Genomic and non-genomic actions of estrogen on synaptic plasticity in SH-SY5Y cells

Estrogen modulates synaptic plasticity, an important mechanism of memory storage. Previously, we have reported that estrogen rapidly increases the expression of Arc (activity-regulated cytoskeleton associated protein), a key protein for synaptic plasticity, via non-genomic phosphoinositide-3 kinase (PI-3K)-, mitogen-activated protein kinase (MAPK)-, and estrogen receptor (ER)-dependent pathways in SH-SY5Y cells. The present study aimed to investigate the role of each ER subtype, α and β, in synaptic plasticity in SH-SY5Y cells. The specific agonist of ERβ (DPN) markedly induced Arc expression that mimics treatment with estrogen, but not ERα (PTT). Determination of subcellular localization of ERβ using immunocytochemistry shows that ERβ was retained in the cytoplasm of the untreated cells. In estrogen-treated cells, the membrane and cytosolic ERβ gradually decreased, while nuclear ERβ progressively increased in time-dependent manner, suggesting estrogen-dependent nuclear translocation of ERβ. Nuclear accumulation of ERβ at 6–12 h post-estrogen treatment, leads to increased PSD-95 and SYP mRNA expression, indicating the classical genomic estrogenic action on synaptic plasticity. However, the block of PI-3K signaling by Wortmannin partially suppressed estrogen (48 h)-induced PSD-95 and SYP expression, suggesting a crosstalk mechanism between genomic and non-genomic actions of estrogen on synaptic plasticity. Therefore, the estrogen-enhanced synaptic plasticity is ERβ-dependent and involves the crosstalk mechanism of non-genomic and genomic estrogenic actions.     

Amyloid-beta at sublethal level impairs BDNF-induced arc expression in cortical neurons

Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive dysfunction that probably due to a deficit in synaptic plasticity. One member of neurotrophins, brain-derived neurotrophic factor (BDNF), is known to be involved in the hippocampal long-term potentiation (LTP), a cellular model for learning and memory. Moreover, activity-regulated cytoskeleton-associated gene (Arc), an immediate early gene, is found to be a downstream effector of the BDNF signaling cascade. Inhibition of Arc protein synthesis impairs both the maintenance of LTP and the consolidation of long-term memory. In addition, the formation of senile plaques is a pathological feature in AD and mainly consists of the deposition of amyloid-beta (Abeta), a proteolytic product of amyloid precursor protein. Several studies concerning neurobehavioral performance have suggested that Abeta at sublethal levels interfere with the signaling cascades critical for synaptic plasticity and thus lead to the cognitive impairment in early stage of AD. Whether the BDNF-mediated Arc synthesis is impaired by sublethal Abeta in early AD is still unclear. Therefore, in the present study, primary cultures of neonatal rat cortical neurons were used to evaluate the effect of sublethal Abeta on the BDNF-induced Arc protein expression. Consistent with the literature, Arc, an indicator of synaptic plasticity, was induced by BDNF (25 ng/ml) in both dose- and time-dependent manners. After treating cultures with sublethal Abeta (5 microM), a significant suppression was observed on the level of BDNF-induced Arc protein expression. This result indicates that Abeta at sublethal level impairs the BDNF-mediated signaling in cortical neurons and thus underlies the deficits of synaptic plasticity occurred at the early stage of AD before significant neuronal loss.     

Effects of stress and adrenalectomy on activity-regulated cytoskeleton protein (Arc) gene expression

Activity-regulated cytoskeletal-associated protein (Arc) is an effector immediate early gene induced by novelty and involved in consolidation of long-term memory. Since activation of glucocorticoid receptors is a prerequisite for memory consolidation, we therefore aimed to study the effect of acute restraint stress on Arc gene expression in adrenalectomized rats. Acute stress produced a significant increase in Arc gene expression in the medial prefrontal cortex, but not in the parietal cortex or in the pyramidal cell layer of the hippocampus. The basal level of Arc mRNA in adrenalectomized animals was high in the medial prefrontal cortex and unaffected by acute stress in these animals. These data are consistent with the role of Arc as an integrative modulator of synaptic plasticity by emphasizing the potential role of stress and glucocorticoids in the control of Arc gene expression.     

