Syntaxin蛋白对神经母瘤细胞分化的影响及机制研究

Syntaxin是特异性地分布在神经细胞突触前质膜上的一种多结构域蛋白,它是细胞质膜融合的关键性蛋白,但Syntaxin在神经细胞分化过程中的作用尚未阐明。本实验旨在探讨Syntaxin蛋白对神经母瘤细胞分化的影响及其影响机制。通过在小鼠的神经母瘤细胞（N2a）中过表达不同的Syntaxin蛋白突变体,统计细胞的分化率、突起分支数和突起总长度等参数,来观察Syntaxin蛋白及其突变体对神经细胞分化的影响。通过实验结果得知Syntaxin蛋白促进神经母瘤细胞分化的作用位点在其氨基端的Habc结构域,主要影响细胞的分化突起数目和突起总长度,对细胞分化率无显著作用。     

大鼠液压冲击脑损伤脑干c—jun mRNA表达的定位观察

目的：研究大鼠中度侧位液压冲击脑损伤时脑干c-jun mRNA及其表达产物Jun变化规律。方法：雄性SD大鼠,随机分为正常对照组、手术对照组和损伤组。损伤组动物均给以0.2MPa液压冲击脑损伤,按冲击后处死时间不同再分为5min、15min、30min、1h、2h、4h、8h和12h组。应用免疫组织化学和原位杂交方法观察c-jun在脑干的表达。结果：脑冲击后15min-12h,Jun阳性细胞数逐渐增多。冲击后5min,c-jun mRNA表达开始增强,2h表达最强,然后逐渐减弱。结论：侧位液压冲击脑损伤后c-jun在脑干表达迅速增强,持续时间较长。     

ERK1/2在高温致神经管畸形神经上皮细胞中的表达变化

目的 探讨高温致神经管畸形(NTDs)作用的分子机制,为防治NTDs的发生提供理论依据.方法 在高温致金黄地鼠NTDs模型的基础上,应用免疫荧光染色技术,观察NTDs发生过程中p-ERK1/2在鼠胚神经上皮细胞中的表达变化.结果 对照组和实验组孕鼠在高温水浴处理后16、24h,p-ERK1/2免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后36、60h,p-ERK1/2表达部位出现了由细胞浆向细胞核的转移;高温处理后,p-ERK1/2在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱.结论 ERK1/2参与胚胎神经管的发育过程,其表达降低在高温致神经管畸形的发生中起重要作用.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运(已撤稿2016-01-13)

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

血小板源性生长因子受体α与高温致神经管畸形发生关系的研究

目的探讨PDGFR-α在高温致神经管畸形(NTDs)中的作用.方法在高温致神经管畸形动物模型上,采用免疫组织化学和图像分析技术,研究血小板源性生长因子受体α(PDGFR-α)在发育不同阶段的神经上皮中的表达,并观察高温对其表达的影响.结果在正常对照组,PDGFR-α广泛分布于神经管及其周围组织中;在高温致畸组,神经上皮中PDGFR-α的表达明显减弱,甚至不表达.结论 PDGFR-α的表达与神经管正常发育密切相关,其表达的减少可能是高温致NTDs机制中的重要环节.     

小鼠局部脑损伤对海马区神经元基因表达的影响

目的:探讨局部脑损伤对小鼠海马区Bax,Bel-2基因表达的影响.方法:40只BALB/c小鼠随机等分成正常组与脑损伤组,用免疫组织化学ABC法检测小鼠海马区Bax,Bcl-2的表达情况.结果:Bax,Bcl-2的免疫性物主要分布于海马区,胞浆染色.小鼠创伤性脑损伤24小时后,神经元Bax,Bel-2的平均灰度分别为(43.6±3.3)和(54.6±4.2),低于正常组,差异有统计学意义(P<0.05).结论:脑损伤致海马区Bax,Bcl-2的表达下降.     

灯盏花注射液抗新生鼠缺氧缺血性脑损伤的作用及对Bcl-2、Bax蛋白表达的影响

目的：观察灯盏花注射液对新生大鼠缺氧缺血脑损伤（HIBD）的保护作用及对Bcl-2、Bax蛋白表达的影响。方法：采用新生7日龄SD大鼠缺氧缺血性脑损伤模型,设立假手术组、缺氧缺血脑损伤模型组、灯盏花注射液治疗大、中、小剂量组、无菌注射用水对照组。采用硫堇染色、免疫组织化学染色的方法测定各组大鼠海马CA1区神经元密度、组织学分级及凋亡相关基因Bcl-2、Bax蛋白表达情况,并计数各时间点Bcl-2、Bax免疫阳性细胞数目及测定积分光密度值。结果：假手术组,大鼠海马CA1区无锥体细胞缺失,未见明显免疫阳性细胞。与假手术组比较,缺氧缺血脑损伤模型组、无菌注射用水对照组Bcl-2、Bax蛋白表达均于3 d时达到高峰（与其余各时间点比较差别有显著意义P〈0.05）,神经元密度明显降低,组织学分级显著增高,积分光密度值增加。灯盏花治疗组,与无菌注射用水对照组比较,Bcl-2蛋白表达进一步增加,积分光密度值增加;而Bax蛋白表达则减少,积分光密度值降低;神经元密度显著高于对照组,组织学分级明显降低。结论：灯盏花注射液可能是通过上调Bcl-2表达,抑制Bax表达,减轻缺氧缺血引起的神经元凋亡及迟发性神经元死亡。     

