79, the Akt inhibitor MK2206, and the Creb inhibitor KG501 to determine the effect of these treatments on the expression levels of the steroidogenic enzymes, Cyp11a1 and Hsd3b, which play critical roles in androgen production, in Leydig cells. Bioinformatic analysis was used to search for ICA-targeted proteins and their associated pathways. We found that icariin interacted with estrogen receptor on the cell membrane, leading to increased phosphorylation levels of Akt and Creb proteins and enhanced transcription of genes encoding steroidogenic enzymes and testosterone synthesis. We further investigated ICA activity in vivo using male mice pretreated with 100 mg/kg ICA and then treated with 750 mg/kg DEHP. ICA pretreatment reversed the reduced protein expression levels of Cyp11a1 and Hsd3b induced by DEHP in Leydig cells in vivo. Furthermore, while the phosphorylation levels of Akt and Creb were decreased in testes of mice exposed to DEHP alone, these effects were reversed by ICA pretreatment. These findings indicate that ICA promotes testosterone synthesis via the Esr1/Src/Akt/Creb/Sf-1 signaling pathway.

Background The tricho-rhino-phalangeal syndrome-1 gene (Trps1) is an atypical GATA family member. Although current studies of Trps1 mainly focus on tumors, whether Trps1 plays a role in the male reproductive system remains unknown. Objectives The purpose of this study was to elucidate the function of Trps1 in Leydig cells, indicating its regulatory mechanism on the cell cycle. Methods Gene-silencing technology, RNA-seq, RT-qPCR, and western blotting were used to evaluate the function of Trps1 in mouse primary Leydig cells and MLTC-1 cells. In addition, ChIP-basesets and ChIP-qPCR were employed to further assess the regulatory mechanism of Trps1 in MLTC-1 cells. Results Knockdown of Trps1 in Leydig cells significantly suppressed phosphorylation of Src and Akt and expression of Ccnd1, which was accompanied by impairment of cell proliferative ability. Trps1 may affect the cell cycle through the Src/Akt/Ccnd1 signaling pathway. In addition, Trps1 may bind to the promoter of Srcin1 to regulate its transcription, thus influencing Src phosphorylation levels and the proliferation of Leydig cells. Discussion and conclusion Src increases in Leydig cells during pubertal development, suggesting its functional involvement in differentiated adult Leydig cells. Inhibition of the Src/Akt pathway would reduce Ccnd1 expression. In the present study, we found that Trps1 may regulate the phosphorylation level of Src and Akt through Srcin1, targeting Ccnd1 to influence mouse Leydig cell proliferation. These findings shed light on the regulation of Trps1 on cell proliferation and differentiation of mouse Leydig cells. This article is protected by copyright. All rights reserved.

Enterovirus A71 (EV-A71) is the major cause of severe hand-foot-and-mouth diseases (HFMD), especially encephalitis and other nervous system diseases. EV-A71 capsid protein VP1 mediates virus attachment and is the important virulence factor in the EV-A71pathogenesis. In this study, we explored the roles of VP1 in the permeability of blood–brain barrier (BBB). Sera albumin, Evans blue, and dextran leaked into brain parenchyma of the 1-week-old C57BL/6J mice intracranially injected with VP1 recombinant protein. VP1 also increased the permeability of the brain endothelial cells monolayer, an in vitro BBB model. Tight junction protein claudin-5 was reduced in the brain tissues or brain endothelial cells treated with VP1. In contrast, VP1 increased the expression of virus receptor vimentin, which could be blocked with VP1 neutralization antibody. Vimentin expression in the VP1-treated brain endothelial cells was regulated by TGF-β/Smad-3 and NF-κB signal pathways. Moreover, vimentin over-expression was accompanied with compromised BBB. From these studies, we conclude that EV-A71 virus capsid protein VP1 disrupted BBB and increased virus receptor vimentin, which both may contribute to the virus entrance into brain and EV-A71 CNS infection.

Preimplantation embryo development is characterized by drastic nuclear reprogramming and dynamic stage-specific gene expression. Key regulators of this earliest developmental stage have not been revealed. In the present study, a “non-classical” nuclear-localization pattern of eIF1A was observed during early developmental stages of mouse preimplantation embryo before late-morula. In particular, eIF1A is most highly expressed in the nuclear of 2-cell embryo. Knockdown eIF1A by siRNA microinjection affected the development of mouse preimplantation embryo, resulted in decreased blastocyst formation rate. CDX2 protein expression level significantly down-regulated after eIF1A knockdown in morula stage. In addition, the mRNA expression level of Hsp70.1 was also decreased in 2-cell embryo. The results indicate an indispensable role of eIF1A in mouse preimplantation embryos.

