Stable expression of amiloid protein precursor gene in SH- EPI cells transfected with nicotinic acetylcholine receptor α7 subtype gene

The senile plaques in the brain are thought to contribute to the pathogenesis of Alzheimer＇s disease （AD） and are mainly consisted of β - amyloid peptide, which is the proteolytic product of amyloid precursor protein （APP）. Previous work suggested that nicotine could effectively reduce β - amyloid peptide aggregation in the brain of animal models and improve the cognition impairment, indicating that the nicotinic acetylcholine receptors （nAChR） might play an important part in the function of memory and cognition and might be a target in the therapy of Alzheimer＇s disease. The α7 nicotinic acetylcholine receptors are highly expressed in hippocampus and in cholinergic neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer＇s disease. To test whether the α7 nicotinic acetylcholine receptors can block the processing of amyloid precursor protein into β - amyloid peptide, we transfected human APP695 into the native nAChR - null SH - EP1 human epithelial cells which had been transfected with the gene of α7nAChR and confirmed their expression by RT - PCR and Western Blot.     

Pharmacological and immunochemical characterization of α2＊ nicotinic acetylcholine receptors （nAChRs） in mouse brain

Aim： α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2＊ nAChRs in these mouse brain regions. Methods： α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing （α2＊） nAChRs in these two regions. Results： [125Ⅰ]Epibatidine （200 pmol/L） autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus （by 4.8±1.7 and 92＋26 fmol·mg^-1 protein, respectively）. Pharmacological characterization using the β2-selective drug A85380 to inhibit [125Ⅰ]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2＊ nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good （〉90% of β2＊ and 〉80% of β4＊ nAChRs were routinely recovered）. Conclusion： Immunoprecipitation experiments indicated that interpeduncular nucleus α2＊ nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits.     

Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors （nAChRs）, they are grouped into either heteromeric （comprising both a and β subunits） or homomeric （comprising only a subunits） receptors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the a-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals： （1） direct calcium influx through the nAChRs, （2） indirect calcium influx through voltage-dependent calcium channels （VDCCs） which are activated by the nAChR-mediated depolarization, and （3） calciuminduced calcium release （CICR） （triggered by the first two sources） from the endoplasmic reticulum （ER） through the ryanodine receptors and inositol （1,4,5）-triphosphate receptors （IP3Rs）. Downstream signaling events mediated by nAChR- mediated calcium responses can be grouped into instantaneous effects （such as neurotransmitter release, which can occur in milliseconds after nAChR activation）, short-term effects （such as the recovery of nAChR desensitization through cellular signaling cascades）, and long-term effects （such as neuroprotection via gene expression）. In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understanding the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.     

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

APP modulator regulates amyloid precursor protein processing in vitro by increasing a-secretase activity： Therapeutic potential in Aizheimer＇s disease？

The beta amyloid cascade has been at the forefront of the hypothesis used to describe the pathogenesis of Alzheimer＇s disease （AD） . It is generally accepted that drugs that can regulate the processing of the amyloid precursor protein （APP） toward the non-amyloidogenic pathway may have a therapeutic potential in AD.     

Reduction of RhoC is involved in the decreased migration of neural stem/precursor cells

OBJECTIVE Alzheimer′s disease(AD) is mainly characterized by a progressive loss of neurons and the deposition of beta-amyloid peptides(Aβ).It was demonstrated that transplanted and endogenous neural stem cells or neural precursor cells can survive,migrate,and differentiate into neurons.Previously we found that Aβ42 could disturb the migratory ability of neural stem/precursor cells.We therefore hypothesized that Aβ42 may affect some important proteins through Rho pathway and result in the decreased migration of neural stem/precursor cells.METHODS AND RESULTS We applied siRNA technology to knock down the expression of RhoC in neural stem/precursor cells and found that the expression of RhoC was down-regulated;Trans well assay was used to measure the migratory ability of the neural stem/precursor cells and the result showed that the migratory ability was disturbed when the expression of RhoC was down-regulated;WRW4 was used for inhibiting the effects of the Aβ42 through the FPRL1.CONCLUSION The reduction of RhoC was involved in the decreased migration of neural stem cells.     

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

Cysteine-rich peptides from the venom of cone snails （Conus） target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors （nAChRs） found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson＇s disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.     