海仁酸致痫大鼠海马组织AMPA受体GluR2表达的变化

目的　为了研究AMPA受体在癫痫发生中的作用。方法　本研究用免疫组织化学方法观察了海仁酸致痫大鼠海马组织AMPA GluR2受体的表达变化。结果　在侧脑室注射海仁酸后 1h ,4h ,12h ,2 4h及 7d ,大鼠海马CA3区及齿状回GluR2的表达明显减弱 ,显微图像分析 :与对照组相比 ,KA 4h ,KA 12h ,KA 2 4h ,KA 7d组大鼠海马组织GluR2阳性神经元平均光密度值降低 ,差异有显著性 (P <0 0 5 )。结论　在癫痫发作过程中AMPA受体 GluR2亚单位表达改变可能与癫痫发作导致的神经元损伤有密切关系。     

新生大鼠慢性脑缺血损伤中氯离子通道ClC2的表达及其与白质发育影响作用的研究

目的探讨新生大鼠缺血缺氧损伤后氯离子(Cl-)通道ClC2表达及其阻断剂DIDS对脑白质发育的影响作用。方法采用新生3-5dSD大鼠,以改良Levine法建立慢性脑缺血缺氧模型。运用RT-PCR、活性氧检测和Western Blot检测结合髓鞘卢卡斯快蓝与免疫组织化学染色等技术,对比观察新生缺血缺氧脑损伤前后白质中ClC2的表达变化;分析其与氧化应激、炎症反应的相关性;对比观察损伤前后及损伤后应用DIDS阻断ClC2表达对脑白质髓鞘发育和细胞凋亡的影响。结果1新生缺血缺氧脑损伤后白质中ClC2mRNA和蛋白表达均显著增高(P0.01);伴白质组织活性氧浓度显著升高(P0.01);和iNOS、TNF-αmRNA的表达增加(P0.01)。在胼胝体等白质区髓鞘染色较正常明显浅淡,促凋亡蛋白caspase-3与ClC2阳性双标细胞数目则明显增多(P0.01)。2损伤早期应用DIDS后,ClC2mRNA和蛋白、活性氧浓度、iNOS、TNF-αmRNA的表达以及caspase-3表达等均较损伤组明显降低(P0.05);白质区髓鞘特异染色逐渐加深。结论新生缺血缺氧脑损伤后白质区Cl-通道表达升高与氧化应激、炎症反应呈正相关;可导致由脑白质中caspase-3介导的细胞凋亡增多和髓鞘发育受阻。伤后早期应用DIDS阻断Cl-通道可降低caspase-3介导的凋亡发生,提示早期使用DIDS可部分保护缺血缺氧损伤后的OLs细胞。     

Altered complexin expression in psychiatric and neurological disorders: cause or consequence?

Complexins play a critical role in the control of fast synchronous neurotransmitter release. They operate by binding to trimeric SNARE complexes consisting of the vesicle protein Synaptobrevin and the plasma membrane proteins Syntaxin and SNAP-25, which are key executors of membrane fusion reactions. SNARE complex binding by Complexins is thought to stabilize and clamp the SNARE complex in a highly fusogenic state, thereby providing a pool of readily releasable synaptic vesicles that can be released quickly and synchronously in response to an action potential and the concomitant increase in intra-synaptic Ca(2+) levels. Genetic elimination of Complexins from mammalian neurons causes a strong reduction in evoked neurotransmitter release, and altered Complexin expression levels with consequent deficits in synaptic transmission were suggested to contribute to the etiology or pathogenesis of schizophrenia, Huntington's disease, depression, bipolar disorder, Parkinson's disease, Alzheimer's disease, traumatic brain injury, Wernicke's encephalopathy, and fetal alcohol syndrome. In the present review I provide a summary of available data on the role of altered Complexin expression in brain diseases. On aggregate, the available information indicates that altered Complexin expression levels are unlikely to have a causal role in the etiology of the disorders that they have been implicated in, but that they may contribute to the corresponding symptoms.     

Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13

Priming of synaptic vesicles (SVs) is essential for synaptic transmission. UNC-13 proteins are required for priming. Current models propose that UNC-13 stabilizes the open conformation of Syntaxin, in which the SNARE helix is available for interactions with Synaptobrevin and SNAP-25. Here we show that Tomosyn inhibits SV priming. Tomosyn contains a SNARE motif, which forms an inhibitory SNARE complex with Syntaxin and SNAP-25. Mutants lacking Tomosyn have increased synaptic transmission, an increased pool of primed vesicles, and increased abundance of UNC-13 at synapses. Behavioral, imaging, and electrophysiological studies suggest that SV priming was reconstituted in unc-13 mutants by expressing a constitutively open mutant Syntaxin, or by mutations eliminating Tomosyn. Thus, priming is modulated by the balance between Tomosyn and UNC-13, perhaps by regulating the availability of open-Syntaxin. Even when priming was restored, synaptic transmission remained defective in unc-13 mutants, suggesting that UNC-13 is also required for other aspects of secretion.     

Identification of the minimal protein domain required for priming activity of Munc13-1

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.     

Multiple functional domains are involved in tomosyn regulation of exocytosis

Tomosyn is a cytoplasmic protein that was shown to bind to Syntaxin1 and SNAP-25 through an R-SNARE domain, forming a complex that is almost identical in structure to the neuronal SNARE complex. Tomosyn inhibits exocytosis in various cell types and these effects were attributed to direct competition between tomosyn's SNARE domain and Synaptobrevin/VAMP. In the present study, we investigated the contribution of different domains of tomosyn to its activity. We show that a tomosyn mutant that lacks the entire SNARE domain is a potent inhibitor of vesicle priming, similar to the full-length tomosyn. The SNARE domain of tomosyn failed to inhibit exocytosis, indicating that this domain is not required for the inhibition. In contrast, over-expression of a N-terminally truncated mutant did not lead to inhibition of exocytosis although this mutant still bound to Syntaxin. Our results indicate that tomosyn can inhibit exocytosis independently of its SNARE interaction with Syntaxin and that the integrity of the WD40-domain is crucial for tomosyn's inhibitory function. Furthermore, we demonstrate that the entire N-terminal region of tomosyn, the WD40-repeats and the linker, is required for tomosyn's inhibitory effect.     

Developmental and spatial expression pattern of syntaxin 13 in the mouse central nervous system

Vesicular transport involves SNARE (soluble- N-ethylmaleimide-sensitive-factor-attachment-protein-receptor) proteins on transport vesicles and on target membranes. Syntaxin 13 is a SNARE enriched in brain, associated with recycling endosomes; its overexpression in PC12 cells promotes neurite outgrowth. This suggests an important role for receptor recycling during neuronal differentiation. Here we describe the spatiotemporal pattern of syntaxin 13 expression during mouse brain development. During early embryogenesis (E12-E15), it was found in the forebrain ventricular zone and in primary motor and sensory neurons in the brainstem, spinal cord and sensory ganglia. In the forebrain at E15, syntaxin 13 was not detected in neuroblasts in the intermediate zone of the embryonic hemispheric wall, while there was labeling in cortical neurons in deeper layers starting at E15-18, and progressively in later-generated neurons up to layer II around P6. Syntaxin 13 reached maximal expression in all brain divisions at about P7, followed by a decrease, with heterogeneous neuron populations displaying various staining intensities in adult brain. While usually restricted to the soma of neurons, we transiently detected syntaxin 13 in dendrites of pyramidal neurons during the first postnatal week. In conclusion, the developmentally regulated syntaxin 13 expression in various neuronal populations is consistent with its involvement in endocytic trafficking and neurite outgrowth.     

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.     

Escape from tolerance of organic nitrate by induction of cytochrome P450.

The mechanism of organic nitrate tolerance is poorly defined. We studied the rat P450-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified P450 isoforms relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. The hypotensive effect of an nitroglycerin (NTG) bolus injection was abolished in rats that had been previously provided a continuous 48 h infusion of NTG. This effect was accompanied by a gradual but marked decrease in plasma and urinary nitrate levels following a peak at 18-24 h. Nitrate tolerance was reversible; the decline in the hypotensive effect and P450 levels observed after 2 d of continuous infusion was followed by restoration to control levels 2 d after cessation of the infusion. Similarly, the hypotensive action disappeared in P450-depleted, and -inhibited rats. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer) -pretreated rats. The appearance and disappearance of P450 paralleled the conversion of organic nitrates to NO. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a strategy to provide relief from ischemic conditions in humans.     

SNARE-dependent signaling at the Drosophila wing margin

The wing of Drosophila melanogaster has long been used as a model system to characterize intermolecular interactions important in development. Implicit in our understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. We have therefore chosen the Drosophila wing to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and we constructed a mutant form of NSF whose expression we directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, we find that the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, we used this phenotype to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. We conclude that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways will act as a sensitive reporter of partial membrane-trafficking disruption.     

Doc2beta is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis

The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2beta as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2beta was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2beta-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2beta in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2beta. The second C2 domain (C2B) of Doc2beta and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2beta complexes by addition of the Doc2beta binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2beta enhanced glucose-stimulated insulin secretion by approximately 40%, whereas siRNA-mediated depletion of Doc2beta attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2beta at the plasma membrane to facilitate exocytosis.     

Filamentous actin regulates insulin exocytosis through direct interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.