The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca2+-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.

The first cell lineage differentiation occurs during the development of mouse 8-cell embryo to blastocyst. Akt is a potent kinase whose role during blastocyst formation has not been elucidated. In the present study, immunofluorescence results showed that the Akt protein was specifically localized to the outer cells of the morula. Akt-specific inhibitor MK2206 significantly inhibited mouse blastocyst formation and resulted in decreased expression of the trophectoderm marker Cdx2 and led to granular distribution of ERα in the cytoplasm. Furthermore, knockdown of ERα by siRNA microinjection can also lead to a decrease in the development rate of mouse blastocysts, accompanied by a decrease in the expression level of Yap protein. We conclude that Akt may be indispensable for the first cell lineage differentiation of mouse.

Infertility caused by environmental pollution is becoming a global problem, but an effective prevention or treatment is lacking. Icariin (ICA) is a flavonoid used in traditional Chinese medicine. The present study investigated the possible roles of ICA in preventing testicular dysfunction caused by di(2-ethylhexyl) phthalate (DEHP), one of the most studied environmental endocrine disruptors. Cultured mouse Leydig cells were pretreated with ICA and exposed to DEHP to determine ICA effects upon cell proliferation, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm), testosterone levels and the expression of transcription factor SF-1 and steroidogenic enzymes (CYP11, 3β-HSD and 17β-HSD), which play critical roles in androgen production. Our results showed that ICA reversed the adverse effect of DEHP on Leydig cell proliferation, and decreased ROS levels and elevated Δψm levels. Also, ICA promoted testosterone production and up-regulated the expression of SF-1 and steroidogenic enzymes. We investigated ICA actions in vivo, using male mice administrated DEHP followed by ICA. Exposure to DEHP decreased epididymal sperm counts and disrupted seminiferous tubules, and both of these effects were reversed by ICA treatment. These results showed that the mechanisms of ICA in protecting mouse testes against DEHP-induced damage involves the prevention of ROS accumulation and promotion of testosterone secretion.

While calpain-1 activation is required for LTP induction by theta burst stimulation (TBS), calpain-2 activation limits its magnitude during the consolidation period. A selective calpain-2 inhibitor applied either before or shortly after TBS enhanced the degree of potentiation. In the present study, we tested whether the selective calpain-2 inhibitor, Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2 (C2I), could enhance learning and memory in wild-type (WT) and calpain-1 knock-out (C1KO) mice. We first showed that C2I could reestablish TBS-LTP in hippocampal slices from C1KO mice, and this effect was blocked by PD98059, an inhibitor of ERK. TBS resulted in PTEN degradation in hippocampal slices from both WT and C1KO mice, and C2I treatment blocked this effect in both mouse genotypes. Systemic injection of C2I 30 min before training in the fear-conditioning paradigm resulted in a biphasic dose-response curve, with low doses enhancing and high doses inhibiting freezing behavior. The difference between the doses needed to enhance and inhibit learning matches the difference in concentrations producing inhibition of calpain-2 and calpain-1. A low dose of C2I also restored normal learning in a novel object recognition task in C1KO mice. Levels of SCOP, a ERK phosphatase known to be cleaved by calpain-1, were decreased in dorsal hippocampus early but not late following training in WT mice; C2I treatment did not affect the early decrease in SCOP levels but prevented its recovery at the later time-point and prolonged ERK activation. The results indicate that calpain-2 activation limits the extent of learning, an effect possibly due to temporal limitation of ERK activation, as a result of SCOP synthesis induced by calpain-2-mediated PTEN degradation.

SummaryA CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous or heterozygous CAPN1-null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knockout (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1-mediated cleavage of PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans.

NSI-189 Phosphate, (4-benzylpiperazin-1-yl)-[2-(3-methyl-butylamino)pyridin-3-yl] methanone is a new chemical entity under development for the treatment of MDD, based upon preclinical data demonstrating stimulation of neurogenesis of human hippocampus-derived neural stem cells in vitro and in mouse hippocampus in vivo. Previous studies have examined the tolerability and efficacy of NSI-189 for treating major depressive disorder (MDD). NSI-189 has shown significant potential as a treatment for MDD, with concurrent improvement of a cognition scale in a small double-blind, placebo-controlled study. The current study evaluated its possible application for the treatment of Angelman Syndrome. Incubation of acute hippocampal slices from wild-type mice with NSI-189 resulted in a time- and dose-dependent increase in the magnitude of long-term potentiation (LTP) elicited by theta burst stimulation (TBS). The same protocol enhanced TBS-induced LTP in acute hippocampal slices from AS mice. A short treatment with daily injections of NSI-189 in AS mice reversed impairments in cognitive and motor functions, while it slightly enhanced performance of WT mice. The effects of NSI-189 on synaptic plasticity and cognitive functions were associated with activation of the TrkB and Akt pathways. These results suggest that NSI-189 could represent a potential treatment for AS patients.