Estrogen protects against amyloid-β toxicity by estrogen receptor α-mediated inhibition of Daxx translocation

Estrogen was shown to promote neuronal survival against several neurotoxic insults including β-amyloid (Aβ). The proposed mechanism includes the activation of the mitogen activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk), phosphatidylinositol 3-kinase/Akt pathways and the upregulation of antiapoptotic proteins. On the other hand, Aβ neurotoxicity depends on the activation of apoptosis signal-regulating kinase 1 (Ask1), and both Ask1 activity and Aβ toxicity are inhibited by thioredoxin-1 (Trx1). Here, we explored the possibility that estrogen could protect cells against Aβ(1-42) toxicity by inhibiting the Ask1 cascade or by modulating Trx1. Cytosolic translocation of death-associated protein Daxx was used as indicator of Ask1 activity. Using human SH-SY5Y neuroblastoma cells, 17β-estradiol (E2) and specific agonists for estrogen receptor (ER) α or β we demonstrated that nM concentrations of E2 protected against Aβ(1-42) by a mechanism depending upon ERα stimulation, Akt activation and Ask1 inhibition. Moreover, this protection would occur independently of ERβ and the induction of Trx1 expression. Our results emphasize the importance of Ask1 cascade in Aβ toxicity, and of ERα and Ask1 as targets for developing new neuroprotective drugs.     

SH-SY5Y细胞高表达糖原合成激酶3β对tau蛋白磷酸化和微管稳定性的影响

目的：构建高表达糖原合成激酶3β（GSK3β）的SH-SY5Y（人骨髓神经母细胞瘤细胞）转基因细胞模型,观察GSK3β高表达对宿主细胞tau蛋白磷酸化、微管稳定性的影响。 方法：构建GSK3β真核表达质粒,转染SH-SY5Y细胞,使GSK3β在SH-SY5Y细胞中得到高表达,应用蛋白免疫印迹方法,观察tau蛋白磷酸化、总tau蛋白、微管蛋白乙酰化水平的变化情况。 结果：GSK3β真核表达质粒转染SH-SY5Y细胞36 h后,GSK3β在SH-SY5Y细胞中的表达达到高峰,tau蛋白Ser199/202、Thr231、Thr205等位点的磷酸化水平在转染后48 h达到高峰; tau蛋白总量未有明显变化。转染后60 h,微管蛋白乙酰化水平明显减低。 结论：高表达GSK3β的SH-SY5Y细胞可使tau蛋白的磷酸化水平增高,从而降低tau蛋白结合和稳定微管蛋白的能力,导致微管蛋白乙酰化水平明显减低。     

“The seven sins” of the Hebbian synapse: Can the hypothesis of synaptic plasticity explain long-term memory consolidation?

Memorizing new facts and events means that entering information produces specific physical changes within the brain. According to the commonly accepted view, traces of memory are stored through the structural modifications of synaptic connections, which result in changes of synaptic efficiency and, therefore, in formations of new patterns of neural activity (the hypothesis of synaptic plasticity). Most of the current knowledge on learning and initial stages of memory consolidation ("synaptic consolidation") is based on this hypothesis. However, the hypothesis of synaptic plasticity faces a number of conceptual and experimental difficulties when it deals with potentially permanent consolidation of declarative memory ("system consolidation"). These difficulties are rooted in the major intrinsic self-contradiction of the hypothesis: stable declarative memory is unlikely to be based on such a non-stable foundation as synaptic plasticity. Memory that can last throughout an entire lifespan should be "etched in stone." The only "stone-like" molecules within living cells are DNA molecules. Therefore, I advocate an alternative, genomic hypothesis of memory, which suggests that acquired information is persistently stored within individual neurons through modifications of DNA, and that these modifications serve as the carriers of elementary memory traces.     