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit： pharmacological properties of ligand binding and function

Aim： To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors （α7 nAChRs） with those of native nAChRs containing α7 subunit （α7＊ nAChRs） in rat hippocampus and cerebral cortex. Methods： We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7＊ nAChRs in rat hippocampus and cerebral cortex. We used [125Ⅰ]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording. Results： The newly established cell line, KXα7R1, expresses homomeric α7 nAChRs that bind [125Ⅰ]-α-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kd values of native rat α7＊nAChRs from hippocampus （Kd=0.28±0.03 nmol/L） and cerebral cortex （Kd=0.33±0.05 nmol/L）. Using whole-cell current recording, the homomeric α7 nAChRs expressed in the cells were activated by acetylcholine and （-）-nicotine with EC50 values of 280±19μmol/L and 180±40μmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, a-bungarotoxin （IC50=19±2 nmol/L） and methyllycaconitine （IC50=100±10 pmol/L）. A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7＊receptors in rat brain, but it also revealed several notable differences. Conclusion： This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7＊ nAChRs.     

Effects of tanshinone on neuropathological changes induced by amyloid β-peptide(1-40)injection in rat hippocampus

INTRODUCTION Alzheimer’s disease (AD) is characterized by neu-ronal loss and extracellular senile plaque, whose majorconstituentis amyloid β-peptide(Aβ), a 39-43 amino acidspeptide derived from amyloid precursor protein (APP).It is widely believed that the cellular actions of Aβ areresponsible for the neuronal cell loss observed in ADand play a causal role in the pathogenesis of AD[1,2].Aggregated Aβ and its activefragmentshave beenshownto have clear neurotoxic effect[3]. …     

Mysterious α6-containing nAChRs： function,pharmacology, and pathophysiology

Neuronal nicotinic acetylcholine receptors （nAChRs） are the superfamily of ligand-gated ion channels and widely expressed throughout the central and peripheral nervous systems, nAChRs play crucial roles in modulating a wide range of higher cognitive functions by mediating presynaptic, postsynaptic, and extrasynaptic signaling. Thus far, nine alpha （α2-α10） and three beta （β2,β3, and β4） subunits have been identified in the CNS, and these subunits assemble to form a diversity of functional nAChRs. Although α4β2- and α7-nAChRs are the two major functional nAChR types in the CNS, α6＊-nAChRs are abundantly expressed in the midbrain dopaminergic （DAergic） system, including mesocorficolimbic and nigrostriatal pathways, and particularly present in presynaptic nerve terminals. Recently, functional and pharmacological profiles of α6＊-nAChRs have been assessed with the use of α6 subunit blockers such as α-conotoxin MII and PIA, and also by using α6 subunit knockout mice. By modulating DA release in the nucleus accumbens （NAc） and modulating GABA release onto DAergic neurons in the ventral tegmental area （VTA）, α6＊-nAChRs may play important roles in the mediation of nicotine reward and addiction. Furthermore, α6＊-nAChRs in the nigrostriatal DAergic system may be promising targets for selective preventative treatment of Parkinson＇s disease （PD）. Thus, α6＊-nAChRs may hold promise for future clinical treatment of human disorders, such as nicotine addiction and PD. In this review, we mainly focus on the recent advances in the understanding of α6＊-nAChR function, pharmacology and pathophysiology.     

Cocaine potently blocks neuronal α3β4 nicotinic acetylcholine receptors in SH-SY5Y cells

Cocaine is one of the most abused illicit drugs worldwide.It is well known that the dopamine(DA)transporter is its major target;but cocaine also acts on other targets including nicotinic acetylcholine receptors(nAChRs).In this study,we investigated the effects of cocaine on a special subtype of neuronal nAChR,α3β4-nAChR expressed in native SH-SY5Y cells.α3β4-nAChR-mediated currents were recorded using whole-cell recordings.Drugs were applied using a computer-controlled U-tube drug perfusion system.We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20μM.Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5μM.Kinetic analysis showed that cocaine acceleratedα3β4-nAChR desensitization,which caused a reduction of the amplitude of nicotine-induced currents.Co-application of nicotine and cocaine(1.5μM)depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value,suggesting a non-competitive mechanism.The cocaine-induced inhibition of nicotine response exhibited both voltage-and use-dependence,suggesting an open-channel blocking mechanism.Furthermore,intracellular application of GDP-βS(via recording electrode)did not affect cocaine-induced inhibition,suggesting that cocaine did not alter receptor internalization.Moreover,intracellular application of cocaine(30μM)failed to alter the nicotine response.Finally,cocaine(1.5μM)was unable to inhibit the nicotine-induced inward current in heterologous expressedα6/α3β2β3-nAChRs andα4β2-nAChRs expressed in human SH-EP1 cells.Collectively,our results suggest that cocaine is a potent blocker for nativeα3β4-nAChRs expressed in SH-SY5Y cells.     