Accumulating evidence indicates that the lysosomal Ragulator complex is essential for full activation of the mechanistic target of rapamycin complex 1 (mTORC1). Abnormal mTORC1 activation has been implicated in several developmental neurological disorders, including Angelman syndrome (AS), which is caused by maternal deficiency of the ubiquitin E3 ligase UBE3A. Here we report that Ube3a regulates mTORC1 signaling by targeting p18, a subunit of the Ragulator. Ube3a ubiquinates p18, resulting in its proteasomal degradation, and Ube3a deficiency in hippocampus of AS mice results in increased lysosomal localization of p18 and other members of the Ragulator-Rag complex, such as RagA, and increased mTORC1 activity. P18 down-regulation by siRNA or shRNA in hippocampal CA1 neurons of AS mice reduces elevated mTORC1 activity and improves long-term potentiation (LTP) and dendritic spine maturation. Our results indicate that Ube3a-mediated regulation of p18 and subsequent mTORC1 signaling is critical for typical synaptic plasticity and dendritic spine development.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deficiency of maternally inherited UBE3A, an ubiquitin E3 ligase. Despite recent progress in understanding the mechanism underlying UBE3A imprinting, there is no effective treatment. Further investigation of the roles played by UBE3A in the central nervous system (CNS) is needed for developing effective therapies.This review covers the literature related to genetic classifications of AS, recent discoveries regarding the regulation of UBE3A imprinting, alterations in cell signaling in various brain regions and potential therapeutic approaches. Since a large proportion of AS patients exhibit comorbid autism spectrum disorder (ASD), potential common molecular bases are discussed.Advances in understanding UBE3A imprinting provide a unique opportunity to induce paternal UBE3A expression, thus targeting the syndrome at its 'root.' However, such efforts have yielded less-than-expected rescue effects in AS mouse models, raising the concern that activation of paternal UBE3A after a critical period cannot correct all the CNS defects that developed in a UBE3A-deficient environment. On the other hand, targeting abnormal downstream cell signaling pathways has provided promising rescue effects in preclinical research. Thus, combined reinstatement of paternal UBE3A expression with targeting abnormal signaling pathways should provide better therapeutic effects.

Epidemiological studies suggest an association between pesticides and the incidence of Parkinson’s disease (PD). Individuals are likely to be exposed to numerous natural or synthetic environmental agents by ingestion, inhalation, or skin contact. Here, we describe a novel environment-contact administration of rotenone model, in which male C57BL/6 mice (15 per group per time-point) were placed in one bedding-free, rotenone-applied cage for 2 h every day over a period of 2–6 weeks, mimicking the common ways a person may be exposed to pesticides. Our results showed that rotenone exposure had no detrimental effect on body weights of mice during 6 weeks, nor did it cause systemic toxicity (HPLC analysis of rotenone in blood and brain, as well as complex I activity measurements in brain and muscle), but it caused significant impairments in motor function (open field test, pole test, and rotarod test) from 4 weeks that were responsive to apomorphine. Accordingly, rotenone caused significant dopamine depletion from the striatum (HPLC analysis), nigrostriatal degeneration (quantitative tyrosine hydroxylase immunohistochemistry and western blot), and accumulation of α-synuclein in the substantia nigra and striatum (α-synuclein immunohistochemistry) in a time-dependent manner. In addition, rotenone-exposed mice also developed deficits in gastrointestinal and olfactory function (fecal pellet output and buried food pellet test) prior to the motor dysfunction. Furthermore, we observed that α-synuclein accumulated in the anterior olfactory nucleus and the enteric nervous system at 2 weeks. In summary, this novel rotenone model was able to reproduce many key aspects of PD progression. Therefore, it provides new insight into how environmental factors could trigger PD and provides a useful tool for studying PD pathogenesis and testing neuroprotective strategies.

Summary Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca 2+ sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function.