线粒体铁蛋白过表达抑制神经肿瘤细胞的增长与细胞铁代谢及细胞周期蛋白表达相关

目的 探讨过表达线粒体铁蛋白（MtFt） 抑制成神经母细胞瘤SH-SY5Y细胞的增殖机制。方法 以过表达MtFt的成神经母细胞瘤细胞MtFt-SY5Y为实验模型,野生型SH-SY5Y和pcDNA3.1-SY5Y（空质粒对照）为实验对照,运用流式细胞术、Western blotting技术等检测了MtFt对SH-SY5Y肿瘤细胞增殖的影响及铁代谢相关蛋白转铁蛋白受体1 （TfR1）,铁蛋白和细胞周期相关蛋白（cyclin）及其依赖性激酶（CDK）、cyclinD1、CDK4、cyclinE、CDK2的表达变化。结果 MtFt过表达通过调节细胞内铁代谢显著抑制了神经肿瘤细胞SH-SY5Y的增殖,与对照组相比,MtFt-SY5Y细胞增殖速度慢了近4倍。MtFt造成了细胞质内铁缺乏, TfR1表达显著上调,而铁蛋白H 亚基（H-ferritin）显著下调。同时 cyclinD1与CDK2蛋白表达显著降低,cyclinE蛋白表达显著上升,CDK4蛋白表达无显著性差异。结论 MtFt过表达能够显著抑制神经肿瘤细胞的生长,其机制可能是通过调节细胞内铁代谢,从而影响细胞周期相关蛋白及其周期蛋白激酶的表达。     

A-kinase anchoring proteins in amygdala are involved in auditory fear memory

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that bind the regulatory subunits of protein kinase A (PKA). AKAP binding to PKA regulates the phosphorylation of various proteins, some of which have been implicated in synaptic plasticity and memory consolidation. Here we show that the regulatory subunits of PKA are colocalized with AKAP150 (an AKAP isoform that is expressed in the brain) in the lateral amygdala (LA) and that infusion to the LA of the peptide St-Ht31, which blocks PKA anchoring onto AKAPs, impairs memory consolidation of auditory fear conditioning.     

Signal transduction and gene expression in cultured accessory olfactory bulb neurons

Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate alpha1 and alpha2 adrenergic receptors in combination with N-methyl-d-aspartic acid and tested expression of the immediate-early gene (IEG) c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by protein kinase C and MAPK.     

Inhibition of lipid peroxidation and protein oxidation by endogenous and exogenous antioxidants in rat brain microsomes in vitro

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic (DAergic) neurons in the substantia nigra and the subsequent depletion of dopamine (DA). This study assessed the protective effects of costunolide on DA-induced apoptosis in human DAergic SH-SY5Y cells, and its regulation of DA metabolism-associated gene and protein expression. Annexin V and propidium iodide (PI) staining using flow cytometric analysis (FACS) revealed that costunolide significantly protected human DAergic SH-SY5Y cells against DA-induced apoptosis. In addition, co-treatment of costunolide with DA in SH-SY5Y cells regulated DA metabolism-associated gene expression, as we observed an increase in both mRNA and protein levels of nuclear receptor related-1 (Nurr1), DA transporter (DAT), and vesicular monoamine transporter type 2 (VMAT2). In contrast, α-synuclein (ASYN) protein levels were decreased. Our findings suggest that costunolide has anti-apoptotic activity, presumably due to its regulatory effects on DA metabolism-associated genes. Therefore, costunolide could be considered as a candidate therapy for the treatment of Parkinson's disease.     

XAV939抑制人神经母细胞瘤细胞的增殖

目的探讨小分子抑制剂XAV939对人神经母细胞瘤(NB)细胞系SH-SY5Y细胞的抗增殖和诱导凋亡的作用,及其可能机制。方法采用MTT法检测XAV939对SH-SY5Y细胞活力的抑制作用,并确定最佳作用浓度。然后采用Annexin V-FITC法检测XAV939处理后早期凋亡细胞百分比,Hoechst33342染色法观察凋亡细胞核形态,克隆形成实验检测细胞体外增殖能力的变化。Western blotting检测Wnt/β-连环蛋白(β-catenin信号通路的关键蛋白和抗凋亡蛋白Bcl-2的变化。结果 MTT结果显示,1μmol/L XAV939作用24h后,SH-SY5Y细胞增殖的抑制率有明显上升。XAV939处理后48h及72h,凋亡细胞百分比显著高于对照组,且细胞呈现出不同程度的凋亡形态。此外,XAV939可显著降低SH-SY5Y细胞的体外克隆形成率,降低Wnt/β-catenin信号通路关键蛋白β-catenin,Cyclin D1和c-Myc及抗凋亡蛋白Bcl-2的表达。结论 XAV939可能部分通过抑制Wnt/β-catenin信号通路而抑制SH-SY5Y细胞的增殖。     

Abnormal expression of synaptic proteins and neurotrophin-3 in the Down syndrome mouse model Ts65Dn