Cellular trafficking of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors （nAChRs） play critical roles throughout the body. Precise regulation of the cellular loca- tion and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs.     

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells

Aim： Somatostatin receptor subtype 2 （SSTR2） is the principal mediator of somatostatin＇s （SST） antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved.
Methods： SSTR2 expression levels were determined by qRToPCR in several tumor cell lines. Then, a plasmid plRES2-EGFP-SSTR2 （pSlG） was constructed and stably transfected into MCF-7 cells （MCF-7/pSIG）. After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor （EGFR） expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT.
Results： The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G1/S cell cycle arrest. Furthermore the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSlG cells.
Conclusion： Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G1/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.     

Nicotinic mechanisms influencing synaptic plasticity in the hippocampus

Nicotinic acetylcholine receptors （nAChRs） are expressed throughout the hippocampus, and nicotinic signaling plays an important role in neuronal function. In the context of learning and memory related behaviors associated with hippocampal function, a potentially significant feature of nAChR activity is the impact it has on synaptic plasticity. Synaptic plasticity in hippocampal neurons has long been considered a contributing cellular mechanism of learning and memory. These same kinds ofcellular mechanisms are a factor in the development of nicotine addiction. Nicotinic signaling has been demonstrated by in vitro studies to affect synaptic plasticity in hippocampal neurons via multiple steps, and the signaling has also been shown to evoke synaptic plasticity in vivo. This review focuses on the nAChRs subtypes that contribute to hippocampal synaptic plasticity at the cellular and circuit level. It also considers nicotinic influences over long-term changes in the hippocampus that may contribute to addiction.     

α-Hederin inhibits growth of lung cancer A549 cells in vitro and in vivo by decreasing c-Myc and HIF-1α dependent glycolysis

OBJECTIVE α-Hederin is an effective component of the traditional Chinese medicine Pulsatilla chinensis,which has been reported to exert many pharmacological activities. However, the effect of α-hederin on metabolism is still unclear. This study aimed to illuminate the role of α-hederin in glucose metabolism in lung cancer cells and investigate the molecular mechanism of α-hederin. METHODS CCK8 and colony formation assays were employed to assess the anti-proliferative effects induced by α-hederin. Glucose uptake, ATP generation, and reduced lactate production were measured using kits, and an A549 tumor xenograft mouse model of lung cancer was used to assess the in vivo antitumor effect of α-hederin(5, 10 mg·kg~(-1)). Glycolytic-related key enzymes were detected by Western blotting and immunohistochemical staining. RESULTS Cell proliferation was significantly inhibited by α-hederin in a dose-dependent manner and that α-hederin inhibited glucose uptake and ATP generation and reduced lactate production. Furthermore, α-hederin remarkably inhibited hexokinase 2(HK2), glucose transporters 1(GLUT1), pyruvate kinase M2(PKM2), lactate dehydrogenase A(LDHA), monocarboxylate transporter(MCT4), c-Myc, and hypoxia inducible factor-1α(HIF-1α) protein expression. Using inhibitors, we proved that α-hederin inhibits glycolysis by inhibiting glycolytic regulators. Moreover, a tumor xenograft mouse model of lung cancer further confirmed that α-hederin inhibits lung cancer growth via inhibiting glycolysisin vivo. CONCLUSION α-Hederin inhibits the growth of non-small cell lung cancer A549 cells by inhibiting glycolysis.The mechanism of glycolysis inhibition includes α-hederin inhibiting the expression of the glycolytic regulatory factors HIF-1α and c-Myc.     

Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum

Aim： There is increasing evidence indicating that embryonic stem （ES） cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is neces- sary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. Methods： One week after the induction of El4 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen stor- age of the differentiated cells was detected by Periodic acid-Schiff （PAS） reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results： In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were αfetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. Conclusion： The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.