Calpain-1 (CANP1) has been shown to play a critical role in synaptic plasticity and learning and memory, as its deletion in mice results in impairment in theta-burst stimulation- (TBS) induced LTP and various forms of learning and memory. Likewise, PHLPP1 (aka SCOP) has also been found to participate in learning and memory, as PHLPP1 overexpression impairs hippocampus-dependent learning. We previously showed that TBS-induced LTP was associated with calpain-1 mediated truncation of PHLPP1.To better understand the roles of these 2 genes in synaptic plasticity and learning and memory, we generated a double knockout (DKO) mouse by crossing the parent strains. Surprisingly, DKO mice exhibit normal TBS-induced LTP, and the learning impairments in fear conditioning and novel object or novel location recognition were absent in the DKO mice. Moreover, TBS-induced ERK activation in field CA1 of hippocampal slices, which is impaired in both single deletion mice, was restored in the DKO mice. These results further strengthen the roles of both CANP1 and PHLPP1 in synaptic plasticity and learning and memory, and illustrate the complexities of the interactions between multiple pathways participating in synaptic plasticity.

Emerging evidence is implicating abnormal activation of the mechanistic target of rapamycin (mTOR) pathway in several monogenetic neuropsychiatric disorders, including Angelman syndrome (AS), which is caused by deficiency in maternally inherited UBE3A. Using an AS mouse model, we show that semi-chronic rapamycin treatment improves long-term potentiation (LTP) and actin polymerization in hippocampal slices, spine morphology, and fear-conditioning learning. Activity of mTORC1 and of its downstream substrate, S6K1, was increased in hippocampus of AS mice. However, mTORC2 activity, as reflected by PKCα levels, was decreased. Both increased mTORC1 and decreased mTORC2 activity were reversed by semi-chronic rapamycin treatment. Acute treatment of hippocampal slices from AS mice with rapamycin or an S6K1 inhibitor, PF4708671, improved LTP, restored actin polymerization, and normalized mTORC1 and mTORC2 activity. These treatments also reduced Arc levels in AS mice. Treatment with Torin 1, an inhibitor of both mTORC1 and mTORC2, partially rescued LTP and actin polymerization in hippocampal slices from AS mice, while partially impairing them in wild-type (WT) mice. Torin 1 decreased mTORC1 and increased mTORC2 activity in slices from AS mice but inhibited mTORC1 and decreased mTORC2 in WT mice. Finally, an mTORC2 activator, A-443654, increased hippocampal LTP in AS mice and actin polymerization in both WT and AS mice. Collectively, these results indicate that events set in motion by increased mTORC1 and decreased mTORC2 activities, including increased Arc translation and impaired actin remodeling, are crucial in AS pathogenesis. Therefore, selectively targeting these two master kinase complexes may provide new therapeutic approaches for AS treatment.

Rhizoleucinoside (1), a unique rhamnolipid-amino alcohol hybrid, was isolated from the rhizobial symbiont bacterium Bradyrhizobium sp. BTAi1. Compound 1 features a rare rhamnolipid core attached to an unprecedented leucinol moiety. Its structure and absolute configuration were determined by spectroscopic analysis, tandem mass spectrometry, chemical degradation, and application of the Marfey's method. Compound 1 possesses moderate cytotoxicity to BV-2 murine microglia and highly aggressive proliferating immortalized (HAPI) rat microglia cells.

Effect of (+)-epi-clausenamide and (-)-epi-clausenamide on synaptic transmission in CA1 region of hippocampal slice was compared in this study. (+)-epi-Clausenamide showed more potency than (-)-clausenamide on either induction or maintenance of long term potentiation (LTP). Effect of (+)-epi-clausenamide on potentiating basic synaptic transmission was also superior to (-)-clausenamide. However, (-)-epi-clausenamide showed only slight effects on synaptic transmission, suggesting that the effect of (+)-epi-clausenamide and (-)-epi-clausenamide on synaptic transmission depended on their chirality. Calcium influx was not involved in (+)-epi-clausenamide facilitating synaptic transmission in this study. (+)-epi-Clausenamide might promote glutamate release through the activation of Synapsin I(Ser9) to activate the downstream effectors which play a key role in synaptic plasticity. Elucidating the mechanism of each optical isomer of clausenamide by electrophysiological methods provided basis for therapeutic strategies for neurological disorders.