Down syndrome (DS) results from triplication of the whole or distal part of human chromosome 21. Persons with DS suffer from deficits in learning and memory and cognitive functions in general, and, starting from early development, their brains show dendritic and spine structural alterations and cell loss. These defects concern many cortical brain regions as well as the hippocampus, which is known to play a critical role in memory and cognition. Most of these abnormalities are reproduced in the mouse model Ts65Dn, which is partially trisomic for the mouse chromosome 16 that is homologous to a portion of human chromosome 21. Thus, Ts65Dn is widely utilized as an animal model of DS. To better understand the molecular defects underlying the cognitive and particularly the memory impairments of DS, we investigated whether the expression of several molecules known to play critical roles in long-term synaptic plasticity and long-term memory in a variety of species is dysregulated in either the neonatal brain or adult hippocampus of Ts65Dn mice. We found abnormal expression of the synaptic proteins synaptophysin, microtubule-associated protein 2 (MAP2) and cyclin-dependent kinase 5 (CDK5) and of the neurotrophin-3 (NT-3). Both the neonatal brain and adult hippocampus revealed significant abnormalities. These results suggest that a dysregulation in the expression of neurotrophins as well as proteins involved in synaptic development and plasticity may play a potential role in the neural pathology of DS in humans.     

Regulation of hippocampal synaptic plasticity by cyclic AMP-dependent protein kinases

Protein kinases critically regulate synaptic plasticity in the mammalian hippocampus. Cyclic-AMP dependent protein kinase (PKA) is a serine–threonine kinase that has been strongly implicated in the expression of specific forms of long-term potentiation (LTP), long-term depression (LTD), and hippocampal long-term memory. We review the roles of PKA in activity-dependent forms of hippocampal synaptic plasticity by highlighting particular themes that have emerged in ongoing research. These include the participation of distinct isoforms of PKA in specific types of synaptic plasticity, modification of the PKA-dependence of LTP by multiple factors such as distinct patterns of imposed activity, environmental enrichment, and genetic manipulation of signalling molecules, and presynaptic versus postsynaptic mechanisms for PKA-dependent LTP. We also discuss many of the substrates that have been implicated as targets for PKA’s actions in hippocampal synaptic plasticity, including CREB, protein phosphatases, and glutamatergic receptors. Future prospects for shedding light on the roles of PKA are also described from the perspective of specific aspects of synaptic physiology and brain function that are ripe for investigation using incisive genetic, cell biological, and electrophysiological approaches.     

miR-449a过表达抑制人神经母细胞瘤细胞SH-SY5Y增殖并促进其凋亡

目的探讨miR-449a对人神经母细胞瘤细胞系SH-SY5Y的增殖和凋亡的影响。方法用Lipofectamine TM2000将miR-449a模似物或miR-449a对照转染至SH-SY5Y细胞,分为空白、miR-449a模似物和miR-449a对照SHSY5Y细胞组;实时荧光定量PCR(q-PCR)检测各组细胞中miR-449a表达;CCK8法检测细胞增殖;流式细胞仪检测细胞凋亡和周期,Western blot检测c-Myc蛋白和Bax/Bcl-2蛋白表达。结果 miR-449a模似物瞬时转染SH-SY5Y细胞后,miR-449a的表达水平明显高于正常对照组(P0.05);SH-SY5Y细胞增殖能力受到明显抑制(P0.05);凋亡率明显增加(P0.05);c-Myc蛋白表达显著降低(P0.05);细胞促凋亡蛋白Bax表达升高;抗凋亡蛋白Bcl-2表达降低(P0.05)。结论 miR-449a可通过c-Myc影响SH-SY5Y细胞的增殖和周期,通过调节Bax/Bcl-2影响其凋亡。     

Regulation and function of immediate-early genes in the brain: beyond neuronal activity markers

Long lasting forms of synaptic plasticity and long-term memory formation require new mRNA and protein synthesis. While activity-dependent expression of immediate-early genes has long been thought to account for such critical de novo macromolecular synthesis, experimental proof has been scarce until recently. During the past few decades, a growing number of genetic and molecular biological studies have started to elucidate essential roles of immediate-early genes in synaptic plasticity and cognitive functions. I here present an overview of the history and recent work on regulation and function of neuronal immediate-early genes, including Arc/arg3.1. This review provides a conceptual framework in which various immediate-early genes underlie several distinct processes required for long-term synaptic changes and memory formation.     

Electrophysiological correlates of adjustment process in anchoring effects

Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10muM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10muM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.