Clausenamide is a chiral compound isolated from leaves of the traditional Chinese herb Clausena lansium (lour) Skeels. It has been shown that (-)clausenamide, but not (+)clausenamide, improved learning and memory in amnesia animal models. However, the precise mechanism of clausenamide's actions remains unknown. Here we used an electrophysiological approach to observe the effect of (-)clausenamide on facilitating field excitatory postsynaptic potential (f-EPSP) in the CA1 area of hippocampal slices from rats. The results showed that (-)clausenamide enhanced synaptic transmission at doses 0.1, 1 and 10 μM. The increase of f-EPSP induced by (-)clausenamide was completely inhibited by preincubation with nimodipine (L-voltage-dependent calcium channel blocker, 10 μM), but there was no change when nimodipine was added after (-)clausenamide application. However, ryanodine (ryanodine receptors blocker, 100 μM) attenuated the slope of f-EPSP before or after (-)clausenamide incubation. The data suggested that (-)clausenamide promoted calcium influx to trigger intracellular calcium release which was responsible for potentiating synaptic transmission. Intracellular calcium release induced by (-)clausenamide promoted the activation of CaMKIIα at concentrations of 0.1, 1 and 10 μM, and pretreatment with KN93 (CaMKIIα inhibitor, 10 μM) completely blocked the enhancement of synaptic transmission induced by (-)clausenamide. cAMP response element-binding protein (CREB) was activated by (-)clausenamide and inhibited by KN93 preincubation, but H89 (PKA inhibitor, 10 μM) had no effect, indicating that (-)clausenamide facilitated synaptic transmission by a PKA-independent pathway. Collectively, (-)clausenamide facilitated synaptic transmission by promoting calcium influx to trigger intracellular calcium release, subsequently activating CaMKIIα-CREB signal pathway.

Growing evidence has implicated glial anomalies in the pathophysiology of major depression disorder (MDD). Gap junctional communication is a main determinant of astrocytic function. However, it is unclear whether gap junction dysfunction is involved in MDD development. This study investigates changes in the function of astrocyte gap junction occurring in the rat prefrontal cortex (PFC) after chronic unpredictable stress (CUS), a rodent model of depression. Animals exposed to CUS and showing behavioral deficits in sucrose preference test (SPT) and novelty suppressed feeding test (NSFT) exhibited significant decreases in diffusion of gap junction channel-permeable dye and expression of connexin 43 (Cx43), a major component of astrocyte gap junction, and abnormal gap junctional ultrastructure in the PFC. Furthermore, we analyzed the effects of typical antidepressants fluoxetine and duloxetine and glucocorticoid receptor (GR) antagonist mifepristone on CUS-induced gap junctional dysfunction and depressive-like behaviors. The cellular and behavioral alterations induced by CUS were reversed and/or blocked by treatment with typical antidepressants or mifepristone, indicating that the mechanism of their antidepressant action may involve the amelioration of gap junction dysfunction and the cellular changes may be related to GR activation. We then investigated the effects of pharmacological gap junction blockade in the PFC on depressive-like behaviors. The results demonstrate that carbenoxolone (CBX) infusions induced anhedonia in SPT, and anxiety in NSFT, and Cx43 mimetic peptides Gap27 and Gap26 also induced anhedonia, a core symptom of depression. Together, this study supports the hypothesis that gap junction dysfunction contributes to the pathophysiology of depression.

Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder characterized by the selective loss of nigrostriatal dopaminergic neurons. However, the molecular pathways leading to the dopaminergic neuron degeneration have remained obscure until recently. Reports demonstrated that reduction of brain-derived neurotrophic factor (BDNF) was involved in the etiology and pathogenesis of PD, but its mechanism has not been elucidated. alpha-Synuclein has a causal role in Parkinson's disease, and could interfere with transcriptional regulation of dopamine neurons. In this study, alpha-synuclein overexpression was found to decrease the expression of BDNF, and also to suppress the transactivation of nuclear factors of activated T-cells (NFAT) and cAMP response element binding protein (CREB), both of which regulate BDNF expression. Furthermore, overexpressed alpha-synuclein could associate with protein kinase C (PKC) and impair its activity. Meanwhile glycogen synthase kinase-3beta (GSK3beta) was activated and extracellular signal-regulated protein kinase (ERK) activity was inhibited by overexpression of alpha-synuclein; both of them were downstream kinases of PKC. Therefore, the impaired PKC signal pathway caused by alpha-synuclein overexpression might account at least partially for the down-regulation of BDNF.

Seven new neolignan glycosides ( 1- 7), two arylglycerol glycosides ( 8, 9), and 18 known glycosides have been isolated from an ethanolic extract of the root of Iodes cirrhosa. Their structures including absolute configurations were determined by spectroscopic and chemical methods. Based on analysis of the NMR data of threo and erythro 8-4'-oxyneolignans and arylglycerols in different solvents, the validity of J 7,8 and Deltadelta C8-C7 values to distinguish threo and erythro derivatives was discussed. In the in vitro assays, compound 4 and liriodendrin ( 17) both showed activity against glutamate-induced PC12 cell damage at 10 (-5) M.

Angelman syndrome (AS) is a neurogenetic disorder caused by deficiency of maternally expressed ubiquitin-protein ligase E3A (UBE3A), an E3 ligase that targets specific proteins for proteasomal degradation. Although motor function impairment occurs in all patients with AS, very little research has been done to understand and treat it. The present study focuses on Ube3A deficiency-induced alterations in signaling through the mechanistic target of rapamycin (mTOR) pathway in the cerebellum of the AS mouse model and on potential therapeutic applications of rapamycin. Levels of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTOR, were increased in AS mice compared with wild-type mice; however, TSC2 inhibitory phosphorylation was also increased. Correspondingly, levels of phosphorylated/active mTOR were increased. Phosphorylation of the mTORC1 substrates S6 kinase 1 (S6K1) and S6 was elevated, whereas that of the mTORC2 substrates AKT and N-myc downstream regulated 1 was decreased, suggesting enhanced mTORC1 but inhibited mTORC2 signaling. Semi-chronic treatment of AS mice with rapamycin not only improved their motor performance but also normalized mTORC1 and mTORC2 signaling. Furthermore, inhibitory phosphorylation of rictor, a key regulatory/structural subunit of the mTORC2 complex, was increased in AS mice and decreased after rapamycin treatment. These results indicate that Ube3A deficiency leads to overactivation of the mTORC1–S6K1 pathway, which in turn inhibits rictor, resulting in decreased mTORC2 signaling in Purkinje neurons of AS mice. Finally, rapamycin treatment also improved dendritic spine morphology in AS mice, through inhibiting mTORC1 and possibly enhancing mTORC2-mediated regulation of synaptic cytoskeletal elements. Collectively, our results indicate that the imbalance between mTORC1 and mTORC2 activity may contribute to synaptic pathology and motor impairment in AS.

To reduce ammonium and nitrite in aquaculture water, an isolate of the denitrifying bacterium Pseudomonas stutzeri, SC221-M, was obtained. The effects of various nitrogen and carbon sources, the ratio of carbon to nitrogen and temperature on bacterial growth, denitrification rates and the expression levels of nirS and nosZ in SC221-M were studied. The following conditions were determined to be optimal for growth and denitrification in SC221-M: NaNO2 as the nitrogen source, sodium citrate as the carbon source, a carbon to nitrogen ratio range of 4-8, and a temperature range of 20-35°C. Subsequently, SC221-M and the Bacillus cereus BSC24 strain were selected to generate microbial preparations. The results showed that addition of the microbial preparations decreased various hydrochemical parameters, including total dissolved solids, ammonium, nitrite, total nitrogen and the chemical oxygen demand. Nitrogen removal rates were highest on day 9; the removal rates of BSC24, SC221-M, a mixed preparation and a 3× mixed preparation were 24.5%, 26.6%, 53.9% and 53.4%, respectively. The mixed preparation (SC221-M+BSC24) was more effective at removing nitrogen than either the SC221-M or BSC24 preparation. Roche 454 pyrosequencing and subsequent analysis indicated that the control and other groups formed separate clusters, and the microbial community structure in the water changed significantly after the addition of microbial preparations. These results indicate that the addition of microbial preparations can improve both the water quality and microbial community structure in an experimental aquaculture system. P. stutzeri strain SC221-M and its related microbial preparations are potential candidates for the regulation of water quality in commercial aquaculture systems.

The mammalian/mechanistic target of rapamycin (mTOR) exists in two complexes, mTORC1 and mTORC2. Both mTORC1 and mTORC2 contain the mTOR kinase and three other common components: mLST8, deptor, and Tti1/Tel. Additionally, mTORC1 contains PRAS40 and raptor, while mTORC2 contains mSIN1, protor, and rictor [1]. Through integration of a variety of signals, including trophic factors, energy statuses, and NMDA and metabotropic glutamate receptors, mTOR plays critical roles in both brain development and synaptic plasticity. Although a wealth of knowledge has been accumulated regarding the function and regulation of mTORC1, much less information is available for mTORC2 due to its insensitivity to rapamycin. Recent studies, performed mostly with cell lines, have suggested that mTORC1 is activated by growth factors, amino acids, oxygen, and energy statuses, while mTORC2 is mainly stimulated by growth factors. Our recent studies have revealed that mTOR signaling is abnormal in the cerebellum of the mouse model for Angelman syndrome (AS) [2]. AS is a genetic neurodevelopmental disorder characterized by severe developmental delay, language and cognition deficits, motor impairment and a happy demeanor. Several lines of evidence have established deficiency in expression of the maternally inherited UBE3A gene as the cause for AS [3]. The AS mice have maternal Ube3A deficiency and display the major phenotypes of AS, including memory and motor deficits, and impairment in synaptic plasticity [4, 5]. In our recent study, we found that while mTORC1 activity is increased in the cerebellum of AS mice, mTORC2 activation is reduced. Moreover, increased mTORC1 activation was associated with inhibition of TSC2, which, together with TSC1 and TBC1D7, forms the major inhibitory regulator of mTORC1. This result was surprising since experiments with cell lines have shown TSC2 undergoes UBE3A-dependent degradation. However, we found that TSC2 inhibition was due to increased phosphorylation of an inhibitory site mediated by a rapamycin-sensitive kinase. The imbalance between mTORC1 and mTORC2 is reminiscent to what has been reported in cells lacking the TSC1/2 complex, where inappropriate overactivation of mTORC1 blocks mTORC2-mediated AKT phosphorylation in response to growth factors. Persistent mTORC1 activation is generally postulated to inhibit AKT via its downstream kinase, S6K1, which phosphorylates and inhibits either insulin receptor substrate-1 or members of the mTORC2 complex, rictor at Thr1135 and mSIN1 at Thr86 and Thr398. However, further studies showed that, in some cases, TSC1/2 deficiency results in mTORC2 inhibition in an mTORC1-independent manner, indicating that the TSC1/2 complex may directly stimulate mTORC2 [6]. We found that S6K1-mediated inhibitory phosphorylation of rictor was also increased in AS mice [2]. Thus, reduced mTORC2 activity in AS mice could be a consequence of either decreased stimulation from TSC2 or increased inhibition of rictor by S6K1. Since rapamycin treatment corrected both lower AKT phosphorylation and TSC2 inhibition, mTORC1-S6K1 overactivation is likely a key step underlying abnormal mTOR regulation in AS mice (Fig. ​(Fig.1).1). How Ube3A deficiency results in abnormal mTOR signaling is not completely clear. It is likely that Ube3A normally imposes a constitutive suppression of mTORC1; lack of Ube3A would then set in motion the abnormal regulation of the mTOR pathway, since rapamycin treatment normalized the activities of both mTORC1 and mTORC2 activities. Rapamycin treatment also corrected abnormalities in dendritic spine morphology of Purkinje neurons and motor function in AS mice [2], indicating that imbalanced mTORC1 and mTORC2 activation contributes, at least in part, to motor dysfunction in AS. Of notice, a recent study has shown that knockout of rictor, either in whole brain or specifically in Purkinje neurons, resulted in changes in neuronal morphology, especially in Purkinje neurons, in a PKC-dependent manner [7]. Another study showed that conditional deletion of rictor in postnatal forebrain excitatory neurons selectively impaired both long-term memory and long-term synaptic plasticity, due to impaired actin polymerization [8]. These results indicate that mTORC2 plays important roles in maintaining neuronal morphology through regulation of the cytoskeletal network. A balanced mTORC1 and mTORC2 activation, with the former controlling protein synthesis and cell proliferation, and the latter regulating cytoskeleton remodeling and cell survival, is necessary for the brain to develop typical circuits and function at optimal levels. Figure 1 Abnormal mTOR signaling in the cerebellum of Angelman syndrome mice Our results also raise additional questions. For instance, it is well known that mTORC2 activates mTORC1 through AKT phosphorylation, yet our results showed mTORC1 overactivation, despite reduced mTORC2-AKT activation. Could this runaway mTORC1 activation due to an ongoing autophosphorylation? Are TSC2 inhibition or other unidentified factors involved? We showed that mTORC2 inhibition was due to mTORC1S6K1-mediated inhibitory phosphorylation of rictor and possibly TSC2 as well. It remains to be determined how general this regulation is in different brain regions, and whether additional mutual interactions occur at different levels of mTOR signaling cascades. Finally, it is noteworthy that mTOR activity is increased in Fragile X, tuberous sclerosis, and Down's syndromes, but decreased in Rett's syndrome (although these syndromes all show various degrees of intellectual disability). However, in all these variants of autism syndrome disorders, it is not clear whether alterations in mTOR signaling are due to modifications of mTORC1, mTORC2, or both. Further studies directed at these issues should provide a better understanding of these developmental neurologic disorders and reveal potential new therapeutic targets.

Parkinson’s disease (PD) is a common neurodegenerative disease, and its etiology remains obscure. Increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing PD and inflammation is the early incident during the process of PD. In this study, we measure the pro-inflammatory cytokines by enzyme-linked immunosorbnent assay and RT-PCR methods; analyze the reactive oxygen species by DCFH-DA; detected nuclear factor κB (NFκB) translocation by western blot and immunofluorescence methods; and analyze the phosphorylation of mitogen-activated protein (MAP) kinase and protein level of Nurr1 by western blot. Results showed that rotenone could induce tumor neurosis factor α (TNFα) and interleukin 1β (IL-1β) release from BV-2 cells, enhance TNFα and IL-1β mRNA levels in substantia nigra lesioned by rotenone; also, rotenone could increase the phosphorylation of inhibitor of κB (IκB), extracellular regulated protein kinase , c-Jun N-terminal kinase, p38 MAP kinases and promote p65 subunit of NFκB translocation to nuclear; at the same time, rotenone could decrease the protein level of Nurr1 in nuclear. So, rotenone exerted toxicity through activating microglia, and its mechanism might be associated with NFκB signal pathway.

This study was conducted to evaluate the effects of Bacillus subtilis SC02 supplement on water quality, microbial community diversity and structure in a grass carp (Ctenopharyngodon idellus) culture. Our selected strain, B. subtilis SC02, significantly reduced ammonia, nitrite and total nitrogen levels in water over an extended period compared with the control group. Pyrosequencing showed that the Shannon diversity index (Shannon) and species richness estimators (Chao) of the treatment group were higher, indicating that bacterial richness was significantly increased in the treatment group. The phyla Proteobacteria, Bacteroidetes and Actinobacteria were dominant in the treatment groups, accounting for 45, 21.9 and 21.9 % of the sequence reads, respectively. However, in sharp contrast, the control fishes were predominantly occupied by Proteobacteria (82.1 %) and Firmicutes (8.4 %). At the genus level, the microbial communities were different between the control and treatment groups, although the two groups shared similar genera. Additionally, some genera (such as Tepidimonas, Variovorax, Roseomonas, Rubritepida, Nitrobacter, etc.) only appeared in the treatment group, and many other genera only existed in the control group. Therefore, we conclude that the addition of the SC02 strain in water improves water quality, which may ultimately be a result of changes in microbial community diversity in grass carp cultures.

To investigate the effect and underlying mechanisms of polygalasaponin F (PGSF), a triterpenoid saponin isolated from Polygala japonica, on long-term potentiation (LTP) in hippocampus dentate gyrus (DG) of anesthetized rats. Population spike (PS) of hippocampal DG was recorded in anesthetized male Wistar rats. PGSF, the NMDAR inhibitor MK801 and the CaMKII inhibitor KN93 were intracerebroventricularly administered. Western blotting analysis was used to examine the phosphorylation expressions of NMDA receptor subunit 2B (NR2B), Ca2+/calmodulin-dependent kinase II (CaMKII), extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB). Intracerebroventricular administration of PGSF (1 and 10 μmol/L) produced long-lasting increase of PS amplitude in hippocampal DG in a dose-dependent manner. Pre-injection of MK801 (100 μmol/L) or KN93 (100 μmol/L) completely blocked PGSF-induced LTP. Furthermore, the phosphorylation of NR2B, CaMKII, ERK, and CREB in hippocampus was significantly increased 5–60 min after LTP induction. The up-regulation of p-CaMKII expression could be completely abolished by pre-injection of MK801. The up-regulation of p-ERK and p-CREB expressions could be partially blocked by pre-injection of KN93. PGSF could induce LTP in hippocampal DG in anesthetized rats via NMDAR activation mediated by CaMKII, ERK and CREB signaling pathway.

The aim of this study was to investigate the cognition-enhancing activity and underlying mechanisms of a triterpenoid saponin (polygalasaponin XXXII, PGS32) isolated from the roots of Polygala tenuifolia Willd. The Morris water maze was used to evaluate the spatial learning and memory of mice. To detect the basic properties of synaptic transmission and long-term potentiation (LTP) in the dentate gyrus of rats, electrophysiological recordings were made of evoked potentials. Western blotting analysis and immunofluorescence assays were used to determine the phosphorylation of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), synapsin I and the expression of brain derived neurotrophic factor (BDNF). When administered at 0.125, 0.5, or 2 mg/kg, PGS32 could significantly prevent scopolamine-induced cognitive impairments in mice. Intracerebroventricular (icv) administration of PGS32 greatly enhanced basic synaptic transmission in the dentate gyrus of rats and induced LTP. In primary hippocampal neurons, as well as in the hippocampus of maze-trained mice, PGS32 activated the mitogen-activated protein (MAP) kinase cascade by promoting phosphorylation of ERK, CREB and synapsin I. The expression of BDNF was also greatly enhanced in the hippocampus. Our findings suggest that PGS32 can improve hippocampus-dependent learning and memory, possibly through improvement of synaptic transmission, activation of the MAP kinase cascade and enhancement of the level of BDNF. Therefore, PGS32 shows promise as a potential cognition-enhancing therapeutic drug.